Cette Thèse

Dedicatoria Dialéctica

A Mi Papá

Cleofas Sánchez

José Luis Meza

Julien Carvalho

Mi

Keywords: Leishmania, LSD1, Macrophage, Épigénétique, Thérapies ciblant l'hôte Macrophage Lysine Demethylase LSD1: Implications for intracellular Leishmania survival and host-directed drug discovery Leishmania, LSD1, Macrophage, Epigenetics, Host-directed therapy

Titre : La Lysine Déméthylase LSD1 du macrophage : implications dans la survie intracellulaire de Leishmania et la découverte de drogues ciblant l'hôte.

RESUME EN FRANÇAIS

Les parasites Leishmania ont développé des stratégies sophistiquées pour modifier les processus cellulaires et mécanismes de défense des cellules hôtes à leur avantage. L'échec actuel de traitements chimio-thérapeutiques contre ces parasites augmente en raison de l'émergence de résistances à ces médicaments. Cette situation appelle à développer de nouvelles stratégies thérapeutiques évitant l'apparition de ces résistances. Ici, nous envisageons une thérapie ciblant l'hôte, notamment les voies des macrophages essentielles à la survie de Leishmania.

Nous avons précédemment identifié par criblage phénotypique et biochimique deux inhibiteurs épigénétiques susceptibles d'agir en ciblant l'hôte, i.e. dirigés contre des cibles de mammifères et réduisant la charge intracellulaire de Leishmania amazonensis tout en étant inefficaces sur les parasites libres. Ces composés ciblent la lysine déméthylase 1 (LSD1) des mammifères, un régulateur clé impliqué dans la polarisation des macrophages et le développement de tumeurs. Son action épigénétique consiste à enlever les groupes méthyle des lysines 4 et 9 de l'histone H3. Mon travail de doctorat, basé sur ces découvertes, a suivi trois objectifs majeurs : Premièrement, j'ai confirmé que le traitement avec ces inhibiteurs réduisait significativement la charge parasitaire dans deux modèles d'infection in vitro (macrophages primaires et une lignée cellulaire de macrophages). Puis j'ai confirmé leur relation composé-cible dans notre système cellulaire en confirmant que i) LSD1 était la cible de ces inhibiteurs (tests d'activité sur des extraits nucléaires de macrophages non infectés) et ii) que ces inhibiteurs interagissaient bien avec LSD1 car ils induisent une stabilisation thermique spécifique à LSD1 dans les échantillons non infectés et infectés (essai d'analyse thermique cellulaire, CETSA).

Mon deuxième objectif visait à évaluer l'influence de Leishmania sur divers aspects concernant LSD1. L'infection n'a modifié ni la quantité ni la localisation nucléaire, ni l'abondance des ARN correspondant à cette démethylase. En revanche, l'infection a modifié (i) sa stabilisation thermique (observée par CETSA), et (ii) ses interactions avec différents partenaires (test de co-immunoprécipitation suivi d'une analyse par spectrométrie de masse, co-IP-MS). Parmi ces partenaires, ont été détectées des protéines hôtes impliquées dans la biogenèse des ribosomes et le traitement des ARNm, ainsi que des protéines de Leishmania, manifestement auparavant ciblées vers le noyau des macrophages.

Enfin, j'ai analysé le protéome nucléaire de macrophages infectés et non infectés, et mis en évidence des différences significatives de contenu et d'abondance en protéines hôtes. L'ontologie génique et l'analyse STRING ont identifié des modulations liées à l'organisation de la chromatine, aux complexes HDAC ou au centrosome. Les prochaines étapes consisteront à étudier individuellement des protéines hôtes dont l'abondance a été modifiée en utilisant la surexpression lentivirale ou ARNsi knockdown. Comme observé dans mes études de co-IP-MS, le protéome nucléaire des macrophages infectés contenait plus de 100 protéines Leishmania, dont des histones et des composants flagellaires.

Les études futures nous fourniront un aperçu mécanistique du rôle de la LSD1 dans la survie intracellulaire de Leishmania. Par exemple, en délétant LSD1des macrophages primaires par CRISPR/Cas9 sur et en étudiant son impact sur la survie du parasite, ou en profilant à l'échelle du génome les changements dépendant de l'infection dans l'occupation des promoteurs de LSD1 et ses sites cibles dans l'histone H3. Des expériences visant à étudier le possible transfert de protéines de Leishmania vers le noyau de l'hôte et évaluer leur rôle dans l'expression génique des macrophages et la survie du parasite, seront réalisées par Western blot, microscopie confocale, des tests de précipitation, des études de suppression génique et expériences d'expression ectopique.

ACKNOWLEDGMENTS

REMERCIEMENTS

AGRADECIMIENTOS

It is hard for me to believe that I am finally writing the acknowledgments for my thesis. I must mention that from the beginning of this journey, a series of unfortunate events unfolded. I suppose the Force was always with me, and thanks to that, I was able to reach this point.

It all began with a multitude of administrative discrepancies between my Mexican fellowship and the stringent French regulations, which led to the establishment of a collaboration between two research units, and resulted in a two-month delay to my starting date. Followed by my first experience of a major SNCF strike (no, really? A strike in France?), which indirectly affected me during my first month in the lab. Only to find myself three months later plunged into a pandemic, confined to a 36-square-meter space, unable to return to my home country with my family due to the prevailing uncertainty, as I had a commitment here. Subsequently, navigating through lockdowns and curfews, along with a reduced workforce in the lab, continued to complicate the progress of my work, including the alternation between two laboratories and even the relocation of my faculty… Bref (a perfect French intervention), while pursuing a Ph.D. is inherently challenging, a series of unfortunate events in a foreign land, with a distinct culture and language, presented a formidable trial.

Yet, here I stand... First, I would like to express my gratitude to the members of the jury. To my rapporteurs, Joachim Clos and Luisa Figueiredo, for dedicating their time to reading this thesis, for their critical evaluations, and for their valuable comments included in the report they provided. To Natalie Fournier for presiding over my thesis defense. To Maria Adelaida Gomez, Isabelle Dimier-Poisson, and Eric Batsché for agreeing to be examiners of my work. To all of them for their questions and comments, which sparked an interesting and constructive scientific discussion enriched by their extensive expertise and knowledge. I would also like to express my deepest gratitude to my thesis director, Gerald Späth. Thank you for the trust you placed in me, for your tenacity and the countless ways you worked tirelessly to ensure my involvement in this project despite the administrative issues I mentioned above. Your dedication and optimism have been a constant source of motivation for me. I have always believed that anyone else might have given up, but your perseverance and optimism did not allow it. I appreciate your constant attention to my progress in the experiments and your consistently relevant feedback, which has been instrumental in improving the work being conducted. Your guidance and invaluable advice throughout these four years have been truly helpful. Thank you for reading, correcting, and improving this thesis. Despite your demanding schedule, you have consistently been there for me at every step. I also want to mention how grateful I am for the significant opportunity you provided for me to present my work at the WorldLeish conference. Being a part of Parsig and having you as my director has been an enormous honor. Thank you for everything.

À Philippe Loiseau, mon codirecteur de thèse, pour avoir accepté d'établir la collaboration qui m'a permis de m'inscrire à l'Université Paris-Saclay, me donnant ainsi l'opportunité de travailler sur ce projet. Mon doctorat n'aurait pas été possible sans toi. Je te remercie du fond du coeur pour ta disponibilité constante et ton soutien inestimable dans toutes les démarches administratives de l'école doctorale.

À Eric Prina, alias mon "jefecito". Si les règles le permettaient, tu serais le troisième codirecteur de cette thèse. Je te remercie pour ta grande gentillesse et pour être devenu une sorte de père adoptif dans le monde de la science. Merci d'avoir écouté toutes mes plaintes du matin (quand j'arrivais à me lever tôt, juste pour les partager avec toi) et d'avoir supporté tous mes soupirs de frustration, que ce soit à cause des problèmes administratifs ou des obstacles expérimentaux. Merci d'avoir partagé tes expériences et astuces pour surmonter ces obstacles, pour les protocoles que tu as réussi à extraire de ta tête et que tu as retranscrits pour que je puisse les comprendre plus facilement, tout comme les protocoles que tu n'as pas réussi à extraire, mais pour lesquels tu as trouvé le moyen de les partager d'une manière ou d'une autre. Je suis reconnaissant que tu n'apprécies pas trop parler anglais, car tu es également devenu mon professeur de français. Merci de me corriger quand c'est nécessaire, de m'apprendre continuellement de nouvelles expressions familières et pour les livres que tu m'as offerts pour mieux comprendre ta langue parfois illogique, dans laquelle tu commets toi-même quelques petites erreurs, hihi. Je te remercie pour tes questions philosophiques du genre « Mais qu'est-ce que la beauté ? », tes blagues XY, ou celles classées comme "marteliennes", tout aussi mauvaises, voire pires, ainsi que pour tes phrases ridicules sur les gens des pays en développement ou ceux de l'altiplano, car tu as toujours réussi à me faire rire de cette manière et à rendre ces années plus agréables. Bien sûr, merci d'avoir lu et corrigé cette thèse, ainsi que pour ton aide dans la préparation du manuscrit et de la présentation. Enfin, même si j'étais prévenue avant ma soutenance et que je savais que je n'obtiendrais plus de réponses de ta part à mes nombreuses questions, j'espère que tu sais que je continuerai à les poser. J'ai définitivement eu de la chance d'être la padawan d'un tel maître Jedi.

À Hervé Lecoeur, alias Don Gordito (qui, soit dit en passant, est devenu plutôt Don Flaquito), mon co-encadrant non officiel. Je tiens à t'exprimer ma profonde gratitude pour avoir été le premier lecteur et correcteur de cette thèse, ainsi que pour avoir supporté ma présentation je ne sais combien de fois et pour m'avoir prodigué de précieux conseils pour l'améliorer, en pensant aux potentielles questions qui pourraient m'être posées. Je te suis également reconnaissante pour tout ton soutien dans la préparation et la rédaction du préprint, car sa publication juste avant ma soutenance n'aurait pas été possible sans toi. Merci aussi pour toutes tes anecdotes amusantes si bien racontées dans la mezzanine, qui ont rendu cette période à Parsig plus agréable. Pour les recommandations de séries, même si nous avons conclu que nos goûts ne sont pas très similaires, il est important de découvrir un peu de tout. Enfin, merci pour les magnifiques housses de coussin, je suis toujours touchée par ce beau et représentatif cadeau. Je n'ai pas encore trouvé l'endroit parfait pour les mettre, j'y réfléchis toujours... À Mme Cheffe Pascale Pescher, alias La Patrona, pour toutes les pauses café et les discussions qui ont suivi, abordant des sujets divers et variés. Merci pour les mélodies qui émanaient de ton coin de bureau, ainsi que pour les conversations que tu avais avec toi-même ou pour ces rares mots un peu crus qui t'échappent de temps en temps. Je considère que tout cela a contribué à créer une ambiance spéciale pendant ces années passées ici. Je te remercie d'avoir toujours été si franche et directe. Tes précieux conseils et recommandations à tout moment, et la grande aide que tu m'as apportée quand tu as rappelé à Eric que je devais arrêter les manips pour que je puisse commencer à écrire cette thèse. Je tiens aussi à te remercier pour cet événement traumatisant qui me permet maintenant de couper le fromage correctement. Sois certaine que dans ma tête, résonnera éternellement : « Mais qui a coupé ce fromaaaaage ?! » N'oublie pas que je t'attends au Mexique après ta retraite ;) Et bien sûr, un grand merci au reste des membres de l'équipe ParSig. Merci à Najma Rachidi pour ton soutien dans l'analyse des données de protéomique, tes conseils et tes commentaires toujours pertinents. À Olivier Leclercq pour les protocoles si clairs que tu m'as partagés, ainsi que pour les astuces qui ont amélioré mes manips. À Laura Piel, pour avoir corrigé cette thèse avec un regard extérieur et pour l'aide que tu m'as apportée à chaque fois que je t'en ai fait la demande. À Isabelle Louradour, pour m'avoir appris à choisir les batailles à mener et à parfois simplement laisser tomber.

Thanks should also go to Ana Murta Santi, for our shared ideologies and engaging discussions, as well as for being part of the project we have in common now. To my PhD mates, Sheng Zhang, for always being willing to help and for the shared laughter while listening to Herve's sneezes from the other side of the mezzanine; Sharvani Shintre and Paul Jenkins, for being my fellow padawans and making the journey more enjoyable.

A Marcela Fuentes, mi doñis, el ingrediente latinoamericano dentro que me faltaba en esta vida alejada de mi país para poder sentir que pertenezco a un lugar. La amistad siempre es una pieza clave. Gracias por tu confianza, por tu apoyo y comprensión incondicionales, por los chambres y por todos los momentos compartidos dentro y fuera de Pasteur que espero no terminen nunca.

À Christel Ricard, pour ton soutien constant dans les questions administratives depuis le début de cette histoire tumultueuse. À Christine Maillet et Sylviane Etiemble, pour votre gentillesse et votre soutien technique, qui sont essentiels au bon fonctionnement du laboratoire. À Geneviève Milon, pour votre passion pour la science qui contamine tout votre entourage. À Juliana Pipoli, Da Fonseca pour avoir partagé avec moi ton expérience dans la réalisation de mes manipulations de ChIPmentation. Et à Charfeddine Gharsallah, pour ta maîtrise experte de Cytoscape, ainsi que pour ta disponibilité et ton soutien lors de la rédaction du pré-print.

To all, thank you for the moments we have shared within and beyond Pasteur, the afterwork gatherings, retreats, and even a journey to the other side of the world. I never cease to realize how fortunate I am.

Je tiens à remercier aussi Florent Dingli et Damarys Loew de la plateforme de spectrométrie de masse de l'Institut Curie, ainsi qu'aux membres du Hub de Bioinformatique et Biostatistique et Biomics, Hugo Varet, Rachel Legendre, Thomas Cokelaer et Rania Ouazahrou, je tiens à exprimer ma gratitude pour votre soutien dans les analyses effectuées. Special thanks to Antonello Mai's team for the chemical compounds, and to Manfred Jung for his collaboration on the ELATION project.

Aux membres de l'équipe Parachem-BioCIS de la Faculté de Pharmacie, Indira Dennemont, Marie-Nadine Hilarion et Sandrine Cojean, je tiens à exprimer ma gratitude pour votre soutien tout au long de mon séjour au laboratoire. Je tiens également à remercier Sébastien Pomel pour les commentaires apportés à mon manuscrit de thèse, contribuant ainsi à son amélioration. À Sameh Obeid et Aurélie Mauras, pour les agréables moments partagés dans le bureau des étudiants, ainsi qu'à Zineb Fechtali-Moute, avec qui j'ai noué une précieuse amitié. Merci Zineb d'avoir toujours été prête à m'aider, à répondre à toutes mes questions, et pour toutes les conversations même en dehors du bureau. Enfin, à Imad Kansau et Audrey Antoine, pour votre soutien précieux dans les processus administratifs au sein de l'école doctorale.

Además, quiero dedicar un espacio a todas las personas que, aunque no participaron directamente en este trabajo, ejercieron una influencia indirecta, sobre todo en la preservación de mi cordura, que, espero, aún persista.

A mi mamá, Mary Sánchez, mi querida "cabecita blanca". Esta es la tercera vez que escribo estas palabras, esperando que sea la última, no porque cambie de opinión, sino porque deseo que este sea el último documento de esta índole que produzca. ¡GRACIAS POR TODO! Sigo sin saber cómo gritarlo más fuerte. En este caso extremo, debería ser capaz de gritarlo tan fuerte como para hacerte llegar el mensaje al otro lado del Atlántico. El tiempo, la distancia y, quiero pensar, que un poco de madurez, solo un poco, me han permitido confirmar lo afortunada que fui al tener una mamá como tú. Quiero que sepas que tienes toda mi admiración. Hoy no estaría escribiendo esto si no fuera por ti. Gracias por darme todo, no hizo falta nada ni nadie.

A Mamá Silvia, mejor conocida como Bombis. Definitivamente, tú también formas parte de la tesis que me construyó. Gracias por cuidarme durante toda mi vida. Gracias por estar al pendiente del teléfono por las tardes porque sabes que te voy a llamar. Por seguir consintiéndome cada vez que voy y por preparar o mandar a hacer lo que sabes que me gusta comer. Por seguir dándome dinero a escondidas de mi mamá (no es el dinero en sí, sino el gesto, lo que hace esto extremadamente tierno). Por ser el pilar de la familia y haber estado presente para todos nosotros. Gracias por ser una de mis razones principales para volver.

A mi familia de allá. A mis tías, tíos y primos/hermanos que cada vez que regreso a México me consienten y me recuerdan, junto con Bombis, que siempre me sobrarán razones para volver a mi país, a mi hogar.

A mis amigos de allá. A Mayte Malagón, por ser mi mejor amiga, por tu apoyo incondicional y por permanecer a pesar del tiempo y la distancia; a José Meneses, por seguir siendo parte del jardín de cronopios; a Rebeca Montero, Babeee, el tiempo sigue pasando y yo sigo confirmando que todo "siempre sale", gracias por haber estado presente incluso a distancia; a Vladimir Loginova, por madrugar para asistir virtualmente a mi defensa y por ser el amigo más antiguo que tengo, todo gracias a Chapingo. Gracias a todas esas bellas personas en México que me ayudan a comprobar que la amistad crece en tres soles y una luna y que permanece a través de los mundos; cada vez que los veo, es como si nos hubiésemos visto un día antes, como si el océano nunca nos hubiera separado.

A mis amigos de aquí, por hacer más llevadera la experiencia de estar tan lejos de casa y ser una migrante más en este país tan distante de mi hogar, ustedes conformaron en cierta forma uno nuevo. À Moïra et Manu du Lèche vin, pour m'accueillir toujours aussi chaleureusement dans ce bar qui me fait me sentir chez moi. Pour tous les weekends où le bar est devenu un refuge après une semaine compliquée.

À ma famille d'ici : Manu, Nico, Elise et Sophie. Merci à toi de m'avoir adoptée comme belle-fille, d'être toujours si gentille avec moi, et d'être présente dans des moments si importants. Pour tous les plats que tu as cuisinés pour me faire découvrir cette autre facette de la France, que l'on ne connaît qu'en faisant partie d'une famille française. Merci aussi pour ta tolérance à comprendre que mes coutumes peuvent être complètement différentes et pour trouver des moyens de nous adapter en conséquence. It is worth mentioning that the majority of individuals infected with a Leishmania parasite do not exhibit any symptoms throughout their lives. As a result, the term leishmaniasis specifically denotes the state of developing clinical pathologies as a consequence of a Leishmania infection, rather than only being infected.

LIST OF FIGURES

GEOGRAPHIC DISTRIBUTION AND CLINICAL FEATURES

Distinct species of Leishmania cause different clinical manifestations, the two main forms being visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL).

The outcome of the disease is influenced by various factors, including parasite characteristics, vector biology and host immune status.

Leishmaniases are found in individuals on every continent except Australia and Antarctica. Geographically, endemic regions can be divided into the New World (the Americas) and the Old World (Africa, Asia and Europe). In 2018, according to the World Health Organization (WHO), 92 and 83 countries were considered endemic for CL and VL, respectively. Currently, over 1 billion people worldwide are at risk of infection as they reside in areas considered endemic. Annually, it INTRODUCTION 4 is estimated that there are over 1 million new cases of CL and around 300,000 new cases of VL.

Cutaneous Leishmaniasis (CL)

According to the WHO, an estimated 700,000 to 1 million cases of cutaneous leishmaniasis are reported worldwide per year. Around 90% of the cases are concentrated in countries such as Afghanistan, Pakistan, Syria, Saudi Arabia, Algeria, Iran, Brazil and Peru (Figure 1). CL is caused by different species of Leishmania parasites. The Old World species, including L. major, L. tropica and L. aethiopica, are prevalent in the Mediterranean Basin, the Middle East, the Horn of Africa and the Indian subcontinent. On the other hand, the New World species, including L. amazonensis, L. mexicana, L. braziliensis and L. guyanensis, are primarily found in Latin America [1].

Figure 1. Global prevalence of cutaneous leishmaniasis in 2020 [2].

CL in the Americas presents a complex epidemiology characterized by the coexistence of multiple Leishmania species, various reservoir hosts and sandfly vectors, as well as diverse clinical manifestations and treatment responses.

INTRODUCTION 5

in the New World, lesions will exhibit diverse severity and healing rates depending on the Leishmania species [3]. While CL is generally considered a milder form of the disease, a small percentage (1-10%) of patients infected with the Leishmania Viannia subgenus may develop mucocutaneous leishmaniasis. MCL is mainly caused by L. braziliensis and is characterized by destructive lesions of the oronasopharyngeal mucosa and cartilage, lips and palate (Figure 2C). These lesions commonly arise from a previous CL episode and are more frequent in immunocompromised people. The advanced stage of the disease can be severely disfiguring and life-threatening, as destructive lesions can affect the larynx, leading to aspiration pneumonia [3].

Figure 2. Patients with cutaneous leishmaniasis. Chiclero's ulcer by L. mexicana (A) [5].

Ulcerative lesion by L. panamensis (B) [START_REF] Knapp | Cutaneous Leishmaniasis[END_REF]. Mucocutaneous necrotic ulcer (C) [START_REF] Crovetto-Martinez | Mucocutaneous leishmaniasis must be included in the differential diagnosis of midline destructive disease: two case reports[END_REF].

INTRODUCTION 6 of visceral leishmaniasis. Nevertheless, the disease is endemic in more than 60 countries (Figure 3). In the past decade, the global incidence showed a significant decrease. The number of new cases reported in 2012 ranged between 200,000 and 400,000 [START_REF] Alvar | Leishmaniasis worldwide and global estimates of its incidence[END_REF], whereas in 2023, it ranged between 50,000 and 90,000. However, it is important to note that only 25 to 45% of these cases are reported to the WHO, as leishmaniasis is not a notifeable disease in various countries, including France.

Figure 3. Global prevalence of visceral leishmaniasis in 2020 [START_REF] Who | Status of endemicity of visceral leishmaniasis worldwide[END_REF].

Visceral leishmaniasis is characterized by persistent irregular fever and splenomegaly (Figure 4A). Patients may also experience pancytopenia, hepatomegaly (an enlargement of the liver beyond its normal size), hyper gammaglobulinemia (the overproduction of more than one class of immunoglobulins per plasma cell) and weight loss. Acute malnutrition is associated with a high parasite burden in young children, although it is unclear whether this is a cause or consequence of poor nutritional status. The onset can be acute or gradual, with an incubation period between 2 weeks to 8 months. Without treatment, the disease is usually fatal within two years due to secondary bacterial infections or anemia. However, some infected people may not develop symptoms until years later once they become immunosuppressed [10]. for visceral leishmaniasis caused by L. donovani. It manifests as a rash with macular, papular or nodular lesions, commonly found on the face, upper arms and trunk (Figure 4B). PDKL is endemic in East Africa, particularly in Sudan but also in Ethiopia and Kenya, and in the Indian subcontinent (India, Nepal and Bangladesh). It is observed in 5-10% of VL cases in Asia, appearing 2-3 years after treatment, and up to 50% of VL cases in East Africa, typically occurring during or within 1 year after treatment. The development is associated with an immune response driven by interferon-γ in the host, which targets the residual dermal parasites [11].

Importantly, the lesions are infectious to sandflies serving as reservoirs of infection during interepidemic periods [3].

DIAGNOSIS

Cutaneous and Mucocutaneous Leishmaniasis

Diagnosing CL and MCL, especially in the New World, can benefit from species identification for clinical care. Currently, there is no single reference test, but amastigotes observation in a clinical specimen, obtained by scraping, fineneedle aspiration or biopsy of a small piece of tissue, confirms the diagnosis.

The biopsy provides a more abundant sample, advantageous when parasites are scarce and can be used for different purposes, such as culturing the parasites or conducting histopathology, but they do not provide species identification [3].

INTRODUCTION 8

Serological tests and the Leishmanin Skin Test (LST), also known as the Montenegro test, have limited utility in diagnosing CL. However, new immunological tests are emerging and show promise in differentiating Leishmania species [START_REF] Al-Salem | Detection of high levels of anti-α-galactosyl antibodies in sera of patients with Old World cutaneous leishmaniasis: a possible tool for diagnosis and biomarker for cure in an elimination setting[END_REF], such as chemiluminescent ELISA to measure anti-αgalactosyl antibodies, or the commercially available cutaneous leishmaniasis Rapid Diagnostic Test (RTD) that detects the parasite peroxidoxin antigen.

Visceral Leishmaniasis

VL, characterized by febrile splenomegaly, requires confirmation through diagnostic tests due to its resemblance to other infectious diseases. The classical approach is microscopic observation of amastigotes in tissue samples or cultures, but sensitivity varies depending on the tissue, with higher sensitivity in spleen and bone marrow aspirates [START_REF] Sundar | Laboratory diagnosis of visceral leishmaniasis[END_REF].

Serological assays exhibit high sensitivity for acute visceral disease but lack specificity for this stage and can yield false-positive results. RTDs using antigens such as rK39 (based on kinesin sequence) and rK28 (comprising kinesin and hydrophilic acylated surface proteins sequences) show good sensitivity but have limitations and are not reliable for diagnosing relapses [START_REF] Mukhtar | Diagnostic accuracy of rK28-based immunochromatographic rapid diagnostic tests for visceral leishmaniasis: a prospective clinical cohort study in Sudan[END_REF]. Antigen detection tests in urine, such as KAtex test and prototype ELISA kits, offer promising diagnostic solutions with high specificity but moderate sensitivity [START_REF] Vallur | Development and comparative evaluation of two antigen detection tests for Visceral Leishmaniasis[END_REF]. While PCR has a role in diagnosis, its complexity and cost often limit its use in resource-limited settings. On the other hand, the LST measures cellular immune response but is not suitable for diagnosing active disease, serving better as a marker of previous exposure in epidemiological surveys [3].

Post-Kala-azar Dermal Leishmaniasis

PDKL diagnosis can be challenging due to its similarity to other conditions such as leprosy, vitiligo, and fungal infections. The diagnostic method normally used is the observation of amastigotes in slit-skin smears or biopsy specimens of lesions, but the sensitivity of these technologies varies depending on the type of lesion. Serological tests are inconclusive as tests relying on antileishmanial antibodies can remain positive even after successful treatment for VL. Molecular methods such as PCR to detect the small subunit ribosomal RNA (SSU rRNA), kDNA or the REPL-repeat region of Leishmania, have shown high sensitivity but low specificity in diagnosing PDKL [START_REF] Adams | Systematic Review into Diagnostics for Post-Kala-Azar Dermal Leishmaniasis (PKDL)[END_REF]. In endemic areas where slit-skin smears and molecular test are not feasible, the WHO recommends a clinical algorithm for diagnosis. However, the sensitivity and specificity of this procedure have not been evaluated yet and there may be a significant number of false-positive results [START_REF] Health | Post-Kala-Azar dermal leishmaniasis: a manual for case management and control[END_REF]. Further research is still needed to improve diagnostic approaches INTRODUCTION 9 and ensure accurate identification of PDKL cases.

LEISHMANIA PARASITE

Leishmania are protozoan parasites belonging to the phylum Euglenozoa, class Kinetoplastea, order Trypanosomatida, and family Trypanosomatidae. Initially, the classification of Leishmania relied on ecobiological and clinical criteria, but this was shown to be inadequate. To improve classification accuracy, biochemical and molecular analyses were introduced. These methods involved studying polymorphisms in kDNA markers or whole genome sequences, proteins and antigens, which allowed a better understanding of the parasite's diversity and evolution [START_REF] Banuls | Leishmania and the leishmaniases: a parasite genetic update and advances in taxonomy, epidemiology and pathogenicity in humans[END_REF].

The Leishmania genus is currently classified into four subgenera: Leishmania, Viannia, Sauroleishmania and Mundinia. The subgenus Sauroleishmania specifically infects reptiles, while the subgenera Leishmania and Viannia are responsible for the majority of human infections. However, the recently identified subgenus Mundinia has also been found to infect humans [START_REF] Espinosa | An appraisal of the taxonomy and nomenclature of trypanosomatids presently classified as Leishmania and Endotrypanum[END_REF]. The subgenus Viannia is primarily found in the Neotropical region of the New World, where it is the main cause of mucocutaneous leishmaniasis, whereas the subgenus Leishmania is present in both the New and Old World [1]. More than 50 Leishmania species have been described. Of these, 31 species are known parasites of mammals or reptiles and around 20 species have been reported pathogenic for human beings ( Leishmania parasites have a digenetic life cycle involving both a vertebrate host and an insect host the latter serving as vector for parasite transmission. They exhibit various cell morphologies and developmental forms adapted to these different hosts, but the two major cell morphologies are the promastigote form (Figure 5A) in the sandfly and the amastigote form (Figure 5B) in the mammalian host. Despite morphological differences, both cell shapes share a conserved basic cellular architecture with unique features, such as a crosslinked, sub-pellicular microtubular network or a single mitochondrion. The kinetoplast, a mass of concatenated mitochondrial DNA, is positioned anterior to the nucleus and is directly linked to the basal body from which the flagellum emerges. At the base of the flagellum, a membrane invagination forms the flagellar pocket, which plays a crucial role as it serves as the exclusive site for endocytosis and exocytosis [START_REF] Sunter | Shape, form, function and Leishmania pathogenicity: from textbook descriptions to biological understanding[END_REF].

INTRODUCTION 10

Leishmania life cycle

Following a blood meal, ingested intracellular amastigotes are released inside the sandflies gut from infected blood cells and transform into promastigotes.

In a subsequent blood meal, infectious metacyclic promastigotes are regurgitated and injected into the vertebrate skin to complete the life cycle (Figure 6).

Stages of the parasite in sandflies

During their blood feeding process on an infected vertebrate host, female sandflies ingest Leishmania amastigote parasites residing in host mononuclear cells. First the sandflies, blood feeding arthropods that severs skin and blood vessels known as telmophages, insert their mouthparts into the host skin, causing a small wound that exposes infected macrophages from skin or blood.

INTRODUCTION 12

Then blood is ingested into the sandfly midgut, characterized by a lower temperature and a higher pH than in the mammalian intracellular environment, which rapidly triggers amastigote differentiation into noninfective, procyclic promastigotes that replicate in the blood meal in the abdominal midgut.

Figure 6. Leishmania life cycle inside the sandfly vector (A) and the vertebrate host (B) [START_REF] Yasmin | Pathogen Interaction in Leishmaniasis: Immune Response and Vaccination Strategies[END_REF].

During the initial "blood meal phase", parasites are enclosed within the peritrophic matrix, a chitin and protein mesh secreted by the midgut epithelium. After a few days, these parasites differentiate into elongated nectomonad promastigotes that go out from the peritrophic matrix with the help of a parasite secretory chitinase. These promastigotes, move towards the anterior midgut, some of them attach to the epithelium microvilli, and eventually reach the stomodeal valve. Once there, they differentiate into shorter, replicative leptomonad promastigotes. This form secretes the promastigote secretory gel (PSG) that plays an important role in transmission. Some leptomonads attach to the cuticle-lined surface and differentiate into haptomonad promastigotes, whereas others will finally develop into infective, nondividing metacyclic promastigotes in a process termed "metacyclogenesis". Metacyclic promastigotes are also capable to de-differentiate inside the sandfly into retroleptomonad promastigotes, increasing parasite load in its next blood meal.

Seven to 14 days are necessary for amastigotes to complete their differentiation into infective metacyclic promastigotes (Figure 6A) [START_REF] Bates | Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies[END_REF][START_REF] Bates | Revising Leishmania's life cycle[END_REF][START_REF] Gossage | Two separate growth phases during the development of Leishmania in sand flies: implications for understanding the life cycle[END_REF][START_REF] Rogers | The role of promastigote secretory gel in the origin and transmission of the infective stage of Leishmania mexicana by the sandfly Lutzomyia longipalpis[END_REF][START_REF] Serafim | Sequential blood meals promote Leishmania replication and reverse metacyclogenesis augmenting vector infectivity[END_REF].

INTRODUCTION 13 1.3.1.

Stage inside host phagocytes

Highly motile metacyclic promastigotes have the ability to migrate through a collagen matrix, facilitating their invasion beyond the site of the insect bite. These metacyclics are taken up by phagocytic cells like neutrophils and macrophages present in the skin and infiltrating the bite site. Parasites are internalized within phagosomes that rapidly transform into parasitophorous vacuoles (PV), specialized compartments with acidic pH and hydrolytic activity. Within these vacuoles, high temperature and low pH trigger differentiation into proliferating amastigotes. In the host, the parasite spreads to new cells via yet unidentified mechanisms possibly involving release from infected cells, phagocytosis of dying infected cells or direct transfer from an infected to an uninfected cell (Figure 6B) [START_REF] Sunter | Shape, form, function and Leishmania pathogenicity: from textbook descriptions to biological understanding[END_REF]. Interestingly, cell types other than macrophages, including dendritic cells or fibroblasts, may also be susceptible to infection; potentially harboring a yet-to-be-discovered quiescent stage specialized for transmission from the mammalian to the insect host.

Vector

Phlebotomine sandflies are responsible of transmitting various pathogens that can affect humans and animals worldwide, including Leishmania. However, besides acting as a vector for Leishmania, phlebotomine sandflies serve as hosts where a complex process of parasite differentiation takes place as described above.

Sandflies are insects included in the order Diptera (two-winged flies), suborder Nematocera, family Psychodidae, and subfamily Phlebotominae. There are around 1000 described species or subspecies of sandflies in the world, categorized into six genera: Phlebotomus, Sergentomyia and Chinius in the Old World and Lutzomyia, Brumptomyia and Warileya in the New World. Only 98 are proven or suspected vectors of human leishmaniasis, all of which belong to the genera Lutzomyia (56 in the New World) and Phlebotomus (42 in the Old World). According to the WHO, they fulfill the criteria for vector incrimination, including i) feeding on humans, ii) feeding on relevant reservoir hosts, iii) being infected with the same Leishmania species found in humans, iv) supporting the complete development of Leishmania parasites, and v) transmitting the parasites to susceptible hosts when they take a blood meal [START_REF] Killick-Kendrick | Phlebotomine vectors of the leishmaniases: a review[END_REF][START_REF] Cecilio | Sand flies: Basic information on the vectors of leishmaniasis and their interactions with Leishmania parasites[END_REF]. INTRODUCTION 14 nocturnal activity and cannot fly long distances (less than 300 m) or at high speed (less than 1 m/s). Both, males and females feed on plant sugars, and only females require a blood meal to develop and lay eggs [START_REF] Maroli | Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern[END_REF].

Figure 7. Lutzomyia longigalpis (left panel) and Phlebotomus papatasi (right panel) [34,[START_REF] Cdc | Phlebotomus papatasi sand fly[END_REF].

Sandflies can be classified as restrictive or permissive vectors based on their ability to disseminate specific Leishmania species. Restrictive vectors have specific interactions with the parasites they transmit, while permissive vectors can transmit different Leishmania species. Parasite attachment to the sandfly midgut is crucial for infection, involving specific interactions between ligands and receptors, particularly in the case of refractory vectors. The mechanisms of permissive vector interactions are not fully understood, but it is suggested that sandfly midgut proteins may play a role. Differences in parasite surface molecules, like the glycolipid lipophosphoglycan (LPG), contribute to the specificity and attachment processes in restrictive vectors [START_REF] Cecilio | Sand flies: Basic information on the vectors of leishmaniasis and their interactions with Leishmania parasites[END_REF].

In addition to their role as vector/host in the parasite life cycle, they may contribute directly to the pathogenesis, since during the feeding process they inject salivary gland content into the host skin, which contains various agents such as anticoagulants, vasodilators, anti-platelet agents, immunomodulatory and anti-inflammatory molecules that affect hemostasis, immune responses and parasite establishment [START_REF] Gomes | The immune response to sand fly salivary proteins and its influence on leishmania immunity[END_REF].

Reservoirs

Leishmaniasis is an anthropozoonotic disease that describes an infection cycle between humans and animals. Hence, Leishmania has the ability to infect a wide range of reservoir animals. The majority of infections are initially zoonotic but there are cases of human-to-human transmission suspected for L. donovani. Depending on the reservoir, leishmaniases have different epidemiological cycles: the primitive or sylvatic cycle, where human infection is accidental and occurs in wild areas; the secondary or peridomestic cycle, where the reservoir is a domestic or peridomestic animal and humans are infected by sandflies that feed on both animals and humans; and the tertiary anthroponotic cycle, where there is no identified animal reservoir and the sandflies feed exclusively on humans [START_REF] Banuls | Leishmania and the leishmaniases: a parasite genetic update and advances in taxonomy, epidemiology and pathogenicity in humans[END_REF].

The presence of infection in a vertebrate is not enough to call it a reservoir host, which are defined according to the WHO by the following criteria: i) the reservoir host should be abundant and long-lived enough to serve as a significant food source for sandflies, ii) there should be intense contact between the host and sandflies, especially in colonial animals (e.g. rodents living in burrows) that provide suitable habitat conditions for sandflies, iii) more than 20% of the individuals within the reservoir host population should become infected during their lifetime, although the prevalence can vary with the season, iv) the infection in the reservoir should have a long enough duration and be nonpathogenic to allow the parasites to survive during no transmission seasons, and v) the parasites should be available in sufficient numbers in the host skin or blood to be taken by sandflies.

The principal reservoir hosts for different species of Leishmania vary depending on the region. The following is a summary of the various reservoir hosts according to the current literature:

INTRODUCTION 16

Sigmodon that participate in the transmission cycle of L. mexicana [37].

INTERACTIONS BETWEEN LEISHMANIA PARASITES AND HOST CELLS

Leishmania parasites have developed strategies to evade and manipulate the immune system and metabolism of their hosts to ensure their survival and growth. The following is a brief description of these strategies used by Leishmania to escape and/or subvert the host immune response.

Leishmania parasites are able to evade complement-mediated lysis, an innate immune defense mechanism of mammalian serum triggered upon their entry into a mammalian host. Leishmania parasites have the ability to activate either the classical or alternative pathway of the complement system. When complement proteins opsonize the parasites, it enhances their phagocytosis by macrophages or neutrophils. The metacyclic form of Leishmania expresses a higher level of the metalloprotease GP63 on its surface compared to procyclic promastigotes. GP63 cleaves the C3b complement component into its inactive form, iC3b. Although iC3b retains its opsonic properties, it also hinders the formation of the C5 convertase, thereby preventing the assembly of the full Membrane Attack Complex (MAC) [START_REF] Geiger | Escaping Deleterious Immune Response in Their Hosts: Lessons from Trypanosomatids[END_REF][START_REF] Hoover | Killing of Leishmania tropica amastigotes by factors in normal human serum[END_REF].

Leishmania metacyclic promastigotes have a thick glycocalyx on their surface containing various molecules. These molecules, such as glycophosphatidylinositol lipids (GIPLs), lipophosphoglycan (LPG), and proteophosphoglycan (PPG) play different roles in infection depending on the Leishmania species. The complement component C3 can bind to LPG, but during metacyclogenesis, LPG undergoes modifications involving the elongation by addition of galactose/mannose repeats. This modification prevents the MAC from reaching the parasite's membrane, thus avoiding lysis. It is worth noting that not all promastigote forms are equally resistant to complement-mediated lysis, and this is largely attributed to the differences in their LPG structures [START_REF] Geiger | Escaping Deleterious Immune Response in Their Hosts: Lessons from Trypanosomatids[END_REF][START_REF] Spath | The role(s) of lipophosphoglycan (LPG) in the establishment of Leishmania major infections in mammalian hosts[END_REF][START_REF] Puentes | Complement binding by two developmental stages of Leishmania major promastigotes varying in expression of a surface lipophosphoglycan[END_REF].

The parasites that survive complement-mediated lysis are efficiently phagocytized. The uptake of promastigotes by macrophages is achieved through receptor-mediated phagocytosis. Various receptors on macrophages, including complement receptors (CR3, CR1), Fc receptors, lectin receptors such as the mannose fucose receptor, and integrins, are involved in this process and in determining the parasite's intracellular fate [START_REF] Ueno | Receptor-mediated phagocytosis of Leishmania: implications for intracellular survival[END_REF].

Once inside, the phagosome undergoes maturation by fusing with endosomes and lysosomes. This process involves the transition from early phagosomes (moderately acidic) to late phagosomes (increased acidity) through the action of Rab5 and Rab7 GTPases. The fusion of late phagosomes with lysosomes forms phagolysosomes or parasitophorous vacuoles, which exhibit membrane expression of lysosomal markers (LAMP-1, LAMP-2). The morphology of PVs differs depending on the Leishmania species. For instance, L. amazonensis amastigotes form large communal PVs, which may aid in diluting toxic nitric oxide. On the other hand, L. donovani resides in tightly enclosed individual PVs [START_REF] Antoine | The biogenesis and properties of the parasitophorous vacuoles that harbour Leishmania in murine macrophages[END_REF][START_REF] Courret | Biogenesis of Leishmania-harbouring parasitophorous vacuoles following phagocytosis of the metacyclic promastigote or amastigote stages of the parasites[END_REF].

Once inside their host cells, the fate of the parasites largely depends on the cellular immune response. This response establishes whether the microbicidal functions of macrophages will be activated or not. A Th1-type response triggers lymphocytes to produce proinflammatory cytokines (such as TNFα,and INFγ), which activate macrophages and promote parasitic control. These activated macrophages produce microbicidal effectors (NOS, ROS), resulting in parasite death and preventing the recurrence of chronic latent infection.

Conversely, a Th2-type response involves the production of anti-inflammatory cytokines (IL-4, IL-10, IL-13, TGF-β), which deactivate macrophages and hinder infection control. Deactivated macrophages produce urea and L-ornithine, crucial for the synthesis of parasite polyamines. This prevents extensive tissue destruction but facilitates intracellular infection. Therefore, the outcome of a Leishmania infection can be summarized as a delicate balance between the activation of microbicidal functions and the subversion of these functions by intracellular parasites [START_REF] Carneiro | The Paradox of a Phagosomal Lifestyle: How Innate Host Cell-Leishmania amazonensis Interactions Lead to a Progressive Chronic Disease[END_REF][START_REF] Chandra | Leishmania donovani infection down-regulates TLR2stimulated IL-12p40 and activates IL-10 in cells of macrophage/monocytic lineage by modulating MAPK pathways through a contact-dependent mechanism[END_REF][START_REF] Olivier | Subversion mechanisms by which Leishmania parasites can escape the host immune response: a signaling point of view[END_REF].

Leishmania parasites have evolved mechanisms to control the fate of host immune cells. They can influence the lifespan of neutrophils, either prolonging it to create a protective intracellular environment or shortening it to dampen the immune response and facilitate parasite growth. Neutrophils, being the first phagocytes recruited to the infection site, play a crucial role in the parasite's establishment and dissemination through a "Trojan horse" strategy. Leishmania delays neutrophil apoptosis and triggers the release of chemotactic molecules, attracting dendritic cells and macrophages to the site. Additionally, this process involves the production of TGF-β, which aids in the silent entry of parasites into macrophages. These modulations are governed by distinct molecular pathways, including the inhibition of procaspase-3 and activation of extracellular kinases (ERK1/2) [START_REF] Geiger | Escaping Deleterious Immune Response in Their Hosts: Lessons from Trypanosomatids[END_REF][START_REF] Gannavaram | Programmed cell death in Leishmania: biochemical evidence and role in parasite infectivity[END_REF][START_REF] Van Zandbergen | Cutting edge: neutrophil granulocyte serves as a vector for Leishmania entry into macrophages[END_REF].

CONTROL, PREVENTION

AND TREATMENT OF LEISHMANIASIS

CONTROL STRATEGIES

Many countries experience delays in initiating leishmaniasis treatment due to a lack of awareness and proactive measures, as they rely on individuals to seek medical attention when they exhibit symptoms, instead of implementing campaigns for early diagnosis. This shortcoming contributes to the prolonged transmission of the parasite within the communities. For this reason, early detection and management of leishmaniasis cases are crucial for control strategies since untreated patients serve as reservoirs for parasites [START_REF] Medley | Health-seeking behaviour, diagnostics and transmission dynamics in the control of visceral leishmaniasis in the Indian subcontinent[END_REF].

Vector control strategies involve indoor residual spraying, long-lasting insecticidal bed nets, and environmental management. Indoor spraying is the main intervention. Emerging resistance to DDT (dichloro-diphenyltrichloroethane) has led to a shift towards synthetic pyrethroid insecticides [START_REF] Coleman | DDT-based indoor residual spraying suboptimal for visceral leishmaniasis elimination in India[END_REF].

Insecticidal bed nets can confer personal protection, but their effectiveness at the population level is not well-established. Selective outdoor spraying may be effective in reducing vector density, but alternative strategies are needed in vast endemic areas [START_REF] Picado | Longlasting insecticidal nets for prevention of Leishmania donovani infection in India and Nepal: paired cluster randomised trial[END_REF]. Environmental management strategies, such as lime plastering of houses, have not demonstrated convincing effects [START_REF] Calderon-Anyosa | Housing Characteristics and Leishmaniasis: A Systematic Review[END_REF]. Further research is needed to better understand and improve vector control strategies, especially in terms of their population-level impact and effectiveness in different geographic regions.

Reservoir control, particularly culling infected dogs, may be relevant in zoonotic foci [START_REF] Quinnell | Transmission, reservoir hosts and control of zoonotic visceral leishmaniasis[END_REF]. Additionally, the use of deltamethrin-treated dog collars has shown effectiveness in limiting infection in children [START_REF] Gavgani | Effect of insecticide-impregnated dog collars on incidence of zoonotic visceral leishmaniasis in Iranian children: a matched-cluster randomised trial[END_REF].

INTRODUCTION 19

inoculation of live and virulent Leishmania major parasites to induce selfhealing lesions. This strategy was successfully employed in Israel, Iran, and Uzbekistan [START_REF] Mohebali | An overview of leishmanization experience: A successful control measure and a tool to evaluate candidate vaccines[END_REF], but has been abandoned due to the risk of recrudescence and acute infection in immunocompromised individuals. Nowadays, there are several candidate vaccines in preclinical development.

First-generation vaccines include killed whole parasite vaccines, which were inactivated by autoclaving or pasteurization. While they have shown success in animal models, they have not yet achieved desired efficacy in human trials.

Examples include Leishvaccine® and Bacillus Calmette-Guérin (BCG). Liveattenuated parasites have also shown potential for canine leishmaniasis prevention, with studies using gentamicin-attenuated strains of L. infantum and L. major [START_REF] Moafi | Leishmania Vaccines Entered in Clinical Trials: A Review of Literature[END_REF].

Second-generation vaccines for leishmaniasis utilize various approaches, including recombinant proteins, purified antigens, synthetic peptides, and genetically modified parasites. Subunits combination vaccines like LEISH-F1 (a recombinant fusion protein made by the thiol-specific antioxidant (TSA) and stress-inducible protein 1 (LmSTI1) genes of L. major and the elongation and initiation factor (LeIF) of L. braziliensis) and LEISH-F3 (containing the nucleoside hydrolase (NH) and sterol 24-c-methyltransferase (SMT) derived from L. donovani and L. infantum, respectively), have advanced to phase II trials and they have been used as therapeutic vaccines, showing efficacy in treating CL and MCL patients. Genetically modified parasite vaccines, such as Biopterin transporter 1 (BT1)-deleted parasites, centrin knockout mutants, and parasites expressing suicidal genes, mimic natural infection and induce protective immune responses [START_REF] Dinc | Leishmania Vaccines: the Current Situation with Its Promising Aspect for the Future[END_REF][START_REF] Zhang | A second generation leishmanization vaccine with a markerless attenuated Leishmania major strain using CRISPR gene editing[END_REF][START_REF] Volpedo | Centrin-deficient Leishmania mexicana confers protection against New World cutaneous leishmaniasis[END_REF][START_REF] Podesvova | Suicidal Leishmania[END_REF]. The intriguing finding that prior exposure to sandfly saliva provides protection against leishmaniasis has opened up the potential for developing a vaccine using salivary proteins such as LJM19 and PdSP15 to induce immune responses that may cross-protect against Leishmania during the initial inoculation [START_REF] Alvar | Case study for a vaccine against leishmaniasis[END_REF]. For canine leishmaniasis prevention, there are currently three commercially available vaccines: Leish-Tec® (recombinant protein A2 from L. donovani), CaniLeish® (composed of purified excretedsecreted proteins (ESP) from L. infantum), and LetiFend® (a chimeric protein composed of five antigenic fragments from different L. infantum proteins). These vaccines have reported prevention efficacy and protection against clinical disease [START_REF] Velez | Commercially approved vaccines for canine leishmaniosis: a review of available data on their safety and efficacy[END_REF][START_REF] Whyte | Cutaneous Leishmaniasis: Update on Vaccine Development[END_REF].

INTRODUCTION 20 effects of DNA vaccines for leishmaniasis have been performed [START_REF] Osman | A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis: First-in-human trial of ChAd63-KH[END_REF].

Overall, while some vaccines have shown success in animal models and canine leishmaniasis prevention, further research and clinical trials are needed to develop more effective and safer vaccines.

TREATMENTS AGAINST LEISHMANIASIS

Although leishmaniasis is an ancient disease, its treatment still remains challenging today. Currently, chemotherapy remains the primary approach for managing the disease. Existing treatments, which involve repurposing drugs not originally developed for leishmaniasis, have various limitations, including adverse effects, high costs, high toxicity, long treatment durations, and the emergence of drug resistance. Therefore, there is a continuous need for research to discover new drugs and alternative therapeutic approaches to overcome these limitations. Figure 8 shows some drugs used in the treatment of leishmaniasis, which will be discussed in the following sections. INTRODUCTION 21 mechanism of action is not fully understood, reports suggest that pentavalent antimonials specifically inhibit Leishmania type I DNA topoisomerase, which affects the purines metabolism, promoting ATP and GTP depletion, reducing parasite energy availability. Also, evidence suggests that Sb V acts as a prodrug that is metabolically converted into its active Sb III form within the body, where the trivalent antimony seem to interact with sulfhydryl containing molecules, affecting the thiol metabolism, thereby inducing DNA fragmentation [START_REF] Frezard | Pentavalent antimonials: new perspectives for old drugs[END_REF].

The use of these compounds is often restricted because of its daily parenteral administration, severe toxicity, side effects (including local irritation, cardiac and hepatic alterations, anorexia, nausea and vomiting) and treatment failure due to the appearance of resistant Leishmania phenotypes [START_REF] Roatt | Recent advances and new strategies on leishmaniasis treatment[END_REF].

Concerning CL treatment, Sb V remains the first option in many regions worldwide, even though its efficacy varies depending on Leishmania species. Intralesional infiltration has been recommended as administration route by the WHO, and while this approach is preferred for most patients in the Old World, in the New World, systemic MA is commonly administered [START_REF] Sundar | An update on pharmacotherapy for leishmaniasis[END_REF].

Drug resistance to Antimonials

Clinical isolates of Leishmania parasites have shown increased resistance to antimonials. Multiple mechanisms of antimonial resistance have been observed, such as decreased cellular entry, reduced drug activation, increased antimony efflux and antimony sequestration into intracellular vesicles. Environmental factors, such as cross-resistance with high levels of arsenic in water in India, have been associated to these phenomena [START_REF] Croft | Drug resistance in leishmaniasis[END_REF]. In addition, antimonial drug resistance involves aneuploidies, gene amplification (e.g., MRPA, an ATPbinding cassette protein implicated in Sb III sequestration), down-regulation of gene expression (e.g., AQP1, transporter having an influence in Sb III uptake), amplification of trypanothione synthetase (TryS), which can increase Sb III sequestration and mutations in specific genes such as multidrug resistance 1 gene (MDR1) and calcium-dependent protein kinase (CDPK1), linked to resistance to paromomycin as well [START_REF] Santi | Impact of Genetic Diversity and Genome Plasticity of Leishmania spp. in Treatment and the Search for Novel Chemotherapeutic Targets[END_REF].

2.3.2 Amphotericin B (AmB) INTRODUCTION 22 adverse effects when administered in its free deoxycholate form, such as fever, hypokalemia, myocarditis, and nephrotoxicity. To reduce side effects, various lipid formulations have been developed, but liposomal AmB (AmBisome®or AmBL) is the only one approved so far [START_REF] Balasegaram | Liposomal amphotericin B as a treatment for human leishmaniasis[END_REF]. Despite its high cost, it is the preferred treatment for VL in HIV co-infected patients, pregnant women, and transplant recipients. Nevertheless, in CL treatment, even if AmB has shown comparable results to antimonial drugs, it is not considered an alternative due to its serious side effects [START_REF] Roatt | Recent advances and new strategies on leishmaniasis treatment[END_REF].

Drug resistance to Amphotericin B

Clinical isolates of L. donovani displaying resistance to AmB showed increased efflux of the drug, which was linked to the overexpression of the MDR1 gene [START_REF] Purkait | Mechanism of amphotericin B resistance in clinical isolates of Leishmania donovani[END_REF]. In the case of L. mexicana, in vitro selection of AmB resistance was linked to altered sterol biosynthesis, involving mutations in the sterol biosynthesis gene sterol C5-desaturase (SC5D), loss of expression of the 24-sterol methyltransferase (SMT) gene [START_REF] Pountain | Genomic instability at the locus of sterol C24methyltransferase promotes amphotericin B resistance in Leishmania parasites[END_REF], and mutations in the sterol 14-demethylase (CYP51) gene, responsible of a resistance phenotype related to loss of drug binding [START_REF] Mwenechanya | Sterol 14alpha-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana[END_REF].

Miltefosine

Miltefosine is an antineoplasic drug that was shown to have antileishmanial activity. It is currently the only oral drug available against leishmanisis [START_REF] Sundar | Oral miltefosine for Indian visceral leishmaniasis[END_REF]. The exact mechanism of action is not fully understood, but it is known to directly impact the parasites by disrupting the synthesis of phospholipids and the metabolism of alkyl-lipids, affecting not only cell membrane synthesis, but also causing mitochondrial depolarization and decreased intracellular ATP levels, thus leading to apoptosis [START_REF] Dorlo | Miltefosine: a review of its pharmacology and therapeutic efficacy in the treatment of leishmaniasis[END_REF]. In early clinical trials, VL treatment resulted in a cure rate of 94%. However, over time, resistance to miltefosine has increased, reducing the effectiveness, and doubling the relapse rate. Currently, miltefosine is recommended only in combination with other drugs [START_REF] Srivastava | Laboratory confirmed miltefosine resistant cases of visceral leishmaniasis from India[END_REF]. Miltefosine effectiveness also depends on the infecting Leishmania species, which is why higher doses are sometimes necessary for leishmaniasis caused by L. donovani in India. Adverse effects of miltefosine include gastrointestinal problems, hepatotoxicity and teratogenic effects [START_REF] Sundar | An update on pharmacotherapy for leishmaniasis[END_REF].

Drug resistance to Miltefosine

Miltefosine resistance is associated with multiple mechanisms. The main mechanism involves reduced drug internalization due to a decrease uptake or increased efflux caused by mutations or deletions in the miltefosine translocation machinery, specifically in the subunit B (LRos3) of the Leishmania miltefosine transporter (LMT), which is responsible for the translocation of phospholipids, observed in resistant strains. Overexpression of ABC transporters ABCB4 (MDR1), ABCG4 and ABCG6 also has been associated to increased drug efflux and resistance. Other cellular modifications, such as changes in fatty acid composition, ergosterol levels, gene expression, oxidative stress response and infectivity have been associated with miltefosine resistance [START_REF] Ponte-Sucre | Drug resistance and treatment failure in leishmaniasis: A 21st century challenge[END_REF].

Paromomycin

Paromomycin is an aminoglycoside antibiotic used as an effective antileishmanial drug. The exact mechanism of action is not fully understood, but some studies suggest that it may bind to the glycocalyx and LPG on the surface of Leishmania parasites, modifying membrane fluidity and permeability [START_REF] Chawla | Paromomycin affects translation and vesicle-mediated trafficking as revealed by proteomics of paromomycin -susceptible -resistant Leishmania donovani[END_REF]. Another proposed mechanism is its binding to the 30S subunit of the parasite ribosome, leading to an accumulation of abnormal initiation complexes and misreading of the mRNA template [START_REF] Sundar | Paromomycin in the treatment of leishmaniasis[END_REF]. It has been used for treatment of VL in a parenteral formulation and of CL in both topical and parenteral formulations. In clinical trials, it has demonstrated cure rates of 94% for VL. However, its effectiveness as monotherapy is limited by parasite drug resistance in East Africa. The most common adverse effect of systemic therapy is pain at the injection, but there is also risk of hepatotoxicity and ototoxicity. Topical formulations of paromomycin have been evaluated in the treatment against CL with mixed clinical results, depending on the Leishmania species involved [START_REF] Roatt | Recent advances and new strategies on leishmaniasis treatment[END_REF].

Drug resistance to Paromomycin

The mechanisms of resistance are not well characterized, but reduced drug uptake and drug accumulation have been observed in resistant L. donovani promastigotes. Also, resistant parasites show a significant reduction in the initial binding of the drug to the cell surface [START_REF] Jhingran | Paromomycin: uptake and resistance in Leishmania donovani[END_REF].

Pentamidine

The exact mechanisms of action of pentamidine is not fully understood, but it has been suggested that it inhibits polyamine biosynthesis by binding to ATrich DNA regions, affecting the transcription and replication process, and affecting the inner membrane potential of mitochondria [START_REF] Bray | Pentamidine uptake and resistance in pathogenic protozoa: past, present and future[END_REF]. Pentamidine was previously used for treatment-refractory cases of VL in India, but it has been abandoned due to its high toxicity, which includes pancreatitis leading to diabetes mellitus, hypoglycemia, hypotension, cardiac alterations, and hyperkalemia (an elevated level of potassium (K+) in the blood). While the use of pentamidine has been documented in some cases of CL and MCL in the New World, its effectiveness varies depending on the Leishmania species, treatment duration and geographical region [START_REF] Sundar | An update on pharmacotherapy for leishmaniasis[END_REF].

Drug resistance to Pentamidine

Pentamidine drug resistance is characterized by reduced drug accumulation in resistant parasites compared to drug sensitive parasites. This resistance is associated with cross-resistance to similar drugs and alterations in carriermediated uptake and increased efflux mechanisms. Additionally, mitochondrial membrane potential plays a role in pentamidine uptake, with reduced potential observed in resistant cells, leading to decreased drug accumulation in the mitochondria [START_REF] Basselin | Resistance to pentamidine in Leishmania mexicana involves exclusion of the drug from the mitochondrion[END_REF].

2.3.6 New strategies for CL and MCL 2.3.6.1 Physical strategies CO2 laser administration and thermotherapy involve delivering heat to infected tissues, leading to the destruction of parasites. Thermotherapy has shown better results compared to antimonial treatment. Cryotherapy, using liquid nitrogen, has also been effective in killing parasites through localized ischemic necrosis. Electrotherapy, involving the application of electric fields, could lead to healing skin lesions [START_REF] Roatt | Recent advances and new strategies on leishmaniasis treatment[END_REF].

Topical drug therapies

Nitric oxide derivatives, such as S-nitroso-N-acetylpenicillamine cream, have been tested and showed healing of lesions for infection with Leishmania panamensis [START_REF] Lopez-Jaramillo | A controlled, randomized-blinded clinical trial to assess the efficacy of a nitric oxide releasing patch in the treatment of cutaneous leishmaniasis by Leishmania (V.) panamensis[END_REF]. Intralesional drug administration of pentavalent antimonials has gained prominence as a way to reduce adverse effects while maintaining efficacy, but side effects such as bacterial secondary infection, erythema, local itching, and pain during administration are still observed [START_REF] Ramalho | Meglumine antimoniate intralesional infiltration for localised cutaneous leishmaniasis: a single arm, open label, phase II clinical trial[END_REF].

Combined therapy INTRODUCTION 25

In regions where resistance to antimonials has emerged, such as Eastern Africa, alternative combination of SSG and paromomycin has been implemented as the first-line treatment for VL, as it showed higher cure rates compared to SSG monotherapy [START_REF] Melaku | Treatment of kala-azar in southern Sudan using a 17-day regimen of sodium stibogluconate combined with paromomycin: a retrospective comparison with 30-day sodium stibogluconate monotherapy[END_REF][START_REF] Musa | Sodium stibogluconate (SSG) & paromomycin combination compared to SSG for visceral leishmaniasis in East Africa: a randomised controlled trial[END_REF]. Exploratory studies have also been performed in these regions to evaluate combinations of AmBL with SSG or with miltefosine [START_REF] Omollo | Safety and efficacy of miltefosine alone and in combination with sodium stibogluconate and liposomal amphotericin B for the treatment of primary visceral leishmaniasis in East Africa: study protocol for a randomized controlled trial[END_REF][START_REF] Wasunna | Efficacy and Safety of AmBisome in Combination with Sodium Stibogluconate or Miltefosine and Miltefosine Monotherapy for African Visceral Leishmaniasis: Phase II Randomized Trial[END_REF].

In India, combinations of AmBL with miltefosine or paromomycin have shown excellent cure rates (>95%) for VL [START_REF] Sundar | Comparison of short-course multidrug treatment with standard therapy for visceral leishmaniasis in India: an open-label, non-inferiority, randomised controlled trial[END_REF][START_REF] Sundar | Recent developments and future prospects in the treatment of visceral leishmaniasis[END_REF].

The WHO also recommends combined treatments for CL and MCL: treatment with antimonial drugs in combination with other agents, such as pentoxifylline (treatment for MCL caused by L. braziliensis), allopurinol (giving higher cure rates in L. major and L. tropica cases), imiquimod cream (treatment for CL in Peru) and with a killed-parasite vaccine for the treatment of leishmaniasis caused by L. amazonensis. A combination of intralesional antimonial plus cryotherapy is also recommended as treatment for CL caused by L. major [3,[START_REF] Sundar | An update on pharmacotherapy for leishmaniasis[END_REF].

Resistance to drug combined therapy

Multidrug therapy is often considered advantageous for overcoming resistance in leishmaniasis treatment. However, resistance to individual drugs can still occur and there is evidence showing that Leishmania parasites can even develop resistance to drug combination due to the plasticity of its genome [START_REF] Laffitte | Plasticity of the Leishmania genome leading to gene copy number variations and drug resistance[END_REF].

Indeed, it has been shown that Leishmania parasites can develop resistance to the drug combinations of miltefosine/paromomycin and SSG/paromomycin [START_REF] Hendrickx | Evidence of a drug-specific impact of experimentally selected paromomycin and miltefosine resistance on parasite fitness in Leishmania infantum[END_REF]. This resistance is associated to metabolic changes in multiple pathways, such as proline and lipid metabolism, activation of stress responses, enhanced ability to neutralize drug-induced reactive oxygen species (ROS), and increased fitness and survival under different stress situations, such as nutrient starvation and pH fluctuations. Drug-combination-resistant Leishmania parasites have been found to show higher ATP and thiol levels, resistance to mitochondrial depolarization, and the ability to maintain DNA and membrane integrity after exposure to drug pressure [START_REF] Garcia-Hernandez | Fitness of Leishmania donovani parasites resistant to drug combinations[END_REF][START_REF] Berg | Experimental resistance to drug combinations in Leishmania donovani: metabolic and phenotypic adaptations[END_REF].

Promising strategies for treating leishmaniasis include exploring new approaches such as immunotherapy or immunochemotherapy. Additionally, the discovery of new drug targets within the Leishmania metabolic pathways and the interactions between Leishmania and macrophages, which are crucial for parasite survival and disease development, could hold potential in combating the disease.

Targeting host-parasite interactions for chemotherapy

The host-parasite interaction between Leishmania and macrophages plays a crucial role in the development of leishmaniasis (as described in Section 1.4 of this chapter). Leishmania has evolved mechanisms to evade and subvert the host immune response, allowing parasite proliferation and persistent infection [START_REF] Geiger | Escaping Deleterious Immune Response in Their Hosts: Lessons from Trypanosomatids[END_REF][START_REF] Shio | Host cell signalling and leishmania mechanisms of evasion[END_REF]. As previously discussed, current antileishmanial drugs have limitations such as high toxicity, high costs, drug resistance and treatment failures, calling for new and improved treatment strategies.

Traditional antileishmanial drug discovery focuses on targeting the parasite directly, but this approach is often limited by the emergence of drug resistance. An alternative strategy is to target and interfere with host cell pathways and processes that are modulated by intracellular Leishmania and that promote parasite survival, proliferation and dissemination.

By focusing on host-pathogen interactions, new therapies for leishmaniases can be proposed that target the host cell and thus are less susceptible to the development of parasite resistance phenotypes. This approach has been successful in treating viral, bacterial, and fungal diseases [START_REF] Zumla | Host-directed therapies for improving poor treatment outcomes associated with the middle east respiratory syndrome coronavirus infections[END_REF]. In addition, ectoproteins released by the parasite are promising new drug targets as they can impact host cell signal transduction, metabolic pathways, or host histonemodifying enzymes [START_REF] Lamotte | The enemy within: Targeting host-parasite interaction for antileishmanial drug discovery[END_REF].

Targeting host cell epigenetics

Intracellular pathogens can significantly influence the host cell transcriptome through epigenetic modifications. Epigenetic factors such as DNA methylation, histone modifications, and noncoding RNAs (ncRNAs), are involved in hostparasite interactions, affecting gene expression without altering the DNA sequence.

Pathogens manipulate these epigenetic mechanisms to inhibit host defense genes and promote genes that support their own growth and survival. For instance, changes in DNA methylation have been linked to altered inflammatory responses, apoptosis, and even changes in host behavior during infection [100][101][102]. Some pathogens also alter chromatin structure by modifying histone proteins or histone remodeling enzymes, which affect the accessibility of certain genes [103,104]. Additionally, secreted pathogen proteins can interfere with host epigenetic control and gene expression, further influencing the hostparasite interaction [105-107]. Particularly, parasites manipulate key host processes involved in immune response, cell cycle, death pathways, metabolism, and signal transduction events, which can affect the host cell regulatory pathways as shown in Figure 9.

Parasites specifically target host transcriptional regulators such as NF-κB, p53, and MAPK to disrupt host cell death and cytokine response pathways. Furthermore, parasites interfere with host metabolism through the HIF1 pathway [108].

However, despite its ability to modify nuclear physiology and potentially influence chromatin remodeling and transcriptional/translational regulation [109] through the alteration of DNA methylation, histone post-translational modifications, and miRNAs expression [110], our understanding of the epigenetic effect of Leishmania infection on the host cell remains limited. Exploring these mechanisms could offer new possibilities for developing INTRODUCTION 28 antileishmanial therapies that are less prone to drug resistance as targeting the parasite only indirectly via the drug effect on the host cell. Such strategies may be able to rescue the host cell immune potential, induce apoptosis in infected cells, or restore normal macrophage metabolic functions that could eliminate the parasites (Figure 10).

CHROMATIN STRUCTURE

Genomic DNA in eukaryotic cells is organized and compacted with proteins to form chromatin. The basic and primary unit of chromatin is the nucleosome, consisting of 147 base pairs of DNA tightly wrapped around a histone octamer composed of two copies of histones H2A, H2B, H3 and H4 [111]. Nucleosomes are connected by H1 histones, and short DNA segments (linker DNA), which helps the formation of chromatin structures and contribute to transcriptional regulation. The nucleosome is stabilized by protein-protein interactions within the histone octamer and electrostatic and hydrogen bonds between proteins and DNA. Structured regions of the histones interact with DNA, while the flexible histone tails, which undergo post-translational modifications (PTMs), extend away from the nucleosome core and interact with neighboring nucleosomes or nuclear factors. The primary "canonical" structure of chromatin, also known as "beads-on-a-string", influences secondary and tertiary organization in a supercoiled organization that limits DNA accessibility (Figure 11). Histone octamers bind to DNA, compacting it into nucleosomes, which then form chromatin fibers and chromosomes. Chromatin is subjected to epigenetic regulations, including DNA methylation (me, yellow bubbles) and post-translational modifications of histones, including acetylation (ac), methylation (me), phosphorylation (P), and ubiquitination (ub). Long noncoding RNAs (lncRNAs) can also play a significant role in epigenetic regulation via interaction with epigenetic modifiers, or influencing transcription through their interaction with DNA [116].

Heterochromatin

Heterochromatin is a chromatin region that is highly compacted, transcriptionally less active and associated with gene silencing. It is typically located towards the nuclear periphery and regions surrounding the nucleolus.

Heterochromatin is divided into two categories: facultative, which assembles on developmentally regulated genes and can be silenced or activated; and constitutive, which occurs in all cell types [117].

MECHANISMS OF TRANSCRIPTIONAL REGULATION

Chromatin compaction is a dynamic process tightly controlled by various mechanisms. A regulatory mechanism, responsible for maintaining distinct phenotypes in eukaryotic cells even when their genetic contents are identical, involves the addition of covalent PTMs of chromatin, which includes modifications of DNA and histones. Additionally, remodeling complexes and noncoding RNAs play a role in regulating the accessibility of the transcriptional machinery to specific regions of the genome.

Chromatin remodeling

The chromatin structure involved in epigenetics is more complex than a simple distinction between euchromatin and heterochromatin. Euchromatin, despite being less densely packed, restricts the access of transcription factors (TFs) and coactivators to the DNA within nucleosomes. Nucleosome positioning, determined by DNA sequence composition and ATP-dependent nucleosome remodelers, is crucial for transcriptional initiation. Remodeler protein families like ISWI and SWI/SNF modify the structure, composition and positioning of nucleosomes. ISWI remodelers ensure equal spacing between nucleosomes, facilitating chromatin assembly and higher-order folding, while SWI/SNF proteins disrupt DNA-histone interactions, leading to nucleosome disorder [115,[125][126][127].

Further chromatin compaction occurs through intra or interchromosomal folding and looping, influencing transcriptional activation or repression. Enhancer-promoter interactions are crucial in chromatin looping, where distant enhancers are brought closer to their target promoters. These interactions then facilitate the recruitment of TFs, coactivators and the transcriptional initiation complex, promoting gene transcription. Key molecules involved in chromatin remodeling that help to establish contact between distal enhancers and target INTRODUCTION 33 promoters include CCCTC-binding factor (CTCF), cohesion and mediator complexes [128].

It is important to mention that histone modifications, which will be discussed in the next section, are interconnected with chromatin higher-order structure. These modifications directly impact DNA accessibility and the recruitment of regulatory proteins, influencing gene expression.

Histone modifications

Histones can undergo over 130 PTMs, including acetylation, methylation, phosphorylation, sumoylation and ubiquitination (Figure 11). These histone modifications define the histone code that controls DNA accessibility by altering chromatin structure and transcriptional regulation affecting the recruitment of the transcriptional machinery. Histone modifications play a crucial role in shaping the epigenetic code, orchestrated by three groups of key enzymatic players: writers, erasers, and readers. It is worth noting that these enzymes are not limited to histone proteins, as they also possess the capability to modify nonhistone proteins (Figure 12).

Figure 12. Histone modifying enzymes. Histone-modifying enzymes, known as "writers," are responsible for catalyzing covalent histone modifications, while "erasers" are involved in their removal. These modifications are categorized based on their enzymatic actions, such as acetylation, phosphorylation, or methylation. Protein domains with specific affinity for a histone tail modifications are termed "readers". Ac, acetylation; HAT, histone methyltransferase; HDAC, histone deacetylase; KDM, lysine demethylase; KMT, lysine methyltransferase; Me, methylation; P, phosphorylation; PAD, peptidylarginine deiminase; PPTase, protein phosphatase; PRMT, protein arginine methyltransferase [129].

Understanding the activity of these players is crucial in the field of epigenetics, as dysregulation of these processes is implicated in disease development. This is why targeting these epigenetics tools for drug discovery is an active area of INTRODUCTION 34 research.

Writers

Writers are a wide variety of enzymes that modify specific amino acid residues on histones by adding different chemical groups. Among the numerous modifications possible, methylation and acetylation are the most extensively studied. Methylation can occur in both, DNA and histones, while acetylation is specific to histones [130].

Histone acetyltransferases (HATs) are enzymes that add acetyl groups from acetyl co-A to lysine residues on histone proteins. Acetylation neutralizes positive charges and reduces the binding of histones to DNA. HATs are categorized into two groups based on their cellular location: type A in the nucleus acetylates chromatin-associated histones, while type B in the cytoplasm acetylates newly translated histones. HATs can be further classified into different families, including GNAT, MYST, p300/CREB binding proteins, general transcription factor HATs that contain the TAF250 domain and steroid/nuclear receptor co-activators (SRC/NCoA). Each family has specific catalytic domains and functions in modifying different histone residues [131].

Histone lysine methyltransferases (KMTs) are enzymes that add methyl groups from adenosyl-methionine to lysine residues on histones. Residues can be mono-(me1), di-(me2) or tri-(me3) methylated, but in contrast to acetylation, none of these modifications alter the electronic charge of the amino acid side chain. As a result, the functions of this methylation marks are primarily carried out by molecules that specifically recognize the methylated site (readers), acting as effector molecules. In contrast to other types of modification that indicate active or repressed chromatin states, lysine methylation can either promote or inhibit transcription depending on their specific positions and methylation states. Specific lysine residues 4, 9, 27, 36 and 79 of Histone H3 and residue 20 of Histone H4 are prone to be methylated by different KMTs. KMTs can be classified based on the presence or absence of the SET domain (Su(var)3-9, Enhancer-of-zeste and Trithorax) [132][133][134].

Table 2 and Figure 14 summarize the methylation marks on specific residues and their location, as well as enzymes that catalyze the transfer of the methyl groups.

INTRODUCTION 35

Readers

Epigenetic modifications carried out by writers require recognition by other cellular proteins to exert their influence. In mammalian cells, specific protein domains have evolved to bind to these modifications, functioning as readers of the epigenome. Many chromatin modifiers serve as epigenetic readers because of their specialized domains that enable them to detect and bind covalent modifications present on the DNA and histones [130].

Histone acetylation and methylation readers are proteins that have specialized domains that enable them to recognize acetylated and methylated residues on histones, respectively.

Histone acetylation readers

Erasers

The PTMs on histones are not permanent. These marks can be erased when the cell needs to modify the expression states of a particular locus. To accomplish this, a group of enzymes called erasers are present, which counteract the activity of the writers. These erasers catalyze the removal of epigenetic marks, leading to the modulation of gene expression (Figure 14).

Histone deacetylases (HDACs) are enzymes that remove acetyl groups from lysine residues on histone proteins, resulting in a compact and repressive chromatin structure. There are two classes of HDAC enzymes: zinc-dependent deacetylases consist of 11 proteins (class I, IIa, IIb and IV) and NAD+-dependent deacetylases containing 7 proteins known as Sirtuins or class III HDACs [138,139].

Table 3 summarizes the characteristics of each HDAC. Class I HDACs contain approximately 400-500 amino acids, with ubiquitous tissue expression and predominant nuclear localization.

Histone demethylases (KDMs) are enzymes that remove methyl groups from lysine residues on histone proteins (Figure 12). There are two families of histone demethylases: the lysine-specific demethylase (LSD), which will be discussed in depth in the following section, and Jumonji C (JmjC) domain-containing iron and α-ketoglutarate-dependent dioxigenases, which are divided into several subfamilies. JmjC enzymes demethylate lysine residues in all three methylated states using 2-oxoglutarate (2OG) and Fe(II) as cofactors [140]. Table 4 summarizes important features about the JmjC demethylase family. HDAC1 HDAC2

HDAC 1 deacetylates lysine residues on the core histones (H2A, H2B, H3 and H4) and is a part of the BRG1-RB1-HDAC1 complex. HDAC2 is a component of RCOR/GFI/KDM1A/HDAC complex. HDAC1 and 2 are almost identical in structure and are part of sin3, NuRD, CoREST and PRC2 repressive complex. HDAC3 Part of NCoR or SMRT complex. HDAC8 Its deacetylase activity is independent of additional co-factors.

IIa.

HDAC4 HDAC5 HDAC7 HDAC9

Contain a long N-terminal region in addition to the deacetylase domain which makes these enzymes responsive to various signaling pathways through phosphorylation by kinases.

IIb.

HDAC6 Contains two deacetylase domains and a unique SE14 repeat domain. HDAC10 Contains a single deacetylase domain in the N-terminal region that is similar to the first catalytic domain of HDAC6, whereas the Cterminal end contains a leucine rich region.

IV.

HDAC11 Shows sequence similarity to class I and II HDACs. Class III (Sirtuins) SIRT1 Deacetylates H1K16 and H4K26, predominantly localized in the nucleus. SIRT2 Deacetylates H4K16, present both in the cytoplasm and nucleus. The catalytic domain of SIRT2 acts both as NAD+-dependent deacetylase (DAC) and mono -ADP-ribosyl transferase (ART). SIRT3 Present in the mitochondria. Its catalytic domain functions as DAC and ART. SIRT4 Mitochondrial protein exhibiting a NAD+-dependent deacetylase activity. SIRT5 Mitochondrial protein that preferentially presents lysine demalonylase and desuccinylase activity compared to lysine deacetylation. SIRT6 Predominantly a nuclear protein that deacetylates H3K9 and H3K56 . SIRT7 Mainly localized in the nucleolus and nucleoplasm, deacetylates H3K18. H3K9me2, H3K27me2 & H4K20me1 KDM7B (PHF8) H3K9me1/me2, H3K27me2 H4K20me1 KDM7C (PHF2) enzymatically inactive, is activated through phosphorylation by PKA acts on H3K9me2 KDM8 (JMJD5) is the only member of this subfamily identified so far. Associated with demethylating the H3K36me2 mark.

LYSINE-SPECIFIC DEMETHYLASES

The lysine-specific demethylase family comprises only two members: LSD1/KDM1A and LSD2/KDM1B. These are flavin adenine dinucleotide (FAD)dependent amino oxidases that specifically target mono-and di-methylated lysine residues in Histone H3.

Their demethylation activity involves two steps: first, they use the cofactor FAD to oxidize the methylated lysine, removing an hydride anion from the methylated ε-nitrogen atom, resulting in the formation of an intermediate imine cation; next, the intermediate undergoes hydrolysis, leading to the formation of a carbinolamine, which decomposes producing formaldehyde as a byproduct and leaves the demethylated residue (Figure 15). The nitrogen of a trimethylated lysine cannot be protonated, an essential step for imine intermediate formation, which restricts the substrate specificity of this enzyme family to mono-and dimethylated lysine residues [142]. LSD2 exhibits similar domain homology to LSD1, although their overall sequence identity is less than 31%. They have similar sizes (852 amino acids for LSD1 and 822 for LSD2) and share key domains essential for enzymatic activity, including the SWIRM domain, a FAD coenzyme-binding motif and a C-terminal amine oxidase (AO) domain, which presents only 45% of sequence identity. However, LSD2 is characterized by two Zn finger domains, ZF and Zf-CW, in its N-terminal region, absent in LSD1 (Figure 16A). Furthermore, LSD1 contains a unique Tower domain extending from the AO domain, a specific feature that is not observed in any other monoamine oxidase. The structural differences among both enzymes (Figure 16B & C) suggest that they have distinct biochemical and biological properties, facilitating association with different protein complexes and different genomic loci [144][145][146][147].

INTRODUCTION 40 section), LSD2 interacts with the NPAC cofactor, which promotes its interaction with the nucleosome and enhances its catalytic activity (Figure 16C). LSD2 specifically targets H3K4me1/2 in the coding sequences of actively transcribed genes, but not at promoters, and maintains low levels of methylation, supporting optimal gene transcription instead of acting as a corepressor [148].

LSD1 STRUCTURE

LSD1, also known as KDM1A (lysine-specific demethylase 1A) or AOF2 (flavincontaining amine oxidase domain-containing protein 2) was the first identified histone demethylase. It demethylates mono-and dimethyl groups on H3K4 and H3K9 and a plethora of nonhistone targets. As described above, LSD1 is composed of three domains: the catalytic AO domain, the Swi3/Rcs8/Moira (SWIRM) domain and the Tower domain [150][151][152].

The AO domain contains a noncovalent FAD-binding site and a spacious substrate-binding pocket capable of accommodating the demethylation target and its surrounding residues. This pocket is notably larger than those found in other amine oxidases, which may explain the broad substrate specificity of LSD1. The presence of negatively charged residues in the AO domain facilitates interactions with positively charged histone tails. The Tower domain serves as binding site for the CoREST transcription repressor complex, which is essential for LSD1 to associate with the nucleosome and exert its demethylase activity.

The interaction between LSD1 and CoREST complex is facilitated by the DNA binding domain of CoREST known as SANT2 (Figure 16B). Moreover, since the Tower domain originates from the catalytic domain, it offers a potential site for allosteric regulation of catalysis and substrate recognition. The SWIRM domain functions as a platform for protein-protein interactions. One notable interaction is between LSD1 and the androgen receptor, which is believed to alter the specificity from targeting H3K4 to H3K9. This shift in substrate specificity enables LSD1 to transform its role as a repressor to act as a coactivator, as the H3K9me1/2 marks are associated with repression [147,153].

LSD1 exhibits both repressive and activation functions, which depend on the specific lysine it targets. It is important to highlight that the manifestation of these functions by LSD1 requires interaction with diverse protein regulatory complexes and transcription factors. These interactions can vary across cell types and activation states.

LSD1 AS A TRANSCRIPTIONAL REPRESSOR

LSD1 only can act as a transcription repressor by interacting with various protein complexes and transcription factors. The most studied LSD1 complexes are described in the following, nonexhaustive list.

LSD1 association with the CoREST (RCOR) protein complex is crucial for the removal of methyl groups from H3K4me1/2 (Figure 17A). The CoREST family consists of three proteins (CoREST1, CoREST2 and CoREST3, also known as RCOR1, RCOR2 and RCOR3, respectively). CoREST1 was first identified as a partner of the RE1-silencing transcription factor (REST). These proteins contain two SANT domains, particularly SANT2 which confers the ability to LSD1 to demethylate residues within the nucleosome. CoREST complexes also include histone deacetylases 1 and 2 (HDAC1/2) and the PHD finger protein BHC80, also known as PHF21A, which also helps to stabilize LSD1 on chromatin. By combining LSD1 and HDAC1/2, the complex acts as a transcriptional inhibitor, since the deacetylation induced by HDACs enhances LSD1 activity, increasing the affinity of the complex for the chromatin. The association of LSD1 with different members of the CoREST family, particularly with CoREST3, results in variation of demethylase activity, suggesting that LSD1 function varies depending on the specific member of the CoREST family it interacts with [154][155][156].

The nucleosome remodeling and histone deacetylase (NuRD) complex is involved in regulating various cellular processes, such as lipid homeostasis (Figure 17A). It consists of core proteins such as ATPases chromo domain helicase DNA-binding proteins (CHD3/4), methyl-CpG-binding proteins (MDB2/3) retinoblastoma-binding proteins (RBBP4/7), corepressors (GATAD2A/B) and metastasis-associated proteins (MTA1/2/3). The MTA proteins in the complex are very similar to CoREST proteins, as they present SANT domains, and recruit HDACs and LSD1 to stimulate gene repression, the LSD1 association with each MTA isoform corresponds to a unique transcriptional outcome [150,157,158].

LSD1 also interacts with Snail/Gfi-1 (SNAG) domain transcription factors, such as SNAI1, SNAI2, GFI1, GFI1B and INSM1. Their N-terminal SNAG domain mimics the Histone H3 tail and binds to the LSD1 catalytic pocket. The formation of a complex involving SNAI1/LSD1/RCOR1 is necessary for the repression of CDH1 (E-cadherin), which affects epithelial-to-mesenchymal transition in various cancers. The interaction between LSD1 and GFI1/GFI1B is relevant in diverse processes such as hematopoietic differentiation control. It has been demonstrated that the disruption of the LSD1 interaction with these transcription factors renders them inactive, suggesting that LSD1 is necessary for the repressive function. The SNAG domain presents a higher affinity for LSD1 than the Histone H3 tail, indicating that SNAG transcription factors can inhibit LSD1 by occupying its catalytic pocket, preventing Histone H3 demethylation [147,[159][160][161].

LSD1 AS A TRANSCRIPTIONAL ACTIVATOR

In addition to its repressive activity, LSD1 also interacts with nuclear receptors to induce gene expression (Figure 17B). The change in LSD1 enzymatic specificity is triggered by the phosphorylation of threonine 6 in Histone H3 (H3T6), which is carried out by the protein kinase C β (PKCβ) activated by hormones, resulting in the interaction of H3K9me1/2 with the catalytic pocket of LSD1. It promotes androgen-dependent transcription by binding to the androgen receptor (AR). Similarly, LSD1 in association with the JMJD2A demethylase and the estrogen receptor (ER), activate estrogen-responsive genes. LSD1 also plays a key role in activating retinoic acid-responsive genes and establishing chromatin loops. However, with progesterone, LSD1 assembles with free receptors to inhibit gene transcription, but upon hormone exposure, it releases the repressor complex, allowing productive gene transcription. LSD1 also has a transcriptional regulatory function mediated by its H2O2 byproduct, which induces DNA oxidation and activates the base excision repair system. These activator functions are associated with the control of cell proliferation and tumor growth, angiogenesis, migration and invasion in different types of cancer [143,152,162,163].

LSD1 NONHISTONE TARGETS

LSD1 can also remove methyl groups from nonhistone proteins. The first nonhistone protein identified as an LSD1 substrate was p53, which is involved in regulating cell cycle progression, programmed cell death, genomic stability, and stemness. One of its lysine residues, K370, can be demethylated, leading to activation of p53 target genes when it binds its cofactor 53BP1. However, when LSD1 removes one methyl group from K370, p53 loses its stimulatory effect. LSD1 affects the activity of the tumor suppressor protein RB1, which controls cell cycle progression. LSD1 demethylates MYPT1 at lysine 442, which affects RB1/E2F1 interaction. Additionally, LSD1 stabilizes E2F1 by demethylating lysine 185, which was previously methylated by Set9 methyltransferase, rendering E2F1 more prone to ubiquitination. LSD1 demethylation activity is also involved in the control of DNMT1 stability. Likewise, it controls the stability of hypoxia-

LSD1 AS TARGET OF POST-TRANSLATIONAL MODIFICATIONS

It is important to consider that LSD1 undergoes various post-translational modifications that impact its function, and localization, leading to changes in its activity and interactions with other proteins (Figure 19). Briefly, LSD1 methylation by EHMT2 and phosphorylation by PKA and PKC affect the recruitment of transcriptional complexes and gene regulation. Phosphorylation by PLK1 and CK2α regulates LSD1 release from chromatin during mitosis and promotes DNA damage repair, respectively. Acetylation by KAT8, also known as MOF, inhibits LSD1 binding to the nucleosomes, contributing to the increase of epithelial markers. Finally, methylation by SUV39H2 stabilizes LSD1 by preventing its degradation [143].

HISTONE MODIFICATIONS IN THE REGULATION OF MACROPHAGE FUNCTION

Macrophage activation plays a vital role in diverse diseases and immune responses. They can adopt different polarization states, such as the canonical M1 and M2 phenotypes, in response to internal and environmental stimuli.

Recent research has unveiled that this polarization process is intricately governed by a complex network of transcription factors and epigenetic modifications. Notably, histone modifications are key regulators of the immune response, macrophage differentiation, maturation, and activation [110,171].

Numerous HDACs and HMTs involved in macrophage function and polarization have been identified. Table 5 and Table 6 summarize these research findings.

HISTONE MODIFICATIONS IN MACROPHAGES DURING LEISHMANIA

INFECTION

Leishmania parasites have the ability to manipulate histone modifications to establish favorable immunometabolic conditions for their survival within host macrophages. Infection leads to a decrease in NOS2 expression, resulting in reduced production of the leishmanicidal molecule nitric oxide. This manipulation involves the activation of HDAC1 and the removal of H3K9Ac activation marks, potentially facilitated by the formation of a repressosome with NF-κB1 homodimers.

Furthermore, infection is associated with decreased expression of proinflammatory genes, which correlates with hypoacetylation of H3K9/14. These genes include those involved in TLR/cytokine receptor signaling. Interestingly, promoters of NF-κB family members, such as IKKB, NFKBIA, NFKB2, and RELA, exhibit hypoacetylation and decreased expression, while the TNFAIP3 gene promoter, encoding a key inhibitor of the TLR/cytokine receptor/NF-κB pathway, displays increased H3K9/14 acetylation and expression.

INTRODUCTION 49

PHD OBJECTIVES

Leishmania infection leads to the subversion of macrophage functions, resulting in an ineffective immune response. Although the understanding of the transcriptomic reprogramming of infected macrophages by Leishmania parasites is limited, recent findings indicate that the infection specifically affects the host cell epigenome, influencing host-pathogen interaction in favor of the parasite.

In previous studies, our lab has identified a series of inhibitors targeting the macrophage epigenetic enzyme LSD1, which eliminated intracellular Leishmania in a host-directed fashion. These results strongly support the major working hypothesis that Leishmania subverts macrophage LSD1 functions to establish permissive conditions for intracellular parasite survival. Investigating LSD1 functions in this system holds the promise of providing new insights into the epigenetic mechanisms underlying intracellular infection and the development of pathological polarization states of macrophages. My thesis project directly contributed to this effort through three major objectives:

The first objective of this PhD thesis was to investigate the effect of the LSD1 inhibitors (LSD1i) on infected macrophages (Results, Chapter 1). This involved assessing the drug's efficacy in infected macrophages, evaluating its antileishmanial activity, and examining the impact on LSD1 activity and target engagement.

The second objective focused on understanding the role of macrophage LSD1 during intracellular Leishmania infection (Results, Chapter 2). This was achieved through the analysis of its subcellular localization, determining protein abundance, identifying qualitative changes in LSD1 complexes, and establishing experimental conditions for genome-wide analysis of H3K4 and H3K9 methylation patterns, as well as the promoter occupancy of LSD1.

The third objective was to investigate the mechanisms underlying Leishmania subversion of host cell epigenetic regulation through the analysis of the nuclear proteome (Outlook).

ANIMALS

Six-week-old female mice (Mus musculus, C57BL/6JRj) from Janvier Laboratories were used as source of bone marrow precursors for macrophage differentiation.

Swiss nu/nu female mice were used for the production of virulent Leishmania amazonensis amastigotes.

Rag2t m1Fwa mice [196], kindly provided by Philippe Bousso, were bred at the Institut Pasteur and used for the isolation of ex vivo lesional Leishmaniainfected macrophages (evLIMs). Also known as RAG2 knockout (RAG2 KO), these mice contain a disruption of the Recombination Activating Gene 2 (RAG2), which is essential for the development of functional B and T lymphocytes. As a result, these mice have severe immunodeficiency and are commonly used as a valuable research tool to study the immune system.

Female Golden hamsters (Mesocricetus auratus RjHan:AURA, weighting between 60-70 g) were purchased from Janvier Laboratories and used for the production of virulent Leishmania donovani amastigotes.

All animals were handled under specific, pathogen-free conditions in biohazard level 3 animal facilities (A3) accredited by the French Ministry of Agriculture for performing experiments on live rodents (agreement A75-15-01). Work on animals was performed in compliance with French and European regulations on care and protection of laboratory animals (EC Directive 2010 /63, French Law 2013-118, February 6th, 2013). All experiments were approved by the ethic committee and the Animal welfare body of Institut Pasteur (dap180091) and by the Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation when needed (project n°19683).

PARASITES

Genetically modified strains of Leishmania amazonensis (L. amazonensis) LV79 (MPRO/BR/1972/M1841) and of Leishmania donovani (L. donovani) Ld1S (MHOM/SD/62/1S-2D) constitutively expressing the gene of the fluorescent molecule mCherry were propagated in Swiss nu/nu mice and Golden hamsters, respectively.

PURIFICATION OF LESION-DERIVED AMASTIGOTES

Leishmania amazonensis amastigotes

Leishmania amazonensis-infected Swiss nu/nu mice were euthanized with CO2. Footpad lesions (<10 mm) were excised and soaked in 70% ethanol at room temperature (RT) for 5 min and then in cold PBS supplemented with antibiotics (100 units/µg per ml of penicillin and streptomycin, [PS]) for 5 min. Skin and necrotic parts were removed and lesions were disrupted in cold PBS (PBS-PS) using a Potter homogenizer. Tissue debris was removed by multiple centrifugations at 30 g for 10 min at 4°C. Parasites present in the supernatant were recovered by centrifugation at 1500 g for 10 min at 4°C. Supernatant was discarded and the parasite pellet was resuspended with 5 ml of Gey's Balanced Salt Solution and incubated for 10 min on ice to lyse red blood cells. Cold PBS-PS was added to stop lysis, and parasites were centrifuged once more at 1500 g for 10 min at 4°C. The pellet was resuspended in 5-8 ml of cold PBS-PS and parasites were counted in a 2.5% glutaraldehyde solution in a Malassez chamber.

Leishmania donovani amastigotes

Leishmania donovani amastigotes were obtained in the laboratory by Pascale Pescher in order to compare the effect of the tested compounds on infection with a different Leishmania species. Briefly, infected hamsters were euthanized with CO2, liver and spleen were recovered and homogenized by grinding in PBS-PS and amastigotes were purified. Live parasites were separated from dead parasites and cellular debris on a 90%/45% Percoll gradient, washed and resuspended in PBS for counting in the same conditions as described for L. amazonensis.

PROMASTIGOTE CULTURE

L. amazonensis promastigotes were differentiated from lesion-derived amastigotes isolated from infected mice and grown in M199 supplemented medium (Ld1spro medium, see Table 7).

MATERIALS & METHODS 55

CELL CULTURE AND INFECTION

PREPARATION OF MURINE PRIMARY MACROPHAGES AND INFECTION

Bone marrow cells were recovered from femurs and tibias of female C57BL6/6JRj mice after their euthanasia with CO2. Femurs and tibias were excised after removal of skin and muscles of both legs, and immersed in cold PBS-PS. Bones were cut at both ends with a scalpel and bone marrow cells were flushed with cold PBS using a syringe with a 25G needle. The cell suspension was centrifuged at 300 g for 10 min at 4°C. The cell pellet was resuspended in 5 ml of Gey's Balanced Salt Solution and incubated for 10 min on ice to lyse red blood cells. Cold PBS was added to stop the lysis and cells were centrifuged again at 300 g for 10 min at 4°C. Cells were resuspended in 1 ml of Complete Medium (C-medium, see Table 8) and living cells were counted using a 0.2% Trypan Blue vital dye solution in a Malassez chamber.

The cell suspension was adjusted to 2.5-3x10 7 cells in 12 ml of C-medium supplemented with 50 ng/ml of recombinant mouse macrophage colony stimulating factor 1 (rmM-CSF1, ImmunoTools), distributed into tissue culture dishes (Falcon® 100 mm TC-treated Cell Culture Dish ref OPTILUX 353003; 12 ml per Petri dish) and placed overnight at 37°C under 7.5% CO2 to discard adherent debris and adherent cells (fibroblasts and macrophages). The optimal concentrations of glucose (4.5g/L) and rmM-CSF1 were empirically defined as the optimal conditions for obtaining a high yield of differentiated macrophages.

The following day, nonadherent cells were collected and the cell suspension was diluted to 1x10 7 per 12 ml of C-medium containing 50 ng/ml of rmM-CSF1 and distributed in bacteriological plates (Greiner Ref. 664161; 12 ml/ Petri dish). After 3 days of culture, 4 ml of C-medium containing 50 ng/ml of rmM-CSF1 were added. After 6 days of culture, the medium was removed and adherent cells were incubated with pre-warmed PBS pH 7.4 containing 25 mM EDTA for 30 min at 37°C. After the addition of cold PBS, macrophages were detached by gentle flushing and collected by centrifugation at 300 g for 10 min at 4°C. The cell pellet was resuspended in C-medium and cell counting was carried out as described above. Bone marrow-derived macrophages (BMDMs) were plated on different tissue culture supports according to the applied procedures, as shown in Table 9.

ISOLATION OF EX VIVO LEISHMANIA-INFECTED MACROPHAGES (EVLIMS) FROM MOUSE LESIONS

evLIMs were isolated from footpad lesions of Rag2 tm1Fwa mice obtained 2 months after intradermal L. amazonensis amastigote inoculation. Mice were euthanized with CO2 and footpads were placed for 2 min in 70% ethanol. Then the skin was removed from the footpads with scissors. Footpads were then placed on a 100 µm cell strainer and incubated in a Digestion Buffer (DB) containing serum-free DMEM supplemented with 50 U/ml DNAse I (Sigma-Aldrich), 100 U/ml collagenase II (Sigma-Aldrich), 100 U/ml collagenase IV (Sigma-Aldrich) and 1 U/ml dispase II (Roche Applied Science) for 30 min in a 6 well plate. Then, one ml of DB was perfused in the footpad at 5 different locations for 30 min at 34°C. This step was repeated two times. evLIMs were recovered and washed (centrifugation at 50 g, 10 min, 4 °C). Then evLIMs were placed for 1 hour at 34°C in Petri dishes (OPTILUX 353003) to remove free amastigotes (released from cells damaged during the procedure) that have the ability to attach to the support. The supernatant containing evLIMs was recovered to perform ex vivo analyses or establish ex vivo cell cultures.

CHEMICAL COMPOUNDS

Lysine Specific Demethylase 1A Inhibitors MC2652 and MC4030 (LSD1i), and TCP were synthesized in the laboratory of Antonello Mai (Facolta di Farmacia e Medicina, Sapienza Universita, Roma, Italy). All tested compounds were solubilized in DMSO at a 10 mM concentration and stored as aliquots at -20°C in 1.5 mL Eppendorf tubes.

CELL TREATMENT

LSD1i were added to uninfected and infected BMDMs, 24 hours after infection, at a final concentration of 10 µM or 15 µM. Subsequently, RNA, protein, or chromatin extraction was performed, as well as imaging (see Figure 20). The detailed procedures for these experiments will be explained in the following sections.

PARASITE LOAD DETERMINATION

GIEMSA ANALYSIS

Lab-Tek Chamber Slide™ were designed for plating, incubating, fixing and staining cells on a single microscope slide with a removable chamber. For a 16well Lab-Tek slide, 5x10 4 BMDMs or 2x10 4 RAW264.7 cells in 100 µl of C-medium were seeded per well. After 4 hours, cells were infected with L. amazonensis (MOI of 4) and treated the following day with LSD1i at 15 µM. Four days post-infection (PI) and three days post-treatment (PT), the medium was discarded, cells were washed with 1x PBS and were fixed with 100 µl of 100% methanol for 3 min at RT. Afterwards, cells were stained with a 10% Giemsa solution for 4 min and finally washed with Milli-Q water. Chamber was removed for counting uninfected and infected cells, as well as the number of parasites per cell.

MULTIPARAMETRIC IMMUNOFLUORESCENCE ANALYSES

This approach was used for both the determination of parasite load (detection of amastigotes with the 2A3-26 mAb) and the localization of LSD1 on fixed cells. 2x10 5 BMDMs were seeded in 500 µl of C-medium per well of 24-well plates containing sterile glass coverslips. They were infected or not with L. amazonensis amastigotes with a MOI of 4 for one day and treated with 10 µM LSD1i. Three days PT, Fixation solution was added to the cells for 30 min at RT after a PBS wash (1 ml/well, 10 min, RT).

Subsequently, Quenching solution was added to block free aldehyde groups (1 ml/well, 15 min, RT). Cells were rinsed twice for 5 min in PBS and incubated with the Blocking and permeabilization solution for 1 hour at RT. Primary antibody solution was added for an overnight incubation at 4°C. The next day, cells were rinsed 3 times for 5 min in 1x PBS and subsequently incubated for 90 min at RT in the dark with the secondary antibody solution. Afterwards, cells were rinsed once in 1x PBS for 5 min, incubated for 15 min in a 1x PBS solution containing Hoechst 33342 (1M) and rinsed one last time with 1x PBS for 5 min.

Finally, coverslips were rinsed in Milli-Q water, dried and mounted using Slowfade Gold mounting agent on clean glass slides. The slides were stored overnight in the dark at 4°C and sealed with transparent varnish. The slides were examined, and images were captured using a Zeiss AxioPhot microscope equipped with an Andor camera. The purpose of this study was to quantify uninfected and infected cells, determine the number of parasites per cell, and identify the subcellular localization of LSD1.

EVALUATION OF COMPOUNDS CYTOTOXICITY BY THE RESAZURIN REDUCTION ASSAY

The Resazurin reduction assay is an enzymatic-based biochemical assay used for assessing the toxicity (cell death) of selected compounds on all types of cells (free parasites and uninfected/infected macrophages). It quantifies the metabolic activity of cells using resazurin, a cell-permeable redox indicator that changes color and emits fluorescence after reduction by diaphorase enzymes (flavoprotein enzymes within the mitochondria, which serve as a catalyst for the reduction or oxidation) (Figure 21). When dissolved in physiological buffers, resazurin has a deep blue color and weakly fluorescent properties. When added to cells in culture, the active metabolism of living cells will reduce resazurin into resorufin (that is pink and highly fluorescent) as depicted in Figure 21. The amount of resorufin produced is proportional to the number of living cells and can be quantified with a microplate fluorescent reader (560 nm excitation/590 nm emission).

The cytotoxic activity of LSD1i was tested in triplicate in 96-well plates using uninfected BMDMs, RAW 264.7 cells, lesion-derived amastigotes, and promastigotes in logarithmic phase. These experiments were conducted in both C-medium and Ld1spro medium, but Ld1spro medium was exclusively used for free, lesion-derived amastigotes and promastigotes.

First, serial two-fold dilutions of compounds (ranging from 200 µM to 100 nM) were prepared in the corresponding medium, and 100 µl of each dilution was distributed by well. Next, cells or parasites, according to the Table 11 were added to the plates in 100 µl of medium per well. The control groups included wells with medium alone and vehicle (1% DMSO as the initial concentration) for all four cell types. Additionally, amphotericin B or miltefosine were included for the lesion-derived amastigotes and promastigotes (dilutions prepared as for compounds), while cycloheximide (10 µg/ml as the initial concentration) was used for BMDMs and RAW 264.7 cells (Figure 22).

Two days after, 10 µl of resazurin were added (5 µg/ml) and the fluorescence intensity was measured 24h later on a Tecan Safire 2 reader. The optimal gain, which determines the amplification of an incoming fluorescent signal and provides better data resolution, was determined automatically for each plate before fluorescence acquisition. The background fluorescence (corresponding to the medium containing resazurin without cells/parasites) is subtracted from the values and the results are expressed as percentage of the negative control (corresponding to the cells/parasites without drugs). The cytotoxicity of the compounds is expressed as Inhibitory Concentration 50% (IC50).

CELLULAR THERMAL SHIFT ASSAY (CETSA)

Uninfected and infected BMDMs were treated or not 1 day after infection with 10 µM LSD1i (MC2652, MC4030, and TCP) or 0.1% DMSO as a control. After one day of treatment, cells were washed with the pre-warmed medium before adding 1.5 ml of cold Extraction Buffer (1x PBS -1x PIC -1 mM PMSF). Then, cells were collected with a cell scraper in two 1.5 ml Eppendorf tubes and subjected to three cycles of freezing in liquid nitrogen and thawing in a thermomixer at 25°C and 400 rpm. Lysates were submitted to 3 sonication cycles (30" on/30" off) at high power, then pooled in one Eppendorf tube per condition and centrifuged at 20,000 g for 20 min at 4°C.

Supernatants containing soluble proteins were transferred to 1.5 ml tubes, then distributed into twelve 0.2 ml PCR tubes and heated at different temperatures (from 37°C to 70°C, ∆T°=+3°C) in a Thermocycler for 3 min. Afterwards, protein solutions were transferred to 1.5 ml Eppendorf tubes and cleared at 20,000 g for 20 min at 4°C. 75 µl of supernatant containing the soluble protein fraction was carefully collected into a new LoBind® Eppendorf tube. The detection of the remaining soluble proteins was performed by Western Blot, where 11 µl of each cleared sample were mixed with 2 µl of NuPAGE® Reducing Agent and 5 µl of NuPAGE® LDS Sample Buffer. The samples for each CETSA were loaded on the same gel, and quantification of soluble protein was performed by measuring the fluorescence intensity emitted by the ECL Plex fluorescent secondary antibodies with an Amersham™ ImageQuant™ 800 device. Normalization was carried out with Amersham ImageQuant TL analysis software taking into account as 100% the band intensity of the sample heated at 37°C. Melting curves were generated with GraphPad Prism software package (Figure 23).

NUCLEAR PROTEIN EXTRACTION & QUANTIFICATION

GENERAL PROCEDURE

Nuclear protein extraction was performed on uninfected and infected BMDMs after two days of infection. The medium was removed from each dish and replaced by 950 µl of Lysis Buffer (LBI or LBII, depending on the following procedure, see Table 12). After 10 min of incubation on ice, cells were detached with a cell culture scraper and collected in a 2 ml Eppendorf tube. The cell homogenate was passed 5 times through a 27G needle to isolate nuclei. The quality of isolated nuclei was assessed by the EVOS Cell Imaging System, as described in Figure 24. Samples were centrifuged for 10 min at 4°C at i) 250 g for activity assay, western blot and nuclear proteomics, or at ii) 500 g for LSD1 co-immunoprecipitation (Co-IP) assay. Supernatants containing cytoplasmic proteins were removed and kept for further analysis, whereas 150 µl of Extraction Buffer (EBI, EBII or EBII, depending on the following procedure, see Table 12) were added to pellets. Tubes were vortexed and incubated for 10 min (a 1 ml tip was used to better dissociate the pellet). MATERIALS & METHODS 66 min (30" on/30" off sonication cycles) at high power. When pellet residues were still present, they were dissociated with a 200 µl tip and 3 more min of sonication. Extracts were cleared by centrifuging at 15,000 g for 10 min at 4°C.

Table 13 summarizes the function of the common components of the Lysis and Extraction buffers used during protein extraction.

Spermidine

Polyamines. Physical quenchers of singlet molecular oxygen 1 [O2], preventing its reactivity with amino acids.

Spermine Protein Inhibitor Cocktail (PIC)

Protects from degradation by proteases released during protein extraction and purification. It inhibits aspartic acid, cysteine and serine proteases as it includes AEBSF, aprotinin, bestatin, E64, Leupeptin and pepstatin A.

NUCLEAR PROTEIN EXTRACTION FOR LSD1 CO-IP

When using EBIII, 1% Digitonin Extraction Buffer, big white pellets were observed in all the tubes after clearing at 15,000 g for 10 min at 4°C. To dissociate the pellets and recover the maximum amount of proteins, an additional 200 µl of EBIII were added. The solution was sonicated for 5 min to further dissociate the proteins. Finally, proteins were cleared by centrifuging at 15,000 g for 10 min at 4°C to remove any debris.

PROTEIN QUANTIFICATION

Nuclear and cytoplasmic protein quantification was performed with the Detergent Compatible (DC) protein assay (Bio-Rad Laboratories), according to manufacturer's instructions. The test is based on the principle of a Lowry assay, using a colorimetric readout to determine protein concentration. The absorbance is read at 750 nm on a scanning spectrophotometer and the concentration of the sample is calculated from a BSA standard curve.

LSD1 ACTIVITY ASSAY

LSD1 activity assay was performed in triplicate with the Fluorometric KDM1/LSD1 Activity Quantification Kit (Abcam), according to the manufacturer's instructions. Briefly, the reaction solution was prepared in strip wells by mixing 1 µg of nuclear protein extracts from uninfected BMDMs, assay buffer and di-methylated lysine 4 of the Histone H3 (H3K4me2) as substrate, with the latter stably coated onto the strip wells (provided in the kit).

LSD1i compounds MC2652, MC4030 and TCP at 30 and 100 µM, were added in order to validate their inhibitory effect on LSD1 activity. Strip wells were incubated at 37°C for 60 min for the enzymatic reaction to take place. The reaction solution was removed from the strip wells that were washed three times. Diluted capture antibody solution (1/1000) was added to the strip wells, covered with aluminum foil and incubated for 60 min at RT. The diluted capture solution was removed, strip wells were washed three times, the diluted detection antibody solution (1/2000) was added, and the strip wells were again covered with aluminum foil and incubated for further 30 min at RT.

Finally, the diluted detection antibody was removed, the strip wells were washed four times and the fluorescent developing solution was added and incubated for 4 min at RT in the dark.

The fluorescence-developing solution causes a color shift (pink) in the presence of LSD1-converted, demethylated products. Relative fluorescence units (RFU) were read with the Tecan Safire 2 microplate reader (530 nm excitation/590 nm emission). The activity of LSD1 is proportional to the intensity of the measured fluorescence. The LSD1 activity was calculated using a standard curve prepared with a standard solution containing non-methylated Histone H3K4 (H3K4me0), following the instructions provided in the kit. Slope calculation of the standard curve (as RFU/ng) was performed using Microsoft Excel and the amount of LSD1-demethylated product (H3K4me0) was calculated using the following formulas:

𝐷𝑒𝑚𝑒𝑡ℎ𝑦𝑙𝑎𝑡𝑒𝑑 𝑃𝑟𝑜𝑑𝑢𝑐𝑡 (𝑛𝑔) = 𝑆𝑎𝑚𝑝𝑙𝑒 𝑅𝐹𝑈 -𝐵𝑙𝑎𝑛𝑘 𝑅𝐹𝑈 𝑆𝑙𝑜𝑝𝑒 𝐿𝑆𝐷 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ( 𝑛𝑔 𝑚𝑖𝑛 * 𝑚𝑔 ) = 𝐷𝑒𝑚𝑒𝑡ℎ𝑦𝑙𝑎𝑡𝑒𝑑 𝑃𝑟𝑜𝑑𝑢𝑐𝑡 (𝑛𝑔) 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 𝑎𝑚𝑜𝑢𝑛𝑡 (µ𝑔) * 60 𝑚𝑖𝑛 𝑋 1000 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 (%) = 1 - 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟 𝑆𝑎𝑚𝑝𝑙𝑒 𝑅𝐹𝑈 -𝐵𝑙𝑎𝑛𝑘 𝑅𝐹𝑈 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑆𝑎𝑚𝑝𝑙𝑒 𝐹𝑅𝑈 -𝐵𝑙𝑎𝑛𝑘 𝑅𝐹𝑈 𝑥 100

WESTERN BLOT (WB) ANALYSES

For each sample, 5-10 µg of proteins were mixed with NuPAGE® Reducing Agent (1X) and NuPAGE® LDS Sample Buffer (1X) and heated at 95°C for 5 min. Samples were loaded onto NuPAGE® Novex Bis-Tris 4-12% polyacrylamide gradient Mini Gels (Invitrogen) and submitted to electrophoresis at 80 V for 10 min and then at 180 V for 40 min.

Proteins in the SDS-PAGE gel were either submitted to Coomassie Staining or electroblotted onto polyvinylidene fluoride (PVDF) membranes (ThermoScientific), previously activated with 100% methanol before assembly of the following "sandwich". From the anode to the cathode: a sponge pad, a Whatman filter paper, the activated membrane, the gel containing the protein samples, a second Whatman filter paper and a second sponge pad. The transfer was performed in transfer solution for 1 hour at 400 mA and 4°C. All subsequent steps were performed in PBS-T (see Table 14).

The membrane was recovered from the sandwich, saturated in the Blocking solution during 1 hour at RT, before incubation with the primary antibodies (Table 15) overnight at 4°C. The following day, three washes of 10 min with PBS-T were performed before incubation with chemiluminescent or fluorescent secondary antibodies for 1 hour at RT (Table 16), followed by three additional washes. Proteins were then revealed with the Amersham™ ImageQuant™ 800 imager.

Two kits were used for Western Blot revelation: Supersignal West Pico Chemiluminescent Substrate (#34080, ThermoScientific) and Amersham ECL Western Blotting Detection Kit.

TOTAL PROTEIN EXTRACTION OF PARASITE PROTEINS

To assess antibody cross-reactivity with parasite proteins, total protein extracts were obtained from 5 x 10 8 L. amazonensis amastigotes or promastigotes. The extraction was performed using 500 µl of RIPA-based lysis buffer, consisting of RIPA 1x (Cell Signaling, #9806), 1x PIC (cOmplete Roche tablets), and 1 mM PMSF. Parasites were lysed with the Lysis Buffer, followed by 10 min of ice incubation, and then subjected to 10 cycles of sonication using Diagenode's Bioruptor® Standard (30" on/30" off sonication cycles) at high power. Subsequently, the lysates were cleared by centrifugation at 15,000 g for 10 min at 4°C.

COOMASSIE BLUE STAINING

The gel was immersed in the Coomassie R250 stain slow solution for 30 min at RT and incubated in the de-staining solution overnight at RT (Table 17).

LSD1 CO-IMMUNOPRECIPITATION (CO-IP)

LSD1 Co-IP was performed using nuclear protein samples from uninfected and infected BMDMs. Dynabeads™ Protein G were resuspended, transferred to Eppendorf tubes (50 µl for sample pre-clearing and 25 µl for immunoprecipitation) and washed three times with 200 µl of PBS-T, pH 7.4. The composition of the buffers used for this Co-IP is indicated in Table 18.

DYNABEADS-ANTIBODY COMPLEXES, SAMPLE PRE-CLEARING & IMMUNOPRECIPITATION

For sample pre-clearing (Figure 25), beads were incubated for 30 min at RT with 5 µg of negative control antibody (IgG from healthy rabbits, C-, Diagenode Ref. Samples (900 µg per sample) were pre-cleared twice with the negative control antibody for 45 min at 4°C as shown in Figure 26. 15 µl of the twice pre-cleared samples were kept as Input for further analysis.

Immunoprecipitation was performed by resuspending the cross-linked Dynabeads-Ab complexes with 300 µg of pre-cleared samples and incubating overnight at 4°C (Figure 26). The following day, supernatants (S) were recovered and transferred to a new tube (for further analysis) and Dynabeads-Ab-Antigen (Ag) complexes were washed 3 times with 1 ml of Wash Buffer.

ANTIGEN ELUTION & MASS SPECTROMETRY ANALYSIS

Denaturing elution for WB and SYPRO Ruby gel staining

During the process of parameters optimization, the antigen (Ag) complexes were eluted with a mix of 20 µl of Elution Buffer, 7 µl of NuPAGE LDS Sample Buffer and 3 µl of NuPAGE Sample Reducing Agent, per tube, for SYPRO Ruby gel staining and WB analysis (Figure 27) (performed as described in Section 10 of this chapter). Input and supernatant samples were also analyzed.

The extremely sensitive, ready to use fluorescent SYPRO Ruby protein gel dye was used to detect total proteins separated by PAGE. The staining sensitivity is as good as or better than the best silver staining techniques.

Half of the eluted samples were loaded on a gel and separated by electrophoresis. The gel was washed twice in a 50% methanol -7% acetic acid solution for 30 min at RT. Afterwards, it was incubated overnight in 50 ml of SYPRO Ruby at RT away from the light. The next day, the gel was washed twice in a 10% methanol -7% acetic acid solution and finally rinsed in Milli-Q water before stained proteins were visualized using the Amersham™ ImageQuant™ 800 imager.

Mass spectrometry analysis

Once the ideal immunoprecipitation parameters were established, antigen complexes were washed four times with 1 ml of 100 mM ammonium bicarbonate. Dynabeads-Antibody-Antigen complexes in 100 µl ammonium bicarbonate were used for mass spectrometry analysis at the CurieCoreTech Mass Spectrometry Proteomics of Institut Curie (Figure 27).

CHIPMENTATION

CHROMATIN EXTRACTION FROM ISOLATED NUCLEI

Following the experimental design shown in Figure 20, medium was removed from the dishes 2 days PI and replaced by 10 ml of serum-free DMEM medium containing 1% paraformaldehyde (Fixation solution) to preserve protein-DNA and protein-protein interactions. After 7 min of fixation at RT, 1 ml of glycine 1.375 M was added to each Petri dish (final concentration of 125 mM) for 5 min at RT, in order to stop formaldehyde cross-linking. The supernatant was removed and dishes were rinsed twice with ice-cold PBS.

Afterwards, PBS was removed and 1 ml of Lysis Buffer was added to each dish to isolate nuclei. Samples were collected using a cell scraper and transferred to a 2 mL Eppendorf tube. Suspensions were passed 10 times through a 27G needle and the quality of nuclear isolation was assessed by the EVOS Cell Imaging System. Tubes were centrifuged at 500 g for 10 min at 4°C. Supernatants were discarded and 300 µl of Shearing Buffer were added to each pellet. Tubes were vortexed and incubated for 10 min, followed by sonication using the Diagenode's Bioruptor® Standard for 15 min (30" on/30" off sonication cycles) at high power (Figure 28). A 60 µl aliquot per condition (Input sample) was used for chromatin quality control tests.

CHROMATIN IMMUNOPRECIPITATION

Dynabeads™ Protein G were resuspended, transferred to Eppendorf tubes (30 µl/tube), washed 3 times with 200 µl of PBS-T, pH 7.4 and 3 times with Blocking Solution (Table 19). Subsequently, they were incubated for 3 hours at RT with 4 µg of antibody (α-LSD1 20752 or IgGs from naïve non-immunized rabbits, as negative control C-) and washed again 3 times with Blocking Solution.

Immunoprecipitation was performed by resuspending the Dynabeads-Ab complexes with 250 µl of chromatin samples and incubating them overnight at 4°C. The following day, supernatants (S) were transferred to a new tube (for further analysis) and Dynabeads-Ab-Ag complexes were washed in RIPA Wash Buffer once, in Low Salt Wash Buffer twice, in High Salt Wash Buffer twice, in LiCl Wash Buffer twice and, finally in 10 mM Tris-HCl, pH 8.0 twice.

During the last wash, samples were separated into two different tubes. The contents of the first tube were used to perform WB as an immunoprecipitation quality control test, together with input and supernatant samples. For this purpose, antigen was eluted with 20 µl of Elution Buffer, 7 µl of NuPAGE LDS Sample Buffer and 3 µl of 1x NuPAGE Sample Reducing Agent. In the second tube, tagmentation on beads was performed (Figure 29).

TAGMENTATION, REVERSE CROSS-LINKING & DNA QUANTIFICATION

Beads were collected in the bottom of the tube by placing it in contact with the magnet and traces of Tris-HCl were removed by pipetting. Beads were then resuspended with 30 µl tagmentation mix per sample. Tagmentation mix was also added in a tube with 2 µl of input sample (chromatin before immunoprecipitation). Afterwards, tubes were incubated for 5 min on a thermomixer at 37°C and 800 rpm.

To stop the tagmentation reaction for input samples, 70 µl of ChIP Elution Buffer were added directly, together with 2 µL of Proteinase K (20 mg/ml). For immunoprecipitated samples, beads were washed twice with Low Salt Wash Buffer and once with 1x TE 50 mM NaCl, then beads were collected in the bottom of the tube, all solution trace was removed as described before, and 100 µl of ChIP Elution Buffer were added, together with 2 µl of Proteinase K (20 mg/ml).

Tubes were incubated in a thermomixer at 65°C with shaking at 1400 rpm overnight for reverse cross-linking. The following day, DNA was purified using the ChIP DNA Clean & Concentrator from Zymo Research, according to manufacturer's instructions.

LIBRARY PREPARATION (PILOT EXPERIMENT)

Different amounts of DNA from input samples and immunoprecipitated (IP) samples (LSD1 and C-) were used for library preparation. Libraries were amplified using Unique Dual Indexes (UDIs) pairs 1 to 10 (Set I, Cat. No. C01011034, Diagenode) as indicated in Table 20, following the procedure described by Xu et al. [198].

Since the tagmentation process creates 9-bp gaps, the first step in the PCR should be at 72°C to allow the polymerase to fill in the gaps. PCR reactions were prepared as indicated in Table 21, and a pre-amplification PCR run was performed following the conditions described in Table 22. After pre-amplification, 9 µl of the reaction were mixed with 1 µl of 10x EvaGreen (in duplicate per sample), and a qPCR was performed using the conditions outlined in Table 23 to determine the optimal cycle number for the whole library amplification. The remaining 32 µl of reaction were kept at 4°C. The cycle number (N) is determined where the amplification curve reaches half of its saturation, and it should be selected during the exponential phase before reaching full saturation. Figure 31 shows an example where N = 11 for one sample and 14 for the second one.

MATERIALS & METHODS

81

Once the optimal cycle number was decided, the remaining 32 µl of the reaction were amplified for N cycles, using the conditions indicated in Table 24.

Subsequently, the library PCR product was purified using Agencourt AmpureXP beads for PCR purification (Beckman Coulter). Quantification of purified DNA libraries was carried using the iQuant™ dsDNA HS Assay Kit and the Qubit 2.0 Fluorometer as previously described.

The quality of the purified libraries was assessed using an Agilent Bioanalyzer 2100 with the Agilent DNA 1000 Kit, following the steps described in the manufacturer's manual.

PROTEOMIC ANALYSIS OF BMDM NUCLEAR PROTEINS

Following the procedure outlined in Section 8.1 of this chapter and utilizing LBI and EBI (composition indicated in Table 12), nuclear protein extracts were obtained from both uninfected and infected BMDMs. This process was conducted for a total of 4 biological replicates. Table 25 provides a summary of the components EBI and their respective functions.

Component Function

DTT

Reducing agent that disrupts intramolecular and intermolecular disulfide bonds. Used to achieve complete protein unfolding and to maintain proteins in their fully reduced states.

Ammonium bicarbonate

Inhibits carbamylation when using urea as a protein denaturant.

Urea

Chaotropic compound that disrupts hydrogen bonds and hydrophobic interactions both between and within proteins. When used at high concentrations, it destroys secondary protein structure and brings otherwise-insoluble proteins into solution.

Samples were quantified following the protocol described in Section 8.3. Next, the proteins were visualized on an SDS-PAGE gel that was stained with Coomassie Brilliant Blue R-250 as described in Section 10.2. Additionally, WB analysis was conducted using LSD1 and GAPDH as nuclear and cytoplasmic markers, respectively, as indicated in Section 10. Subsequently, 30 µg of each sample was sent to the CurieCoreTech Mass Spectrometry Proteomics platform, located at the Curie Institute, for mass spectrometry analysis.

RESULTS

83

RESULTS

RESULTS

CHAPTER 1. PHARMACOLOGICAL CHARACTERIZATION OF MACROPHAGE LSD1 INHIBITORS (LSD1I) 1 INTRODUCTION

As I begin this thesis, it is crucial to acknowledge both the current and former team members for their significant contributions in laying the foundations of my experimental work. This introduction aims to summarize the knowledge acquired thanks to their efforts. Their research has laid the groundwork for my experiments and forms the basis of my scientific activities.

HIGH CONTENT ANALYSIS FOR ANTI-LEISHMANIAL DRUG DISCOVERY

The current intervention strategy for leishmaniasis relies primary on chemotherapy due to the lack of effective reservoir and vector control strategies, as well as preventive and therapeutic vaccines. Existing treatments have limitations and drawbacks such as high cost, poor compliance, safety concerns, treatment failure, and drug resistance. Efforts to develop combined therapies have also faced challenges due to the emergence of drug resistant strains. Therefore, there is a need for the discovery of novel strategies, drugs and drug targets for the treatment of leishmaniasis.

Despite the availability of screening methodologies and compound libraries, the translation of these resources into new therapeutics has been limited, raising questions about the suitability of current protocols and pipelines.

Common screening approaches have limitations due to the types of parasites and host cells used. Cultured promastigotes and axenic amastigotes do not accurately reflect the biology and environment of the parasites inside macrophages [200]. Additionally, the malignant background of macrophage cell lines results in significant variations in gene expression, immune activation and cellular characteristics compared to authentic macrophages [201], thus compromising the interpretation of screening results. Furthermore, combining host cell lines with cultured-derived parasites leads in general to an abundance of extracellular promastigotes and a lower rate of cells hosting infectious, proliferating amastigotes.

To overcome these limitations, Aulner and colleagues, developed a reliable assay using mouse primary macrophages (BMDMs) and highly virulent lesionsderived amastigotes of L. amazonensis [202]. With these parasites, macrophage infection results in the development of large PVs housing cell-cycling amastigotes, a characteristic of successful intracellular infection for the parasites belonging to the L. mexicana complex. In this assay, the presence or absence of large communal PVs stained with a specific acidotropic fluorescent probe served as readout to measure the leishmanicidal effect of the tested compounds. Based on this biologically relevant infection system, a semiautomatic phenotypic screening pipeline was established. Subsequently, this pipeline was adapted for screening with L. donovani, where the measurement relied specifically on the quantity of individual, immuno-stained parasites [203].

Leishmania is capable of modulating the TF landscape in infected cells and promoting changes in macrophage epigenetic regulation to establish a disease-promoting state. Furthermore, it has been demonstrated that parasites remodel the chromatin of host cells during infection by causing Histone H3 demethylation at activating H3K4 and repressive H3K9 marks [194]. Based on these findings and in the context of the FP7 A-PARADDISE (Anti-Parasitic Drug Discovery in Epigenetics) project, our team applied a dual screening assay to evaluate a selected library of 475 mammalian epigenetic inhibitors originally developed for cancer treatment (Table 26 and Figure 33A). The screening assay tested both the survival of free promastigote and intramacrophagic amastigote forms (Figure 33B).

TCP-DERIVATIVES WITH HIGHLY SPECIFIC ANTI-LSD1 ACTIVITY

LSD1 possess a catalytic domain that exhibits structural similarity to amine oxidases, a class of flavin-dependent enzymes involved in the metabolism of biogenic amines. Among these enzymes, monoamine oxidases (MAOs) A and B have been extensively studied, particularly for their key role in inactivating neuroactive amines such as serotonin, dopamine, and norepinephrine, proposing MAOs as drug targets for the treatment of different nervous system disorders, including depression and Parkinson's disease. Due to the catalytic and structural similarities between MAOs and LSD1, the possibility to use anti-MAO drugs as inhibitors of LSD1 has also been explored. Tranylcypromine (TCP), a major MAO inhibitor used for over 50 years has been identified as a LSD1 inhibitor. Based on the TCP scaffold, the team of our collaboration partner Antonello Mai (UNIROMA2, La Sapienza, Italy) designed compounds with increased selectivity towards demethylases [146].

Our previous screening efforts identified the two TCP-derivatives MC4030 and MC2652 (LSD1i) (Figure 34), synthesized in Antonello Mai's laboratory, as interesting hit compounds with strong efficacy against intramacrophagic amastigotes. It was observed that these compounds did not directly target the parasites themselves, i.e. they do not kill Leishmania promastigotes in culture suggesting that a macrophage-mediated mechanism is responsible of the decrease of intracellular parasite load. This hypothesis was also supported by the absence of a conserved LSD1 ortholog in Leishmania [204,205] and the demonstrated inhibitory activity against host LSD1 in enzymatic assays (detailed in the following section). Subsequently, MC4030 (Figure 34) was designed as an analog of MC2652 by replacing the 3-phenyl ring with heterocyclic thiophene moieties. The thermal Chapter 1. Pharmacological characterization of macrophage LSD1i 91 stability and inhibition of human LSD1-CoREST, as well as the levels of H3K4me2 and H3K9me2, were evaluated to assess the activity of the inhibitors. Additionally, their antiproliferative potential was tested against U937 AML and prostate LNCaP cancer cell lines, demonstrating its efficacy against mammalian LSD1 in cellula [207].

The two compounds identified in these prior studies served as the foundation of my investigations. I used them to (i) elucidate their potential host-directed mode of action, (ii) assess the mechanisms of their anti-leishmanial activity, and (iii) pharmacologically validate the Leishmania epigenetic subversion strategy on its host macrophage. Through our research, we aimed to gain a deeper understanding of the interplay between these compounds and the Leishmania parasite within the host macrophage, with the ultimate goal of developing more effective and targeted treatments for leishmaniasis.

TRANSCRIPTOMICS ANALYSES

In order to gain first insight into how inhibiting LSD1 leads to the macrophagedependent elimination of intracellular L. amazonensis, a series of RNAseq analyses was performed by team members. The analyses were conducted in quadruplicate with BMDMs that were untreated or treated with 10 µM of MC4030 for 24h following two days of infection with lesion-derived L. amazonensis amastigotes. Infection alone had a significant impact on the gene expression of macrophages, with 6,436 differentially expressed genes (DEG) (adjusted p value < 0.05): 3,241 and 3,195 genes showing a statistically significant increased or decreased in mRNA abundance, respectively (Figure 35A).

Among the modulated genes, a list of LSD1 indirect and direct target genes (color dots) was compiled from bibliography (Table 27). Following 24 hours of treatment with MC4030, uninfected and infected macrophages showed different profiles that also affected the expression levels of many putative LSD1 target genes (Figure 35B & Figure 35C), thus validating the direct effect of the inhibitor on macrophage epigenetic regulation and further supporting its possible host-directed mode of action.

A principal component analysis (PCA) of the expression changes across the various conditions Figure 36 indicates that treating infected macrophages with MC4030 resulted in a partial restoration of normal macrophage functions, as indicated by the reduced distance between drug-treated uninfected and infected samples compared to the distance observed for the corresponding untreated samples. Green and red dots represent LSD1 direct and indirect target genes that were down and up modulated, respectively. Grey dots correspond to modulated genes that have not been identified as LSD1 targets (Table 27). We used the STRING application implemented in the Cytoscape software package to better assess and visualize the reversion of the expression profile upon treatment of infected BMDMs. In this analysis, the differential expression values observed between infected and uninfected BMDMs are represented by the border color of the bubbles, while the p-value is represented by the border thickness. The fill color of the bubbles represents the differential expression changes observed in infected and treated BMDMs compared to infected untreated control BMDMs, with the bubble size corresponding to the p-value (Figure 37).

From a total of 456 genes that responded to the treatment, 377 showed counter-regulation of the Leishmania-induced expression changes as indicated by the bubbles with opposing border and filling colors, i.e. 293 transcripts showed decreased abundance caused by infection (green filling), which showed increased abundance (i.e. normal expression was 'rescued') by the treatment (red border) while the opposite was observe for 84 transcripts (increased transcript abundance upon infection, which was reduced by treatment). Treatment had no effect on 70 genes that showed infection-induced expression changes (33 up corresponding to red filled bubbles with gray border and 37 down corresponding to green filled bubbles with gray border). For 9 genes we observed similar expression changes between the conditions as indicated by the bubbles with the same border and fill color (Figure 37). Together these data demonstrate that MC4030 treatment of infected BMDMs counteracts 83% of infection-induced expression changes, which likely renders infected BMDMs unsuitable for intracellular parasite survival thus further supporting the host-directed mode of action of this compound.

Indeed, closer inspection of this counter-regulation identified various pathways that may be linked to host-directed parasite elimination either by restoring low expression levels (e.g. sterol biosynthesis, regulation of protein kinase activity, steroid and sterol biosynthetic processes (Figure 38A), or by rescuing higher expression levels (e.g. response to lipopolysaccharide, stress, hypoxia, and oxidative stress were significantly downregulated during the infection (Figure 38B). Hence, the host-directed killing may be explained by a lack of essential sterols that need to be scavenged by growing parasites to fulfill the increased requirement of membrane synthesis, and by the induction of innate macrophage responses with anti-microbial activities.

In my thesis, I followed up on these findings by setting up the experimental conditions for a genome-wide analysis of H3K4 and H3K9 methylation patterns and the promoter occupancy of LSD1 in infected and uninfected BMDMs (see Section 5 of Chapter 2). The primary aim of these analyses is to elucidate an explanation for the DEGs observed in our RNAseq analysis, and to provide insights into the role of LSD1 in modulating gene expression during the Leishmania infection process. Chapter 1. Pharmacological characterization of macrophage LSD1i 97

EFFECT OF LSD1I ON PARASITE LOAD

To validate the results obtained through previous HCA screening campaigns, I performed various experiments to verify the ability of MC2652 and MC4030 (LSD1i) to decrease the parasite load in infected macrophages. These validation tests included analyses of both fixed and living cells. It is important to mention that my thesis was conducted as collaboration between Philippe Loiseau's and Gerald Spaeth's labs. The results obtained using the RAW 264.7 macrophage cell system were acquired during my stay at the Faculty of Pharmacy in the Loiseau laboratory, whereas those obtained on BMDMs were conducted in the Spaeth unit.

During the 4 years of my PhD project, I worked with three different batches of inhibitors. These batches were sent to me sequentially, once each previous batch was exhausted. As a result, it should be noted that the inhibitors were not synthesized simultaneously, and unfortunately, we noticed some variations in toxicity and anti-leishmanial activity against intramacrophagic parasites between the different batches.

Nevertheless, the majority of the data presented in this introduction, which also formed the basis of my project, were produced using batches that demonstrated reproducible anti-leishmanial activity and low host cell toxicity.

ASSESSING DRUG EFFICACY IN FIXED CELLS

Giemsa analysis

The determination of the anti-leishmanial activity of selected compounds on different host cell types was performed, i.e. the assays on BMDMs and RAW 264.7 macrophages infected with lesion-derived amastigotes of L. amazonensis (MOI of 4). After one day of infection, LSD1i and TCP were added at a concentration of 15 µM. At 4 days post-infection (PI) (3 days PT), cells were fixed with methanol and stained with a Giemsa solution to determine the parasite load (Figure 39A-D). Giemsa is a differential stain consisting of a mixture of dyes, including methylene blue, eosin, and Azure B. The basic nature of Azure B reacts with acidic components like nucleic acids, resulting in a deep purple coloration of the nucleus. Eosin, an acidic dye, interacts with the cytoplasmic components producing a pink coloration. This staining technique is used in histopathological diagnosis of Leishmania and other protozoan parasites. Leishmania amastigotes are observed as oval or round organisms measuring between 2 to 4 micrometers. The nuclei and kinetoplast of amastigotes display a dark red or purple color, while the cytoplasm appears as a lighter shade of pink (Figure 39A-D).

The data presented in Figure 39 confirmed that the infection could be conducted in a similar manner in both BMDMs and RAW 264.7 macrophages attaining at least 90% of infected cells. They also demonstrate a similar effect of the different compounds tested. In this case, the inhibitor MC2652 exhibited the strongest anti-leishmanial effect at 15 µM, resulting in the cure of more than 50% of infected cells after 3 days of treatment in both cell types.

At the same concentration, the inhibitor MC4030 showed a lower antileishmanial effect in these experiments, which could be attributed to differences in its mode of action compared to the other inhibitors. While TCP inhibits LSD1 enzymatic activity, our two LSD1i hit compounds may affect its non-enzymatic scaffolding function or protein interactions. This difference in mode of action could explain the varying degrees of anti-leishmanial activity I observed between TCP and the hit compounds, as well as in-between the compounds.

Immunofluorescence analysis

In order to observe the effect of LSD1i during infection with L. donovani, BMDMs were infected with liver/spleen-derived amastigotes of L. donovani (MOI of 8). One day PI, DMSO as a vehicle control, and LSD1i at 15 µM, were added. At 4 days PI and three PT, cells were fixed with 4% PFA and parasites were counted after immunofluorescence staining with a hamster anti-Leishmania immune serum. In both untreated and the vehicle control (DMSOtreated) infected macrophages, more than 90% of cells were infected. After treatment, MC2652 resulted in nearly 50% cure of infected BMDMs (Figure 40). Thus, the anti-leishmanial effect of the inhibitors was similar to that observed in the case of L. amazonensis infection, with MC2652 showing greater effectiveness than MC4030.

ASSESSING DRUG EFFICACY IN LIVING CELLS

BMDMs infected with L. amazonensis

The impact of LSD1i on parasite load and its potential toxicity in living L. amazonensis-infected BMDMs was analyzed using epifluorescence microscopy.

LSD1i was added at a concentration of 10 µM after one day of infection, and epifluorescence microscopy was performed three days PI. Macrophage nuclei and parasites were revealed using Hoechst 33342 staining (blue fluorescence signal) and mCherry expression (red fluorescence signal), respectively. Additionally, we used the cell-impermeant nucleic acid stain oxazole yellow (YO-PRO®-1) that produces a green fluorescence signal in the nuclei of dead macrophages thus informing on compound toxicity for host cells (Figure 41A).

Similar to the results obtained on fixed cells, those obtained on live infected cells also exhibited varying degrees of anti-parasitic activity for LSD1i. Both inhibitors are capable to significantly reduce the parasite load (Figure 41B). However, in this case, MC4030 demonstrated greater effectiveness. It is important to note that this experiment and the previous one conducted on fixed cells, were not performed using the same batch of inhibitors. This further supports the earlier statement regarding potential variations between batches.

It is also worth mentioning that the almost complete absence of green-stained cells by the YO-PRO dye suggests that the inhibitors do not exhibit cytotoxicity towards macrophages at the concentration used (Figure 41A). However, additional cytotoxicity tests were conducted with different LSD1i concentrations, and the results are presented in the following section.

BMDMs infected with L. donovani

The effect of inhibitors at 10 µM on live BMDMs infected with L. donovani was also examined using epifluorescence microscopy. In this case, only the presence of parasites was visualized by the red fluorescence generated by mCherry transgenic expression (Figure 42A). Once again, a significant reduction in parasite load was observed in presence of both LSD1i as observed for L. amazonensis infection (Figure 42B).

EVALUATION OF LSD1I MACROPHAGE CYTOTOXICITY AND ANTI-LEISHMANIAL ACTIVITY

To assess host cell cytotoxicity and anti-parasitic activity of the LSD1 inhibitors MC4030 and MC2652, resazurin assays were performed using a range of concentrations from 100 µM to 50 nM, to determine the half-maximal inhibitory concentration (IC50) values. The IC50 is a measure that quantifies the concentration of a compound needed to reduce the cell viability by 50% when compared to cells grown in the absence of the tested compound. Cytotoxicity was assessed in uninfected BMDMs and RAW 264.7 macrophages, while antileishmanial activity was tested on lesion-derived free amastigotes and promastigotes of L. amazonensis.

Firstly, it was important to verify the nontoxic nature of the inhibitors for the macrophage host cells in C-medium. Both inhibitors from the 2019 batch consistently showed no toxicity towards both BMDMs and RAW 264.7 macrophages, even at a concentration of 100 µM. The cytotoxicity assessment of the 2022 and 2023 batches was conducted exclusively on BMDMs, as RAW 264.7 macrophages were not used for any other experiments during this period. MC2652, in these last two batches, continued to demonstrate the absence of cytotoxicity even at 100 µM. However, the inhibitor MC4030 batches from 2022 and 2023 exhibited cytotoxicity with IC50 values of 31.4±3.5 µM and 58.2±3.3 µM, respectively (Table 28). Nevertheless, in all experiments, the maximum concentration of the inhibitors used was 15 µM, which did not induce any toxicity in BMDMs. Subsequently, my aim was to verify the activity of the inhibitors against lesionderived free amastigotes, which had not been previously tested in the dual screening assay pipeline. Obviously, the previous activity tests performed on Chapter 1. Pharmacological characterization of macrophage LSD1i 105 free promastigotes used the Ld1spro medium. For amastigotes, I used Cmedium and Ld1spro medium to assess potential effects of the culture medium on compound activity.

Surprisingly, the initial results obtained with the 2019 batch were not as consistent as those observed in the host cells. While the inhibitor MC4030 showed no activity even at a concentration of 100 µM in both culture media, MC2652 displayed activity towards amastigotes only in C-medium, with an IC50 of 2.6 µM (Table 29). The same effect was also observed in the tests using promastigotes, where MC4030 had no activity in either of the two culture media, while MC2652 exhibited activity only in C-medium, with an IC50 of 4.9 µM (Table 30). Furthermore, when the activity from the 2022 and 2023 batches of inhibitors was assessed in amastigotes and promastigotes, different results were obtained. MC4030, which had previously shown no activity at the tested concentrations, now exhibited activity at very low concentrations, below 10 µM in Ld1spro medium and 3 µM in C-medium, not only on amastigotes, but also on promastigotes, as depicted in Figure 43. On the other hand, MC2652 proved to be highly active in C-medium at concentrations below 10 µM, while displaying moderate activity at around 20 µM in Ld1spro medium, in both amastigotes and promastigotes (Table 29 & Table 30).

The variation in results among different batches further indicates a problem in their synthesis, which could be attributed to a lack of standardization in the chemistry laboratory or variations introduced by different individuals involved in the synthesis process.

The observed increase in the activity of LSD1 inhibitors in C-medium (Figure 43) suggests that the composition of C-medium has the potential to enhance the activity of LSD1 inhibitors or that certain components in the medium may react with these compounds (and/or potential contaminating chemicals), leading to a non-specific, toxic effect on the Leishmania parasite. A non-specific toxicity is supported by the microscopically observed disintegration of the parasites, suggesting a chemical rather than a target-based activity. Currently, the challenge lies in distinguishing between leishmanistatic and leishmanicidal LSD1i effects due to short evaluation periods. To understand mechanisms better, manipulating interaction durations and using flow cytometry with viability markers like YO-PRO can help to differentiate viable/non-viable parasites.

In addition, the unexpected outcomes highlight challenges in mimicking LSD1i effects on intramacrophagic amastigotes using ex vivo parasites due to intracellular environment complexities (outside their PV). I observed variations in compound effectiveness against amastigotes and promastigotes; however, I did not test lower concentrations in infected cells. Future studies could explore various concentrations in infected macrophages to determine the compounds selectivity index, where a high toxic concentration and very low active concentration characterize an optimal drug.

Alternative LSD1 inhibitors will be explored in the near future as part of the ANR ELATION project in collaboration with the team of Manfred Jung at the University of Freiburg, renowned for their expertise in Medicinal Chemistry and Chemical Biology of epigenetic targets. The main objective of the project is to synthesize epigenetic probes targeting LSD1 using ChemBio approaches. This involves utilizing biotinylated LSD1 inhibitors and implementing chemical knockdown through Proteolysis Targeting Chimeras (PROTACs) technology.

LSD1 ACTIVITY ASSAY

To further validate the potential host-directed mode of action of the LSD1i, we directly assessed their effect on LSD1 activity in nuclear protein extracts from uninfected macrophages, determining the enzymatic activity of LSD1 to demethylate H3K4me2 residues. This analysis was conducted using the fluorometric KDM1/LSD1 Activity Quantification Kit (Abcam), following the manufacturer's instructions, as described in the Materials & Methods Section 9.

Currently, there are limited methods for quantifying LSD1 activity. These methods rely on measuring the release of H2O2 or formaldehyde, which are byproducts of the LSD1 enzymatic reaction. However, these methods have several drawbacks, such as the need for large amounts of substrate and enzyme, the inability to use nuclear extracts, the unsuitability for certain LSD1i, the bias associated with the use of DMSO and thiol-containing chemicals, and a low accuracy compared to the direct measurement of LSD1-converted demethylated product.

In the chosen assay, dimethylated Histone H3K4me2 LSD1 substrate is stably coated onto the strip wells. Active LSD1 enzyme binds to the substrate and removes methyl groups, resulting in demethylated products (H3K4me0). These products are detected by a specific antibody and the fluorescence of the demethylated products is then measured, the signal intensity being proportional to the activity of the LSD1 enzyme.

Firstly, to calculate the demethylated product of LSD1 in the tested conditions, a standard solution containing demethylated lysine 4 of Histone H3 (H3K4me0) was used to establish the standard curve, as shown in Figure 44. Concentrations ranging from 0 to 5 ng/µl and their respective fluorometric measurements (RFU) were employed for this purpose.

Subsequently, the amount of LSD1 demethylated product (in ng) for each tested condition was determined using 1 µg of nuclear proteins from BMDMs, with the uninfected/untreated sample serving as the control. The measurements were conducted for two doses of MC2652, MC4030, and TCP. The calculations were performed using the formulas described in Materials & Methods Section 9. Furthermore, the LSD1 activity and the percentage of inhibition were calculated, taking into account a reaction time of 60 minutes.

The results are presented in Table 31. Since the kit had a limited number of wells to carry out the enzymatic reaction, I was unable to test a range of different concentrations and only used a random concentration around the average and the highest one recommended by the manufacturers. Due to this limitation, it was difficult to standardize the assay, and I couldn't test different amounts of nuclear extracts or different reaction times, which prevented me from achieving 100% inhibition of LSD1 activity. However, the assay demonstrated inhibition of LSD1 demethylase activity, notably when the nuclear extracts were treated with our two LSD1i hit compounds, showing an inhibition of around 40% at a concentration of 30 µM and reaching an inhibition of around 50% at 100 µM. This demonstrated a greater inhibition compared to the original TCP inhibitor, which showed a maximum inhibition of about 30% at 100 µM, as observed in Figure 45.

CELLULAR THERMAL SHIFT ASSAY (CETSA)

In order to evaluate the binding of the inhibitors specifically to their target protein LSD1 in cellula, a Cellular Thermal Shift Assay (CETSA) was conducted.

The CETSA method enables the measurement of a compound interaction with a target protein in living cells. The underlying principle of this assay relies on protein stabilization as a result of ligand binding. Hence, following interaction between an inhibitor and its target protein that generally results in protein stabilization, the melting temperature or melting point (Tm), which determines the temperature at which 50 % of the protein is soluble, is shifted (Figure 46). One of the important advantages of CETSA is that it allows protein analysis in a biologically relevant cellular context. This means that the protein of interest is studied within its native environment, alongside its natural partner proteins, and with physiological concentrations of cofactors and substrates [216,217]. The assay consists of treating cells with a compound of interest, followed by cell lysis, protein denaturation and precipitation by increasing temperatures, and separation of denatured/precipitated proteins from the soluble fraction through centrifugation. While unbound proteins precipitate at increasing temperatures, compound-bound proteins remain in solution. Subsequently, the detection of the remaining thermostabilized target protein in the soluble fraction of the samples is accomplished through quantitative Western blot analysis.

Chapter 1. Pharmacological characterization of macrophage LSD1i 112

SETTING UP CETSA CONDITIONS

Fluorescent detection for Western blot analysis

The WB fluorescence detection method employs a secondary antibody linked to a fluorophore, eliminating the need for additional detection reagents. Fluorescent western blotting antibodies address the need for accurate, quantitative protein analyses. Advantages over conventional WB detection methods include multiplex detection, higher sensitivity, brighter signal, and a more efficient workflow. Given the advantages offered by the fluorescent system, I set up the conditions for using fluorescent secondary antibodies. By combining suitable rabbit and mouse antibodies, I enabled multiplex detection on a single blot, which allowed me to simultaneously reveal additional proteins aside LSD1 (e.g. other potential targets, negative control proteins) that could offer insights into the selectivity and off-target effects of the LSD1i (further elaborated in the following section).

Evaluation of LSD1i selectivity and off-target effects

The presence of enzymes with catalytic monoamine oxidase domains was analyzed to assess the selectivity and potential off-target effect of the LSD1i. To achieve this, attempts were made to identify MAO-A, MAO-B, and LSD2 through WB. Out of the three proteins analyzed, only MAO-A could be identified by this technique. Additionally, it was decided to evaluate a protein that did not have a monoamine oxidase domain as a negative control, represented in my analyses by GAPDH.

Determining the sample volume to establish a melting curve

Under various experimental conditions, the superoxide dismutase 1 (SOD1) value could potentially serve as a reference in CETSA. This is due to its constant melting point of 80°C, which is stable regardless of the target-inhibitor interaction being assessed, and its convenient detection by WB, facilitated by its molecular weight of 18 kDa. However, our previous transcriptomic analyses revealed a substantial difference in the SOD1 transcript levels between infected and uninfected conditions. As a consequence, SOD1 is not suitable as a proper control for my experiment.

Typically, normalization is not required in CETSA assays as the half-maximal effective concentration (EC50) value serves as the primary readout. The EC50 represents the concentration of the ligand (LSD1i) at which half of the target (LSD1) is in the bound state, and it is determined through the establishment of a melting curve. This curve is generated by measuring soluble protein levels at various temperatures ranging from 37°C to 70°C, with the abundance of soluble protein determined by WB and intensities normalized using the signal obtained at the lowest temperature.

It is crucial to establish the initial sample volume for constructing the melting curve. Therefore, working within the linear range is essential to effectively build a melting curve. Within this range, there is a direct relationship between the amount of blotted protein on membrane and the observed WB signal intensity.

To determine the linear range of the LSD1, MAO-A and GAPDH protein signals, I assessed different volumes of protein samples heated at 37°C.

The LSD1 band fluorescent intensity was found to be linear up to 11 µl of protein lysate obtained from uninfected BMDMs treated with either DMSO, as negative control, or with LSD1i and TCP (Figure 47). This linearity was also observed in the case of MAO-A and GAPDH (data not shown). Furthermore, as there was no signal saturation, it was decided to use 11 µl of sample to generate the melting curves. (11,[START_REF] Alvar | Leishmaniasis worldwide and global estimates of its incidence[END_REF]5, and 2 µl) obtained from uninfected BMDMs treated with DMSO, LSD1i, or TCP. The data was analyzed using regression analysis in Excel to determine if the relationship between the measured variables was linear. The calculated R 2 values demonstrated that the data points followed a linear relationship.

CETSA RESULTS

The CETSA assay was performed on soluble protein fractions obtained from both infected and uninfected BMDMs. The proteins were subjected to increasing temperatures, and the thermostability of LSD1, MAO-A, and GAPDH was assessed using western blot analysis.

Briefly, to conduct CETSA assay, BMDMs were either infected or left uninfected.

After 24 hours PI, the cells were treated with the LSD1i or TCP at a concentration of 15 µM for 24 hours. Subsequently, total proteins were extracted; soluble proteins were separated in fractions and subjected to various temperatures ranging from 37 to 70°C for 3 min. Finally, a high-speed centrifugation was performed to remove denatured proteins. Subsequently, 11 µl of each heattreated sample, containing the remaining soluble proteins, was used to quantify the remaining soluble target (LSD1) through western blot analysis.

Figure 48A & Figure 49A show respectively representative WBs for each of the analyzed proteins using protein lysates from uninfected and infected BMDMs. In these blots, only the control (treated with DMSO) and the LSD1 inhibitors MC2652 and MC4030 are presented. However, it is worth noting that the TCP treatment was also analyzed, and this information is shown only in the melting curves.

To construct the melting curves (Figure 48B & Figure 49B), I used the percentage of fluorescent signal detected at 37°C, the lowest temperature. Notably, the thermal stabilization observed in the presence of both LSD1i and TCP is specific to LSD1 and is evident in both infected and uninfected samples. These findings suggest that the effects of the inhibitors are likely due to the interaction with LSD1 and are not affected by the infection status of the BMDMs.

Table 32 & Table 33 show the observations in both infected and uninfected samples. The most significant thermal shift is induced by the treatment with MC2652, reaching 5.5°C in uninfected samples and 6.5°C in infected samples, followed by the TCP treatment and then MC4030. Additionally, a slight thermal stabilization of MAO-A is observed in the TCP treatment (ΔTm=2.1), as expected due to the lack of specificity of this inhibitor for LSD1. It is worth mentioning that TCP was initially developed as a monoamine oxidase inhibitor. The results indicated that both LSD1i (MC2652 and MC4030) and TCP treatment specifically stabilized the LSD1 protein in both infected and uninfected samples. The thermal shift induced by MC2652 was the most significant, reaching 5.5°C in uninfected samples and 6.5°C in infected samples. TCP treatment also showed a slight thermal stabilization of MAO-A. Surprisingly, comparison between uninfected and infected conditions treated with DMSO indicated that infection alone seems to lead to LSD1 thermal stabilization, an intriguing finding that will be further discussed in the Chapter 2, Section 3. Overall, these findings validate LSD1 as a target of our LSD1i hit compounds. To investigate whether LSD1 localization is affected by Leishmania infection, I performed an immunofluorescence analysis in uninfected and infected BMDMs.

CHAPTER 2. CHARACTERIZING THE EFFECT OF LEISHMANIA INFECTION ON MACROPHAGE LSD1

To enable the detection of intracellular antigens, it is necessary to first permeabilize cells, particularly after fixation with cross-linking agents such as formaldehyde and glutaraldehyde.

Permeabilization facilitates access to intracellular and intraorganellar antigens.

There are two kinds of reagents commonly used for this purpose: organic solvents such as methanol (as shown in Chapter 1, Section 2.1.1) or detergents including saponin, Triton X-100 and Tween-20. These reagents disrupt or create pores in the lipid bilayer of cell membranes, allowing antibodies to penetrate and bind to their intracellular targets. However, the use of solvents can denature proteins and modify antigens or certain epitopes and consequently hamper their immunodetection [223].

Saponin is a plant-derived glycoside that interacts selectively with cholesterol membranes and has the ability to create pores by removing cholesterols. Nevertheless, its effect is reversible and it is not able to permeabilize cholesterol-poor membranes such as mitochondrial and nuclear membranes, whereas non-ionic detergents such as Triton X-100 and Tween-20 are able to permeabilize irreversibly all types of lipid bilayers [223]. Chapter 2. Effect of Leishmania infection on macrophage LSD1 123 Initially, I followed the protocol established in the lab, in which 0.1 mg/ml saponin is used as the permeabilizing agent as it is known to maintain the integrity of protein surface antigens. However, the results of LSD1 immunostaining showed a diffuse distribution throughout the cell (data not shown). It should also be mentioned that controls without primary antibody were performed in parallel in all experiments to ensure an absence of secondary antibody background staining.

Based on these findings and considering that the literature suggests LSD1 is mainly localized in the nucleus, it was decided to switch to Triton X-100 as permeabilizing agent for subsequent experiments.

A colocalization of LSD1 with Hoechst 33342-stained nuclei was observed, along with an absence of signal in the cytoplasm, in both uninfected and infected BMDMs, as depicted in Figure 50. This suggests that the global nuclear localization is not affected by Leishmania infection at the time point examined. However, it should be noted that this result does not exclude the possibility of changes in LSD1 sub-nuclear localization or its localization at different stages of infection.

DETERMINING LSD1 PROTEIN ABUNDANCE

Our previous transcriptomics analyses did not reveal any significant difference in the amount of LSD1 mRNA between infected and uninfected BMDMs. Therefore, I decided to investigate whether infection affected the protein level of LSD1. Given the nuclear localization of LSD1 in the macrophage shown by the immunofluorescence assay above (and absence of any signal in the cytoplasm), we decided to quantify LSD1 in only nuclear protein extracts by Western Blot analysis. To achieve this goal, several exploratory experiments were carried out in order to identify the best conditions for separating nuclear and cytoplasmic fractions and performing quantitative WB analyses of LSD1.

NUCLEI ISOLATION AND NUCLEAR PROTEIN EXTRACTION

Traditionally, the isolation of nuclei is done by centrifugation through highdensity glycerol or sucrose. Even if this method can yield clean nuclei, it is time consuming and sucrose viscosity can lead to protein degradation and/or modification [224]. To limit protein deterioration, the protocol reported by Schreiber et al. [225] was selected for nuclei isolation, as it uses a lysis buffer that minimizes protein degradation, modification or loss.

The development of an effective lysis buffer for protein extraction involves several key considerations with respect to pH, ionic strength, detergents and denaturants, and the inclusion of specific components to enhance protein stability and prevent proteolysis [226]. Mild detergents such as NP-40 or Triton X-100 are very useful for nuclei isolation as they can selectively dissolve the plasma membrane without damaging the nuclear membrane, and allows minimal cytoplasmic contamination [227].

In order to assess the various protocols, I used phase contrast microscopy (EVOS) to test for the purity of the nuclear preparation and visualize possible contamination with plasmatic membrane or endoplasmic reticulum. Figure 51 depicts a nuclear preparation in which remnants of cytoplasmic contents are evident in both uninfected and infected samples. However, following protocol optimization, I obtained a nuclear preparation, as shown in Figure 52, exhibiting minimal to no contamination of cytoplasmic remnants. The upper part of the panel displays uninfected (left) and infected (right) BMDMs, while the lower part illustrates the corresponding isolated nuclei. In the case of infected samples, the debris observed surrounding the nuclei corresponds to remnants of parasites (parasite ghosts) that have also been lysed, as well as parasite nuclei. It is worth mentioning that the centrifugation speed used to sediment the BMDMs nuclei (250 g) is relatively low to avoid precipitating the parasite nuclei. Therefore, the risk of this type of contamination is minimized.

Once a "clean" preparation of nuclei was obtained as determined by EVOS observation as described above, the optimal detergent for the nuclear protein extraction buffer had to be chosen. First, I tested 1% Sodium dodecyl sulfate (SDS) following the protocol by Schreiber et al. However, it was replaced by 1% Triton X-100 for the reasons listed below:

 SDS is a strong anionic detergent that disrupts the non-covalent bonds between and within proteins, and causes denaturation and loss of native conformation, leading to loss of activity and antigenicity.  Triton X-100, on the other hand, is a milder non-ionic detergent, which makes it less disruptive to protein-protein interactions and protein structure. This is important for nuclear proteins, which are often in complex and have multiple subunits that need to remain intact for proper function.  Triton X-100 is, in addition, more effective at solubilizing membrane proteins, which are present in nuclear membranes.  Triton X-100 is less likely to interfere with downstream applications such as enzyme assays, protein quantification and gel electrophoresis, whereas SDS can interfere with these assays due to its strong negative charge

LSD1 QUANTIFICATION BY WESTERN BLOT ANALYSIS

Determination of a reference protein for normalization

A reference protein had to be first identified as normalization control to ensure that differences in band intensities on a Western blot analysis are not only due to technical factors such as protein loading variability and/or transfer across the gel lanes and to the membrane.

For this purpose, I tested the expression levels of various proteins that are known to localize to the nucleus and did not show significant differences in mRNA abundance between uninfected and infected macrophages 48 hours after infection as judged by our previous RNAseq analyses (p<0.05) [229]. As shown in Figure 54, strong signals were only obtained with BMDMs nuclear protein extracts, while no signal was detected when using parasite extracts, thus indicating the specificity of the antibodies used in my study.

Evaluation of Nuclear Protein Extracts Purity

To assess the proper separation of nuclear from cytoplasmic extracts, Western Blot analyses were performed using GAPDH and YY1 as cytoplasmic and nuclear markers, respectively.

As depicted in Figure 55, robust signals of GAPDH were exclusively observed in the cytoplasmic extracts, while the nuclear extracts exhibited nearly complete signal absence, indicating minimal cytoplasmic contamination. Conversely, for YY1, strong signals were evident in the nuclear extracts.

It is important to consider the challenge faced by any fractionation method based on mechanical disruption with detergent in completely avoiding crosscontamination, which can vary between experiments. Similarly, nuclear damage is inevitable, which is why small quantities of the transcription factor YY1 can be observed in our cytoplasmic extracts. Nonetheless, this method is considered one of the most effective ways to obtain an almost intact nuclear fraction with minimal contamination [227]. Furthermore, to evaluate the quality of the protein extracts, 5 µg of each sample were resolved on an SDS-PAGE gel and stained with Coomassie Blue. A remarkable distinction was evident in the protein band patterns between nuclear and cytoplasmic extracts, as well as to total parasite extracts. Even more intriguingly, differences in protein band patterns between the nuclear protein samples of uninfected and infected BMDMs were observed (Figure 56). The Outlook Section 1 is dedicated to discussing this phenomenon in more detail. Chapter 2. Effect of Leishmania infection on macrophage LSD1 130

LSD1 protein quantification

After determining YY1 as the optimal normalization reference protein, verifying the absence of antibody cross-reactivity, and evaluating the purity of nuclear protein extracts from uninfected and infected macrophages, I proceeded with the quantification of LSD1 protein by WB.

This analysis was carried out with nuclear protein extracts from uninfected and infected macrophages at 24, 48 and 72 hours after infection. Figure 57A shows a representative WB with samples obtained at different time points of the infection. Normalized LSD1 content was calculated using the YY1 signal for each sample, with the uninfected sample value set to 1.0. The quantitative values obtained for LSD1 content of nuclear extracts at 24, 48 and 72 hours after infection were obtained from at least two biological replicates (Figure 57B). They indicate that L. amazonensis infection does not modify the amount of LSD1 protein in macrophages.

VISUALIZING LSD1 THERMAL STABILIZATION BY LEISHMANIA INFECTION

In Chapter 1 Section 5.2, we observed a clear thermal stabilization of LSD1 indicative of LSD1i target engagement. Additionally, and quite surprisingly, we observed a comparable thermal stabilization of LSD1 following L. amazonensis infection compared to uninfected BMDMs treated with the DMSO vehicle (Figure 58A) as judged by the differences in their corresponding melting curves (Figure 58B).

Indeed, we revealed a ΔTm for LSD1 of 3.3°C in infected compared to uninfected samples, which is equal to the ΔTm obtained by its interaction with MC4030 (Table 32). A similar behavior was also noticed for MAO-A (ΔTm=3.0°C) but not for GAPDH (Table 35). These findings suggest the presence of specific interactions for LSD1 and for MAO-A in response to Leishmania infection. One of the advantages of CETSA method is that it evaluates the interaction with a target in its natural state, as it is endogenously expressed in physiologically relevant samples.

Our analysis revealed intriguing implications of Leishmania infection on LSD1, suggesting that the infection might influence LSD1 complex formation and its interaction partners, resulting in enhanced thermal stability.

Another possibility is that a ligand from the parasite itself could be interacting with macrophage LSD1, leading to an increase in its thermal stability. To further investigate these two possibilities, I performed co-immunoprecipitation experiments that are described in the following.

INVESTIGATING MACROPHAGE LSD1 COMPLEXES DURING LEISHMANIA INFECTION

Up to this point, I have demonstrated that Leishmania infection neither modulates LSD1 transcript nor protein abundance in BMDMs. Instead, it influences the thermal stability of the protein, as indicated by the CETSA analysis described above.

LSD1 is a scaffolding protein for multiple protein complexes and interacts with various proteins (Introduction,Section 4). In order to understand how Leishmania impacts LSD1 interactions in macrophages, I performed LSD1 coimmunoprecipitation (co-IP) experiments using nuclear extracts of infected and uninfected BMDMs. This was accomplished through the use of validated α-LSD1 antibodies, magnetic bead-based enrichment of the complexes, their separation and visualization by SDS-PAGE using SYPRO Ruby staining, and LSD1 Western Blot analysis employing an immunofluorescence-based antibody detection method.

During the optimization of the co-IP procedure, I tested numerous conditions, including the use of different detergents/buffers for nuclear protein extraction and protocols, protein-specific antibodies and controls from different sources, incubation times and sample pre-clearing conditions. The different conditions tested in the various co-IP experiments are compiled in Table 36.

SETTING UP CO-IMMUNOPRECIPITATION (CO-IP) ASSAY CONDITIONS

Detergent selection

Non-ionic detergents are employed to dissolve biological membranes and the choice of a specific detergent depends on the stability of the protein complex under investigation. Digitonin is among the mildest detergents and is suitable for identifying physiological protein-protein interactions without using any chemical cross-linking. It is used to isolate supramolecular associations of multiprotein complexes. Dodecylmaltoside (DDM) is a mild neutral detergent that is suitable for isolating membrane proteins and individual complexes. However, it may be more aggressive in removing lipids compared to digitonin and can dissociate weak hydrophobic interactions. In order to select the detergent that would best preserve protein interactions prior to performing the co-IP assay, I attempted to visualize LSD1 under nondenaturing conditions using a blue native gel. Nuclear protein samples were extracted using either digitonin or DDM. The band corresponding to LSD1 on the electro-transferred membrane was only observed in samples extracted with digitonin (in both uninfected and infected BMDMs, data not shown).

Additionally, since I had previously prepared nuclear extracts with Triton X-100, I also tested those samples by Western blot after LSD1 co-IP. Triton X-100 proved to be less efficient than digitonin (data not shown). Therefore, digitonin was selected as the detergent for subsequent co-IP assays.

Protocols

The Rapid Immunoprecipitation Mass spectrometry of Endogenous protein (RIME) is a technique in which protein complexes are first fixed with a crosslinking agent like formaldehyde allowing the study of protein complexes and their interactome. A good example of RIME application was described in the article of Mohammed and colleagues that focused on the study of chromatin and transcription factors complexes [231].

I first used the RIME protocol that uses a particularly efficient lysis buffer (LBII, see Table 12) to best preserve LSD1 complexes. This protocol was not optimal since the nuclear preparations were contaminated by cytoplasmic debris, probably as a consequence of the protein fixation procedure (Figure 59). Consequently, I decided to continue with an internal protocol that was established in the Parsig laboratory based on the general guidelines for using Dynabeads™ Protein G. The main difference in this protocol (Section 11 of Materials and Methods) is the absence of the fixation step, which permitted the gradual removal of the cell membrane as well as the cytoplasmic content after a few minutes of contact with LBII (Figure 60A). Figure 60B shows the purified nuclei after being passed through a 27G needle.

Internal controls

In protein-protein interaction analysis, it is crucial to incorporate a negative control, typically normal IgGs, to evaluate non-specific background purification, detect false-positive interactions, establish specificity, and ensure the accuracy of the findings. The IgG negative control should be derived from the same species as the primary antibody used for precipitating the protein of interest. By immunoprecipitating samples with the IgG negative control, potential contaminants (non-specific binders) can be identified and subsequently excluded from the list of specific interactors obtained with the α-LSD1 antibody, effectively filtering out non-specific binding events [232].

Before performing the mass spectrometry analysis, I confirmed by WB that the band corresponding to the bait protein (LSD1, in this case) was revealed with the specific α-LSD1 antibodies only in the eluate of the immunoprecipitated sample (lane 3 and 4) and not in the IgG negative control eluate (C-, lane 5) (Figure 61).

Chapter 2. Effect of Leishmania infection on macrophage LSD1 137 A second positive control consists in the detection of a known partner of the bait protein, e.g. RCOR1, a known LSD1 protein interactor. Indeed, this protein was only detected by WB in the eluate of the LSD1-specific co-IP (lane 3 and 4) but not the IgG control eluate (lane 5) (Figure 61B). This result also showed that the purification procedure maintained the protein interactions between RCOR1 and LSD1, thereby validating the experiment protocol I applied.

Antibodies (Ab)

The success of endogenous protein immunoprecipitations largely depends on the quality, affinity and specificity of the used antibodies [231]. To select the best antibody for LSD1 co-IP, I tested both commercial and "homemade" LSD1 antibodies.

I initially used the LSD1 antibody from Cell signaling (CS) given the good results obtained with this antibody for LSD1 detection in Western blot analyses (see Section 2.2.4 of this Chapter). However, it should be taken into account that an antibody could be able to recognize an antigen in denaturing conditions but not in native conditions, i.e. when the epitopes recognized by the antibodies can be hidden by the protein three-dimensional structure and/or by its location within molecular complexes.

The main problem I encountered with the CS antibody was the absence of information in the data sheet concerning the exact amount of the antibody, the only data provided being the dilution to be used in case of a co-IP assay. I therefore decided to use an antibody provided by another company, Proteintech (Ptt). In this case, the information about the antibody content in milligrams was specified in the data sheet. Unfortunately, this antibody also did not yield the expected results, as the band corresponding to LSD1 was not observed in the eluate by WB analysis (data not shown).

Consequently, I contacted Eric Metzger from the Roland Schüle team at the University of Freiburg in Germany, whose research focuses on LSD1 regulation. They kindly shared with me an aliquot of two of their previously characterized and validated homemade α-LSD1 antibodies (Ab1, 20752;Ab2, 3544) [233].

These homemade antibodies were found to be the most effective option, as illustrated by the SYPRO Ruby stained gel and WB results from experiment 6 (see Table 36), performed only with nuclear protein samples from uninfected BMDMs (Figure 61). First, the nuclear protein enrichment observed in the input using SYPRO Ruby staining and LSD1 detection by WB (lane 2, Figure 61A andB) was validated by the different band pattern observed between the cytoplasmic extract (CE) and nuclear proteins (Input, sample before co-IP).

In Figure 61A, it is evident that the band patterns in lanes 6, 7 and 8, showing the proteins contained in the supernatants (samples recovered after co-IP), are quite similar. However, in lanes 3, 4 and 5, slight differences can be observed in the protein bands obtained in the eluate, as expected, indicating distinctions between the IgG control and specific α-LSD1 Abs.

In Western Blot analysis, LSD1, RCOR1 and H3 were detected in the Input sample (lane 2) and to a lesser extent in the CE (lane 1) (Figure 61B). The capacity of both α-LSD1 antibodies, Ab1 and Ab2, to co-IP LSD1 were demonstrated in the eluate, as evidenced by the expected WB signal for this protein at approximately 110 kDa in lanes 3 and 4. Conversely, no band was observed in the negative control (lane 5, C-). However, not all LSD1 is captured, as a band corresponding to LSD1 is still observed in supernatant samples (lanes 6 and 7).

The presence of Histone H3 in all samples suggests its non-specific enrichment.

At this stage, I confirmed that Ab1 and Ab2 antibodies were able to immunoprecipitate LSD1 from uninfected BMDMs nuclear extracts. The next question was whether they could do so with the same efficiency in samples coming from infected cells, as LSD1 could have different interactors that may impede its recognition by the antibodies.

Due to the unavailability of biological material obtained in the same conditions for infected samples at that time, nuclear extracts in Triton X-100 from uninfected and infected cells were utilized to co-immunoprecipitate LSD1 partners (Figure 62).

The SYPRO Ruby-stained SDS-PAGE gel (Figure 62A) exhibited protein patterns highly similar to the previous experiment depicted in Figure 61A. Western blot analysis (Figure 62B) revealed successful IP of LSD1 in both uninfected and infected samples using Ab1 (lanes 3 and 5, respectively) and Ab2 (lanes 4 and 7, respectively). The desired LSD1 band at the expected molecular weight was evident with both antibodies.

Notably, a stronger band signal was obtained with Ab1 (lane 3 for uninfected and lane 6 for infected), indicating its higher efficiency compared to Ab2.

Preliminary Analysis by Mass Spectrometry of co-IP samples

Once the ability of the antibodies to immunoprecipitate LSD1 in both uninfected and infected samples was confirmed, a preliminary mass spectrometry test was performed using a single biological replicate (experiment Chapter 2. Effect of Leishmania infection on macrophage LSD1 141 8). This pilot experiment was carried out at the CurieCoreTech Mass Spectrometry Proteomics of Institut Curie, with the two antibodies, Ab1 and Ab2, following the approach reported by Duteil et al. [234].

Figure 63 shows the Venn diagrams of this pilot experiment for the immunoprecipitated proteins that were identified by at least 3 peptides. The intersection of the two interactomes (groups B and C of Figure 63) revealed 60 proteins in uninfected samples and 167 proteins in infected samples, representing a significantly enriched group. Among these, several proteins commonly reported in the literature as LSD1 interactors, including RCOR1, RCOR3, HDAC1, HDAC2, ZNF516, GSE1, ZMYM2, and others, were identified in both uninfected and infected samples. Moreover, as expected, a series of Leishmania proteins were immunoprecipitated in the infection condition with the two α-LSD1 specific antibodies.

Proteins found in Group A can be considered as background noise as they are immunoprecipitated with the IgG negative control antibodies. Of particular interest was the significant number of proteins found in the overlapping region of the three conditions (622 in uninfected and 165 in infected), implying nonspecific interaction with LSD1-specific antibodies. Further optimization of sample pre-clearing was still necessary to reduce this background noise before carrying out the co-IP assay with 4 biological replicates to obtain statistically significant results.

Pre-clearing conditions

The following pre-clearing assays were carried out with the nuclear extracts: a first step with beads alone followed by a second step with beads in presence of control IgGs. Although the band pattern detected in the SYPRO Ruby stainedgel showed improvement, non-specific bands were still observed with this procedure (data not shown).

Consequently, to reduce the background noise as reported by Sciuto et al,[235] we used two-steps of pre-clearing with beads + Rabbit IgGs in further experiments. This procedure would allow to amplify the signal arising from specific protein-protein interactions and to eventually improve the sensitivity of the assay.

The co-IP experiment 10 (Table 36) was carried out with a nuclear extract from uninfected BMDMs. As shown in Figure 64A, the band pattern, of the precleared eluate in the SYPRO-stained gel is reduced between step 1 (lane 2) and 2 (lane 3). These bands represent proteins that adhered to the rabbit IgGs (C-). This result suggested that the background noise could be reduced after each pre-clearing step.

The WB analysis (Figure 64B) further confirmed that neither the pre-clearing (lanes 2 and 3) nor the co-IP with the IgG negative control (lane 5) precipitated LSD1, as no band was observed. Furthermore, Ab1 was more efficient than Ab2 as evidenced by i) the stronger LSD1 band intensity in the co-IP eluate (lane 6 compared to lane 7 LSD1 bands), and ii) the nearly undetectable level of LSD1 in the Ab1 supernatant (lane 9). Based on these findings, it was concluded that two pre-clearing steps with beads + IgGs would be performed prior to the analysis of the 4 biological replicates by mass spectrometry.

RESULTS OF THE CO-IMMUNOPRECIPITATION ASSAY

The final LSD1 co-immunoprecipitation procedure was performed as described in Material and Methods Section 11. Two specific α-LSD1 antibodies (Ab1 and Ab2) and rabbit IgGs as negative control (C-) were used to immunoprecipitate proteins from 4 biological replicates of nuclear extracts from uninfected and infected BMDMs.

Normalization of the raw data was performed using the Prostar software tool, designed to process quantitative data from mass spectrometry-based label-free proteomics [236]. As described in Section 4.1.3 of this chapter, the negative control was crucial to reveal non-specific protein interactions. Only specific and statistically significant binding events (p<0.01) were considered. This procedure was performed for the four conditions UI-Ab1, UI-Ab2, Inf-Ab1 and Inf-Ab2 (Figure 67).

The initial rationale behind using two different antibodies was to enhance the enrichment of LSD1-specific interactors. The Venn diagrams in Figure 65 illustrate the comparison of immunoprecipitated proteins from uninfected and infected samples using both antibodies, Ab1 and Ab2. A significant number of proteins were identified commonly with both antibodies in uninfected (192 proteins) and infected (79 proteins) samples, which was the purpose of this experiment. However, it can be observed that in both cases, there are significantly more immunoprecipitated proteins with Ab1 than with Ab2. In the uninfected condition, Ab1 immunoprecipitates more than double the number of proteins compared to Ab2 (489 vs 223 proteins, respectively). In the infected condition, the difference is even more pronounced, with a total of 330 proteins identified with Ab1, compared to only 87 proteins detected with Ab2. Notably, Ab1 is more performant compared to Ab2. This significant difference was also observed when directly comparing uninfected and infected conditions for each of the two antibodies (Figure 66). Only 34 proteins were found to be common in the case of Ab2, in contrast to the 259 shared proteins in the case of Ab1. This suggested that Ab2 is suboptimal and would limit the results obtained with the more performant Ab1. Therefore, I decided to only consider the data set obtained with Ab1 for the subsequent analyses.

Figure 67 displays the volcano plots depicting the removal of background interactors of uninfected (upper panel) and infected (lower panel) conditions.

In both cases, LSD1 is detected, along with its known interactors, such as RCOR1, ARID1A, POLR2B, among others. Interestingly, only in the infected condition, specific LSD1 interactors included histone proteins from the parasite.

In the comparison between uninfected and infected LSD1 interacting proteins (Figure 66, left panel), three different groups were identified: (i) 259 proteins present in both conditions, (ii) 230 and 71 proteins unique to the uninfected and infected conditions, respectively, and (iii) 24 out of these 71 proteins identified as parasite proteins (Table 37), suggesting that Leishmania can transfer parasite proteins to the macrophage nucleus (see Outlook Section 1).

STRING analysis was performed on the 47 macrophage proteins unique to the infected condition using the Cytoscape software tool (Figure 68). The first cluster of proteins is mainly associated with GO terms related to ribosomal biogenesis; the second cluster is related to GO terms linked to mRNA processing, binding, splicing, and metabolic processes, while the third cluster is Chapter 2. Effect of Leishmania infection on macrophage LSD1 146 formed by histone proteins and associated with the GO term 'nucleosome'. These findings suggest that Leishmania alters the macrophage gene expression profile both at the transcriptional and post-transcriptional levels through its influence on LSD1 protein-protein interactions.

LSD1 CHIPMENTATION

ChIP-seq is a technique that combines chromatin immunoprecipitation (ChIP) with next-generation sequencing to determine genome-wide distribution of chromatin-associated proteins, their post-translational modifications and their interactions with DNA. However, current protocols for ChIP-seq are still timeconsuming, expensive and complex, despite optimization efforts. An alternative approach is ChIPmentation, which uses a hyperactive Tn5 transposase that allows simultaneous DNA fragmentation and adaptor tagging (tagmentation) directly on immunoprecipitated bead-bound chromatin. This can result in much faster library preparation from purified DNA [237].

As depicted in Figure 69, both ChIP-seq and ChIPmentation workflows involve a cell fixation step with formaldehyde, followed by cell lysis, chromatin sonication and immunoprecipitation with specific Abs bound to beads. For standard ChIP-seq, DNA is reverse cross-linked, purified and subjected to a multi-step library preparation process. In contrast, ChIPmentation directly performs tagmentation on the immunoprecipitated bead-bound chromatin, allowing chromatin proteins to protect DNA from excessive tagmentation. This method avoids the production of sequencing adaptor dimers and requires only one step of DNA purification, resulting in a fast and convenient protocol. Additionally, ChIPmentation requires a lower input (number of cells) compared to standard ChIP-seq, while still obtaining high-quality data, which in my experimental system is ideal given the use of a primary macrophage culture.

I therefore decided to use ChIPmentation to analyze the genomic binding sites of LSD1 during Leishmania infection in order to identify specific genes and pathways that are regulated by LSD1. This information can help to understand the role of LSD1 in regulation of gene expression and biological processes during infection.

SETTING UP CHROMATIN IMMUNOPRECIPITATION CONDITIONS

Chromatin Isolation and Shearing

Initially, I compared the lysis and shearing buffer composition established in the lab, i.e. a shearing buffer containing 0.2% SDS, based on the NEXSON procedure which includes a gentle but delicate sonication step as described by Arrigoni et al. [238]. However, I found that the buffer composition reported by Park et al.

Chapter 2. Effect of Leishmania infection on macrophage LSD1 150

[239] using a shearing buffer containing 0.1% sodium deoxycholate and 0.5% N-lauroylsarcosine resulted not only in higher quality DNA fragments (as demonstrated by the electrophoresis gel, data not shown), but also in higher DNA quality and quantity (as revealed by the Nanodrop technology, data not shown).

Quality control of sheared chromatin

To improve specificity and enhance the quality of the resulting data, I followed an optimized procedure that involved verifying two critical steps: assessing sonication efficiency and conducting preliminary antibody tests, as described previously by Xu et al. [198].

Sonication has two functions: to decompact chromatin and fragment DNA into an appropriate size range for next-generation sequencing. However, certain protein epitopes can also be damaged during this process. Therefore, the goal is to achieve a balance between obtaining the correct DNA size range while preserving protein integrity. This is the aspect that may vary the most between protocols, since it relies entirely on the equipment used. Thus, I had to determine the ideal conditions for my experimental system using a bath sonicator (Bioruptor® Standard, Diagenode).

Cell concentration and shearing buffer volume can influence sonication results too. I found that 300 µl of shearing buffer -the maximum volume per tube recommended for bath sonicators -per 7-9 x 10 6 cells was most suitable.

Then, in order to find the appropriate number of sonication cycles, I conducted a sonication time course of 5, 10, 15, 20 and 25 min with 30" on/30" off cycles with uninfected samples. At the end of each time point an aliquot was extracted for protein and DNA size analysis.

Antibody specificity test and protein integrity

Figure 70 presents a Western blot analysis in which I evaluated the specificity of the homemade antibody, α-LSD1 Ab1 (20752). This assessment was performed using chromatin samples subjected to different numbers of sonication cycles (indicated at the top of the blot). The selection of this antibody for testing was based on its superior performance in the co-IP assay (see Section 4.2 of this chapter). The consistency of the no sonication input (0 min) was extremely viscous, making it difficult to load the sample onto the gel for electrophoresis. However, in the sample corresponding to 5 min sonication, a single clear band at the expected size for LSD1 is revealed and it remains detectable during sonication at 20 min. α-HDAC1 antibody was used to compare levels of stability. Both proteins remain stable up to 20 min and at 25 min a beginning decay is observed as judged by a noticeable decrease of signal intensity. DNA from different time points was purified after reverse cross-linking, then quantified and separated by agarose gel electrophoresis. Ideally, the shortest sonication time that results in DNA fragments within the desired range of 100-800 bp should be selected for library preparation. However, the decision is based on both, the Western blot analysis shown above and the DNA gel results.

In addition, ChIPmentation involves a transposase to attach sequencing adapters to the DNA after immunoprecipitation, which results in further DNA fragmentation. Therefore, having slightly larger DNA fragments at this stage is acceptable, as long as the majority of the DNA is below 1000 bp and there is fragment above this size.

The agarose gel in Figure 71 shows purified DNA fragments from diverse chromatin samples, which were subjected to an increasing number of sonication cycles. It is apparent that DNA fragments become smaller with higher sonication cycles. Notably, starting from 15 cycles of sonication, fragments below 1000 bp are obtained. I selected 15 min as the sonication time as it gave the desired DNA fragmentation size pattern (Figure 71) and maintained good protein detection (Figure 70). Furthermore, it resulted in an increased yield and improved quality during quantification, as shown in Table 38. By WB analysis (Figure 72), I showed the ability of the specific antibody (Ab1) to immunoprecipitate LSD1 even under cross-linking conditions. Indeed, I evidenced i) a single clear signal at the predicted size for LSD1 in the input sample (lane 1), ii) a single signal at the same size in the LSD1 immunoprecipitated eluate (lane 2) but not in the rabbit IgG negative control (C-) eluate (lane 3), and iii) absence of a visible LSD1 signal in the LSD1 supernatant (lane 4), compared to the single band in the C-supernatant (lane 5). These results collectively indicate the successful immunoprecipitation of the majority of LSD1. YY1 and GAPDH were used as controls and were not detected in the IP fraction but were present in both input and supernatant fractions.

Strong band signals in lanes 4 and 5 correspond to denatured antibody heavy chains. Chapter 2. Effect of Leishmania infection on macrophage LSD1 155

PRELIMINARY CHIPMENTATION EXPERIMENT

I prepared three biological replicates of chromatin from uninfected and infected BMDMs, as well as chromatin from evLIMs obtained from lesions of two RAG2 KO mice (Rag1 and 2), as indicated in Material and Methods, Section 3.3. All these chromatin samples were obtained after 15 min of sonication.

The protein integrity and DNA fragment size tests were conducted on all the samples mentioned above. Figure 73 shows the WB analysis using a chromatin aliquot from each sample, where a clear single band at the predicted size for LSD1 is observed, as well as for RCOR1, which was used as an additional positive control for a known partner of LSD1. The agarose gel in Figure 74 displays the purified DNA fragments after reverse cross-linking. It was confirmed that in most of the samples, except for the one derived from Rag1, proper DNA fragmentation was achieved as they presented a size smaller than 100 bp, which is ideal for library preparation. These purified fragmented DNA samples were quantified (Table 39), demonstrating appropriate yield and quality.

However, for this preliminary experiment, I only immunoprecipitated LSD1 from the chromatin of both uninfected and infected samples from Replicate 3, using rabbit IgGs as negative control (C-). For this purpose, 2 µg of α-LSD1 Ab1 or rabbit IgGs (C-) were used to immunoprecipitate the sheared chromatin from around 8x10 6 uninfected and infected BMDMs. Table 39. Quantity and quality of purified DNA from 3 biological replicates (uninfected and infected) and evLIMs from two Rag2-KO mice.

After overnight immunoprecipitation, washes were performed to maintain specific interactions and reduce background noise (see Material and Methods, Section 12.3). During the final wash in 10 mM Tris-HCl, the contents of the tubes (Beads-Ab-Ag) were divided into two parts used for conducting WB analysis (Section 5.2.1) and the tagmentation reaction (Section 5.2.2).

Sample ng/µl

Western blot analysis of LSD1 chromatin immunoprecipitation

WB analysis confirmed the successful IP as judged by the presence of a protein band corresponding to LSD1. In Figure 75, a single clear band at the expected size for LSD1 can be observed in the Ab1-immunoprecipitated eluate lanes of both uninfected and infected samples (lane 3 and lane 5, respectively), with a more pronounced band in the infected sample.

However, this band is absent in the negative control (lane 4, uninfected sample, and lane 6, infected sample). Unfortunately, it was not possible to identify the same band in the input (lanes 1 and 2) and supernatant lanes (lanes 7, 8, 9, and 10) as a very small amount of sample was deposited per lane in these cases.

YY1 and GAPDH were used as controls and were not detected in the IP fraction, but they were present in both input and supernatant fractions.

Tagmentation and DNA Purification

The tagmentation reaction was performed directly on the beads. Here, Tn5 transposase will add sequencing adapters to the immunoprecipitated DNA while it is still bound to the protein, protecting the DNA from over-tagmentation. Figure 76 depicts the schematic view of the process. As a control, the tagmentation reaction was also performed with 2 µl of input sample, which consisted of sheared chromatin before immunoprecipitation. The reaction was stopped, and reverse cross-linking with proteinase K digestion was performed overnight. The next day the DNA was purified, eluted in a final volume of 22 µl and quantified using Qubit 2.0, as described in Material & Methods (Section 12.2.1). The quantification results are presented in Table 40.

LIBRARY PREPARATION

As an initial step, my intention was to familiarize myself with the procedure, given the limited amount of immunoprecipitated chromatin samples (totaling only 1.4 ng for the UI IP-LSD1 sample). Therefore, I decided to initiate the library preparation using the input samples and conducted a preliminary test with various input quantities to simulate less concentrated sample and identify the Chapter 2. Effect of Leishmania infection on macrophage LSD1 159 detection limits.

Consequently, I experimented with three different initial amplification amounts using the input chromatin samples, obtained from both uninfected and infected BMDMs: 6, 3, and 0.6 ng. The PCR reaction was set up using the Unique Dual Indexes (UDI) 1 to 6, as specified in Table 20 andTable 21 (Final reaction volume=50µl). These UDIs consist of barcode combinations, each exclusively assigned to a single sample. These UDI sets prove to be indispensable for detecting errors during index sequencing. The primer indexes employed for the tagmented libraries include 8-nucleotide long i7 and i5 indexes. A preamplification of 4 cycles was carried out, following the cycling conditions outlined in Table 22, beginning with a 5 min cycle at 72°C to fill the 9-bp gap created by the tagmentation process (Figure 77). Subsequently, 9 µl of this reaction were mixed with 1 µl of EvaGreen 10x (in duplicate), and qPCR was performed using the conditions described in Table 23 to determine the optimal number of cycles (N) for library amplification. The value of N is determined where the amplification curve reaches half of its saturation, and it should be selected during the exponential phase before reaching full saturation.

Figure 78 displays the qPCR amplification plots of the various input samples. The value of N is represented by black lines crossing the X-axis at the cycle number corresponding to the midpoint of saturation on the Y-axis. N varies Chapter 2. Effect of Leishmania infection on macrophage LSD1 160 between about 12 cycles (for reactions started with 6 ng of sample) and around 17 cycles (for reactions started with 0.6 ng). To find a balanced value for all samples, N=15 cycles was chosen for the amplification of the libraries. The remaining 32 µl of the reaction (18 µl were used for qPCR) were subjected to 15 cycles of amplification, following the cycling conditions listed in Table 24.

The libraries were purified using AMPure XP beads, as described in Section 12.5 of Materials and Methods. The purified libraries were quantified using the Qubit 2.0 (Table 41), and their quality was assessed using the Agilent Bioanalyzer.

Figure 79A shows the virtual gel obtained with the Agilent Bioanalyzer for the six purified libraries amplified from tagmented input samples. Figure 79B displays the six corresponding electropherograms, each representing a specific library. Both the gel and the electropherograms reveal that the majority of the library fragments exhibit sizes within the 200-300 bp range, as indicated by the blue arrow on the gel and the vertical blue lines on the electropherograms. This size distribution is suitable for proceeding with the sequencing process. After observing this positive result from the libraries prepared with the input samples, I proceeded with the library preparation of the immunoprecipitated samples (those precipitated with the specific α-LSD1 antibody and the negative control). For this step, I used the entire sample of tagmented purified DNA, described in Table 40.

The PCR reaction was set up using the UDI 7 to 10, as specified in Table 20 and Table 21 (Final reaction volume=50 µl). A pre-amplification of 4 cycles was Chapter 2. Effect of Leishmania infection on macrophage LSD1 162 carried out, following the cycling conditions outlined in Table 22, beginning with a 5 min cycle at 72°C to fill the 9-bp gap created by the tagmentation process (Figure 77). Subsequently, 9 µl of this reaction were mixed with 1 µl of EvaGreen 10x (in duplicate), and qPCR was performed using the conditions described in Table 23 to determine the optimal number of cycles (N) for library amplification.

Figure 80 displays the qPCR amplification plots of the immunoprecipitated samples. The value of N is represented by black lines crossing the X-axis at the cycle number corresponding to the midpoint of saturation on the Y-axis. N varies between 7 cycles (Inf IP-LSD1 sample) and around 10-11 cycles (for the rest of the samples). To find a balanced value for all samples, N=10 cycles was chosen for the amplification of the libraries. The remaining 32 µl of the reaction (18 µl were used for qPCR) were subjected to 10 cycles of amplification, following the cycling conditions listed in Table 24.

The libraries were purified using AMPure XP beads, as described in Section 12.5 of Materials and Methods. The purified libraries were quantified using the Qubit 2.0 (Table 41), and their quality was assessed using the Agilent Bioanalyzer.

Figure 81A shows the virtual gel obtained with the Agilent Bioanalyzer for the purified libraries amplified from tagmented immunoprecipitated samples. Figure 81B displays the four corresponding electropherograms, each representing a specific library. In this case, the gel and electropherograms show that the IP samples UI-LSD1 (lane 1), UI-C-(lane 3), and Inf-C-(lane 4) predominantly exhibit library fragments within the 200-300 bp range, as indicated by the blue arrow on the gel and vertical blue lines on the electropherograms. However, the Inf-LSD1 sample (lane2) displays an additional, discreet peak of fragments between 500-600 bp in the electropherogram. This observation suggests the possibility of fragment dimerization during amplification, as further supported by the qPCR amplification plot in Figure 80, where the Inf-LSD1 sample approached saturation at 10 cycles.

I consulted with the experts from the sequencing platform at the Institut Pasteur to determine if the situation with the Inf-LSD1 library could potentially pose any issues during the procedure. According to their assessment, it should not be problematic since the average fragment size of 406 bp (Table 41) meets the necessary criteria for sequencing. The average fragment size of all the samples was obtained with the Bioanalyzer, and its corresponding concentration was quantified with the Qubit 2.0 (Table 41). These data are crucial for calculating the sample concentration in nM, which is later used during the library pooling step in the sequencing process.

Currently, I am at this stage within the ChIPmentation procedure, and the libraries are ready for sequencing. If all goes well, the next steps would involve performing immunoprecipitation on the remaining two biological replicates (UI and Inf BMDMs) as well as the two biological replicates of Rag2 knockout mice, described in Section 5.2 of this chapter.

NUCLEAR PROTEOME ANALYSIS

As described in Chapter 1 Section 2.2.3, differences in protein band patterns were observed between the nuclear protein samples of uninfected and infected BMDMs following electrophoretic separation and Coomassie staining.

Figure 82 shows a Coomassie-stained gel loaded with 5 µg of nuclear protein extracted from both uninfected and infected BMDMs, obtained from two independent biological replicates. In this gel, a distinct band exclusively appears in the infected samples, which is absent in the uninfected samples, as highlighted by the red box. Conversely, the blue boxes indicate bands that exhibit reduced intensity in the infected samples. These observations, which reveal only a fraction of the molecules present in the gels, already indicate that the infection induced alterations in the nuclear protein composition of macrophages. We next conducted a proteomic analysis of nuclear proteins to compare the differences in protein abundance induced by intracellular amastigotes in BMDMs. This analysis was performed at the CurieCoreTech Mass Spectrometry Proteomics platform, located at the Curie Institute.

Outlook 168

To accomplish this, nuclear proteins were extracted from four biological replicates of both uninfected and infected BMDMs. The procedure for isolating cell nuclei remained the same as described in Chapter 1 Section 2.1. However, the extraction buffer used was modified based on recommendations from the mass spectrometry platform engineers, and its composition (EBI) is provided in Table 12 of the Materials and Methods.

Prior to sending the protein samples to the platform for analysis, they were quantified by the DC protein assay, as indicated in Material & Methods Section 8.3, and their quality was assessed by separating 5 µg of each sample onto an SDS-PAGE gel and staining it with Coomassie Blue, a non-specific protein dye that binds to almost all proteins (Figure 83A). Outlook 169 Furthermore, to verify the proper separation of nuclear and cytoplasmic proteins, as mentioned in Section 2.2.3 of Chapter 2, a WB was conducted using LSD1 and GAPDH as markers for the nuclear and cytoplasmic fractions, respectively. Figure 83B shows a minimal amount of LSD1 observed in the cytoplasmic fraction, while no detectable GAPDH is present in the nuclear extracts. These findings suggest that the nuclear extracts have minimal to no contamination of cytoplasmic proteins.

These two results confirmed the quality of the nuclear extracts. Therefore, 30 µg of each sample were taken to the platform for subsequent analysis by mass spectrometry.

The preliminary results of this experiment were obtained using the web server for management and validation of mass spectrometry-based proteomic data, termed myProMS, which has been developed be the Institut Curie mass spectrometry platform. At this stage, it is evident from the volcano plot (Figure 84) that there are significant differences (adj. p<0.05) in the protein content of the nuclei when comparing the infected condition (B) to the uninfected condition (A). Moreover, there are proteins exclusively present in the uninfected samples (Only in A) as well as proteins exclusively found in the infected samples (Only in B). Outlook 170 Gene ontology (GO) analysis for cellular components, describing the protein location within cells, was conducted for both sets of proteins that either showed reduced or increased abundance. The statistically highly significant GO enrichment for the terms 'nucleus', 'nuclear lumen', 'nucleoplasm', and 'nucleolus', among others, confirms the specificity of our nuclear protein extraction protocol (Figure 85A). A classic STRING analysis was also performed to assess the impact of infection on protein-protein networks that showed GO enrichment in terms such as 'chromatin organization', 'HDAC complex', and 'centrosome', among others. In these networks, green and red colors represent respectively decrease and increase of proteins in the infected state (Log2FC), with dot size representing the p-value (Figure 85B). This analysis has unveiled significant alterations in macrophage nuclear protein networks induced by Leishmania infection. These changes could potentially elucidate the extensive expression variations observed by our RNAseq analysis.

The most striking aspect of this dataset is the presence of proteins identified "Only in B" (Figure 84). Remarkably, approximately 300 of these proteins correspond to Leishmania proteins, which were visualized in REVIGO, a Web server that simplifies and summarizes complex lists of GO (Figure 86). Assuming there is proper removal of cytoplasmic proteins and parasite debris during the nuclei isolation before protein extraction, this result suggests a transfer of Leishmania proteins into the macrophage nucleus during infection. Outlook 172

FUTURE STUDIES

The preliminary data described in this outlook set the stage for exciting future studies. To better understand the biological validity and significance of these data, we will explore the functions of individual nuclear host proteins whose abundance changed during infection, for example by lentiviral overexpression or siRNA-mediated knockdown in order to comprehend their implications in intracellular parasite survival.

A fascinating aspect of our nucleome analysis of infected BMDMs is the presence of a significant abundance of Leishmania nuclear proteins. The possible trafficking of Leishmania histone proteins into the host nucleus opens the exciting possibility that expression changes observed during infection may be caused by mixed nucleosomes containing both host and parasite histones as suggested by Dacher et al [240].

Likewise, the nucleome of infected macrophages also contained parasite flagellar components. The flagellum is an organelle that has been extensively associated with motility, but it has recently been recognized to possess sensory and secretory functions as well [241,242]. How this can be linked to their identification in the macrophage nucleus remains to be elucidated.

During the parasite life cycle, the flagellum exhibits an extensive ability to undergo structural changes. In the case of amastigotes, the flagellum shrinks and barely protrudes from the flagellar pocket. Strikingly, this retracted flagellum is commonly associated with the host cell parasitophorous vacuole membrane, and it has been suggested that it may receive or transmit signals to and from the host, and is possibly involved in the release of parasite effectors into the infected macrophage [243,244].

The following experiments will be conducted to confirm the transfer of parasite proteins and their potential effects on macrophage phenotype and parasite survival:

 WB analysis of macrophage nuclear and cytoplasmic extracts to detect the presence of the tagged protein.  Confocal microscopy to identify the subcellular localization of the tagged protein.  Reciprocal pull-down experiments using macrophage nuclear extracts to isolate endogenously tagged parasite proteins and investigate their interactions with macrophage proteins.

Outlook 175  Knockout of parasite proteins from the list to study their impact on intracellular parasite survival.  Ectopic expression of the protein by lentiviral expression in J774 or THP-1 cell lines to assess its influence on macrophage expression.

Together these experiments will allow us to distinguish between specific targeting of parasite proteins to the macrophage nucleus versus their nonspecific presence due to cytoplasmic contamination. Intracellular pathogens have evolved intricate strategies to survive within their host cells by exploiting the host cellular metabolism and subverting their defense mechanisms. Epigenetics plays a crucial role in these processes, enabling intracellular bacterial, viral or protozoan infections to manipulate the host cell gene expression and immune responses. Given its intracellular lifestyle inside host macrophages, the protozoan parasite Leishmania represents an interesting model system to elucidate mechanisms underlying the subversion of immuno-metabolic functions of these important innate immune cells.

My PhD research employed a multi-disciplinary approach to investigate this process, combining techniques in pharmacology, proteomics and genomewide profiling of DNA-binding proteins, to unravel the complex interactions between pathogen and host at the epigenetic level.

My research provided important new insight into the mechanisms underlying Leishmania subversion and opened a new venue for anti-parasitic drug discovery. Using two distinct macrophage cell types, I validated the antileishmanial effect of the two inhibitors MC2652 and MC4030 designed to target mammalian LSD1. I demonstrated a direct effect of these inhibitors on LSD1 activity in macrophage nuclear extracts and validated their interaction with LSD1 by Cellular Thermal Shift Assay (CETSA). These results define LSD1 as a potential target for host-directed, anti-leishmanial therapy.

The negative impact of LSD1 inhibition on parasite survival indicates an important role of this epigenetic eraser enzyme in promoting Leishmania infection.

Investigating the mechanisms by which the parasite engages with host LSD1, I demonstrated that infection neither alters LSD1 nuclear localization nor its expression at RNA or protein levels. However, infection had a significant impact on the macrophage nuclear protein content and LSD1 complex formation. These alterations not only involved host proteins but also Leishmania proteins that seem to be targeted to the host cell nucleus.

Together my results open up a series of important questions concerning (i) the possibility to target the macrophage for host-directed, anti-leishmanial therapy, (ii) the nature of LSD1-regulated macrophage pathways that are exploited by Leishmania to ensure parasite survival within the host cell, (iii) the molecular mechanisms by which infection modifies the various LSD1 activities such as scaffolding and demethylase functions, and (iv) the strategies evolved by Leishmania to alter the host cell epigenome.

Exploring host-directed therapy as a promising approach for targeting intracellular Leishmania -Insights from anti-leishmanial drug discovery

Host-directed therapy is a novel approach in infectious diseases for overcoming antimicrobial resistance that is currently explored across many viral, bacterial and eukaryotic pathogens [245][246][247]. This emerging strategy has already received clinical validation, exemplified by the utilization of Maraviroc, in HIV treatment [248], and more recently, Bulevirtide or Myrcludex-B, in the case of Hepatitis B Virus infection [249]. Notably, these medications are now accessible for treating patients affected by these diseases.

Our team pioneered a host-directed approach towards anti-leishmanial drug discovery, proposing mammalian epigenetic regulation as a possible target [START_REF] Lamotte | The enemy within: Targeting host-parasite interaction for antileishmanial drug discovery[END_REF], based on several key observations, including the following:

First, my host lab and others showed that Leishmania affects transcriptional and epigenetic regulation in infected macrophages using various ways. For example, infection enhances STAT6 activity that promotes macrophage polarization towards a permissive M2 phenotype [250], while at the same time dampening anti-microbial M1 polarization interfering in various ways with NF-κB signaling and activity [229,[251][252][253][254]. The parasite further manipulates macrophage miRNAs to further limit proinflammatory gene expression, for example of TLRs [255,256]. Additionally, Leishmania modifies DNA methylation, affecting host cell signaling pathways, oxidative phosphorylation, and cell adhesion [257].

Finally, histone modifications can be exploited to dampen proinflammatory response and enhance anti-inflammatory genes, using HDACs and altering H3K9/14 acetylation, ultimately promoting parasite survival within host macrophages [193]. Our lab recently correlated the anti-inflammatory phenotype of infected macrophages with Histone H3 demethylation at lysine residues K4 and K9, which are the key targets for LSD1 activity [194]. This result provided first evidence that Leishmania may exploit this epigenetic regulator for host cell subversion, and that interfering with LSD1 activity may be a promising path to establish a first proof-of-principle for host directed, antileishmanial therapy.

Second, our initial drug screening campaign using an epigenetic inhibitor library provided by the FP7 A-PARADDISE project (https://cordis.europa.eu/project/id/602080/fr) identified a series of antileishmanial compounds that showed potential host-directed mode of action given that they specifically reduced intracellular load of Leishmania amastigotes without affecting parasite viability in culture. From all classes of inhibitors tested, the inhibition of macrophage demethylase activity showed the strongest anti-leishmanial effect, with the LSD1-specific compounds MC4030 and MC2652 pointing us to this particular demethylase enzyme as a first possible target for host-directed, anti-leishmanial intervention.

Third, transcriptomic profiling previously conducted in our lab further supported the possibility to discover host-directed, anti-leishmanial drugs. Comparison of uninfected and Leishmania-infected macrophages uncovered important changes in gene expression that contribute to the creation of a favorable environment for parasite survival and replication within the host cells. Treatment of such infected macrophages with LSD1i restored a largely normal immuno-metabolic expression profile (despite the presence of the parasite at 24h post-treatment), with increased transcript expression of innate immune functions and reduction of cholesterol biosynthesis likely interfering with Leishmania intracellular survival. The significant expression changes observed even in uninfected macrophages treated with LSD1i, and the absence of a Leishmania LSD1 ortholog further support a host-directed drug action mechanism.

The epigenetic subversion of macrophage immuno-metabolomic functions by Leishmania is reminiscent to other intracellular pathogens known to target histone modifications and enzymes responsible for modifying chromatin structure. Notable examples include Mycobacterium tuberculosis, which modifies the SWI/SNF protein complex by perturbing IFN signaling [258]; Listeria monocytogenes, which affects MAPK signaling and alters Histones H4, H3, and H2A through its Listeriolysin [259]; and Toxoplasma gondii, which disrupts STAT transcriptional regulation and MAPK pathways, while binding to the host nuclear factor via the parasitic protein factor ROP16 [260].

In conclusion, these results identify host cell epigenetic regulation and their downstream pathways as a fertile ground for discovering macrophage drug targets that impact parasite survival. This strategy aligns with the emerging field of host-directed therapy in infectious diseases, offering promise for combating Leishmania and other pathogens.

LSD1-regulated macrophage pathways exploited by Leishmania for intracellular parasite survival

The identification of specific pathways affected by infection and counteracted by the LSD1i can inform on additional macrophage pathways that may represent potential host targets for anti-leishmanial intervention, such as sterol biosynthesis or innate immunity.

The significance of sterol and polyamine synthesis in the context of Leishmania macrophage infection has been underscored in various transcriptomic studies, correlating the upregulation of these pathways to parasite intracellular infection and growth [261][262][263]. Our previous transcriptomics analysis indeed revealed a coordinated upregulation in infected macrophages of nearly all enzymes catalyzing sterol biosynthesis, which we linked to an equal upregulation of the transcription factor SREBP2, one of the master regulators of this pathway [264].

Similar to SREBP2, the paralog SREBP1 has been linked to lipid homeostasis via its activity on lipogenic gene activation. Even though the expression of this gene is not modulated in infected macrophages in our system, its known interaction with LSD1 opens the possibility that altered LSD1 activities in these cells may affect SREBP1 downstream-regulated pathways. LSD1 has been linked to SREBP1-driven gene expression, both indirectly via interaction with the deacetylase SIRT1 or directly as demonstrated by its essential role in SREBP1-FAS promoter activation. The role of LSD1 in lipogenic gene control suggests it as a lipid metabolism intervention target [265]. In the context of my work, subsequent studies could explore the formation of regulatory complexes containing LSD1 at the SREBP2 promoter itself and its interactions with SREBP2 at downstream regulated target promoters as observed for SREBP1.

Our RNAseq analysis further uncovered macrophage innate immune response pathways as key targets for Leishmania immune subversion during infection, which are rescued by LSD1i treatment, and thus regulated at the epigenetic level. Indeed, epigenetic control of gene expression during infection plays an important role in the development of the anti-microbial, inflammatory response. LSD1 is one of the key regulators of the macrophage inflammatory response, influencing the expression of inflammatory genes via distinct mechanisms.

For example, in smooth muscle cells from a diabetic model, LSD1 downregulation was correlated with increased H3K4me2 near NF-κB response elements, enhancing the expression of the proinflammatory genes . In contrast, in the cancer cell lines MDA-MB231 and HepG2, LSD1 decreases H3K4 methylation at the promoters of proinflammatory cytokine genes , leading to their repression. Additionally, LSD1 collaborates in a synergistic manner with HDAC1 to decrease the global acetylation level of H3K9/14, which is a mark of transcriptional activation, thus reducing proinflammatory cytokine expression [267].

In the context of our parasitic system, we have demonstrated changes in methylation pattern for H3K4 and K9 sites in the promoters of proinflammatory genes [194]. We have further shown that Leishmania subverts the macrophage inflammatory response in a dual way by simultaneously reducing proinflammatory gene expression while at the same time activating the expression of anti-inflammatory genes. The dual activity of LSD1 to demethylate both the activation mark H3K4 (hence reducing gene expression) and the repressive H3K9 mark (hence inducing gene expression) makes it a perfect candidate for Leishmania-mediated subversion of the macrophage inflammatory response. We will explore this possibility in the near future conducting genome-wide analyses of LSD1 promoter occupancy and H3K4/K9 methylation levels in uninfected and infected macrophages.

LSD1 serves not only as a suppressor of the inflammatory response but also exhibits the potential to exacerbate it. For example, LPS stimulation of BMDMs results in LSD1 phosphorylation by PKCα. This phosphorylation is crucial for the activation of NF-κB target genes and operates independently of LSD1 demethylase activity on H3K4me2 and H3K9me2 [182], representing a key example for the role of LSD1 in demethylating nonhistone proteins, in this case facilitating the removal of methyl groups from NF-κB. Indeed, in recent years, LSD1 was attributed four distinct activities: canonical enzymatic and scaffolding functions on histone targets, as well as non-canonical enzymatic and scaffolding functions on nonhistone targets. The role of such non-canonical LSD1 function in our infection system will be assessed in the future, for example by Western blot analysis, comparing the levels of phosphorylated LSD1 between infected and uninfected BMDMs.

The molecular mechanisms by which infection could modify LSD1 activity

I demonstrated that infection neither altered LSD1 localization nor RNA or protein expression levels, suggesting that Leishmania engages this essential macrophage regulator in a more complex fashion. The CETSA analysis I conducted provided crucial insight into the possible mechanisms how Leishmania infection affects LSD1, which ultimately may explain the changes in macrophage gene expression and phenotype. From a pharmacological point of view, the CETSA showed that the LSD1i effectively engage the target, binding to the LSD1 protein regardless of the infection status of the cells. This indicates that the drug interacts with its target regardless of potential infection-induced changes.

From a cell biological point of view, the CETSA analysis provided first evidence that infection itself leads to an important increase in LSD1 thermal stability, suggesting that infection impacts LSD1 molecular interactions and likely complex formation, prompting me to conduct an in-depth molecular interaction study to assess changes in LSD1 scaffolding function during infection.

It is now well established that acetylation and methylation of lysine residues within the tails of histone proteins not only control the recruitment of regulatory factors, but also influence the architecture of chromatin itself. Previous studies have indicated that the activity and substrate specificity of LSD1 could be controlled by its interacting proteins. For instance, the regulatory protein CoREST enhances the binding of nucleosomes and the demethylase activity of LSD1, while also protecting it from proteasomal degradation [152,154]. As a second example, at the core of the CoREST complex, two histone modification enzymes, HDAC1/2 and LSD1, are linked by RCOR1/2/3, establishing a connection between these two enzymes [268]. Although these enzymes are positioned at opposite ends of the complex, there is a remarkable interplay between their enzymatic activities. Inhibitors of one enzyme significantly impact the kinetics of the partner enzyme, with only one active site capable to interact with a substrate at any given time [156].

The co-IP studies I conducted indeed revealed that Leishmania alters LSD1 interactions, potentially influencing the expression of infection-related genes. Among the 47 macrophage proteins identified as LSD1 interactors in only infected BMDMs, several caught our attention: LSD1 interacted in an infection-specific way with the heterogeneous nuclear ribonucleoprotein K (HnRNPK) (displayed in Cluster 2 of Figure 68), that has been reported as a nonhistone target of LSD1 in THP-1 cells [269], demonstrating an indirect role in stabilizing Cxcl10 mRNA, a well-known chemokine associated with resistance to Leishmania infection [270]. HnRNPK serves various functions in both the nucleus and cytosol, including the regulation of transcription, splicing, mRNA silencing, mRNA stability, and translation [271]. The interaction between HnRNPK and LSD1 in infected macrophages, and its known role in stabilizing Cxcl10 mRNA, suggests a potential link between epigenetic regulation, chemokine expression, and host resistance to Leishmania infection. However, further investigation is required to precisely determine the extent and mechanisms of HnRNPK involvement in this context.

Infection further triggered novel LSD1 interactions with the splicing factor proline-and glutamine-rich (SFPQ), the ATP-dependent RNA helicase A (DHX9), and Matrin 3 (MATR3), (observed in the Cluster 2 of Figure 68), as well as the non-POU domain-containing octamer-binding protein (NONO). These proteins have recently been identified as SARS-CoV-2 RNA interactors where they could promote viral RNA amplification [272]. In other studies, it has been shown that NONO forms a heterodimer with SFPQ. This interaction might contribute to a nuclear process responsible for retaining defective RNAs, immature RNAs not ready for export, or RNAs intended for nucleus retention [273]. The SFPQ-NONO heterodimer might also participate in DNA non-homologous end joining (NHEJ) required for double-strand break repair and V(D)J recombination, possibly stabilizing paired DNA ends [274]. Moreover, HnRNPK, SFPQ, and DHX9 have been identified as components of a network that modulates transcription and translation processes in a colorectal adenocarcinoma model [275].

The identified LSD1 interactions with SFPQ, DHX9, MATR3, and NONO could hold significance for Leishmania survival strategies. Considering their roles in processes like RNA binding, splicing, and metabolic regulation, it is conceivable that Leishmania could exploit these interactions to modulate host cell RNA dynamics in infected macrophages.

The LSD1 interactors within infected macrophages, specifically those in Cluster 1 of Figure 68, are enriched in ribosomal proteins. LSD1 indeed has been shown to be involved in demethylating the ribosomal protein Rpl29 [276], suggesting that similar processes might affect other ribosomal proteins in our infection system. These particular interactions between LSD1 and ribosomal proteins in Leishmania-infected macrophages could influence ribosome formation and protein synthesis, potentially changing the macrophage phenotype to favor parasite survival.

Pathogen strategies to alter the host cell epigenome.

The discovery during my thesis of Leishmania proteins acting as LSD1 interactors, along with the identification of around 300 Leishmania proteins within the nuclear proteome of infected macrophages reveals a potential new parasite subversion strategy. The presence of these proteins holds the potential to disrupt nuclear dynamics, thereby enhancing parasite survival.

Recent research has emphasized the microbial subversion of host cell functions through the direct engagement of pathogen-derived proteins with nuclear components. These microbial proteins, known as nucleomodulins in the context of bacterial infection [277], are involved in influencing nuclear processes. My research proposes the existence of Leishmania nucleomodulins, raising questions about (i) their release mechanism and (ii) their impact on the host cell epigenome.

Most likely these Leishmania nucleomodulins are released via exosomes, which may fuse with macrophage endomembranes to allow their delivery via host trafficking systems (such as the ER). Exosomal protein release indeed emerges as a potential mechanism for transporting pathogenic effector molecules into host cells. Parasite-derived exosomes that may target the host have been described for Leishmania,Trypanosomes,and Trichomonas [278,279]. Intracellular pathogens further employ specialized secretory vesicles and protein export systems that allow to shuttle their proteins through parasitophorous vacuoles into the host cell. For example, microvesicles from Plasmodium-infected erythrocytes can induce macrophage activation and proinflammatory cytokine transcription [280].

Similar to other intracellular parasites, Leishmania effectors may impact the host epigenetic landscape. This host-Leishmania interaction can significantly influence epigenetic patterns and chromatin structure, consequently having an effect on host cell transcriptional regulation phenotype. This manipulation could occur through various mechanisms, which will be briefly described in the following.

First, Leishmania nucleomodulins could indirectly modulate host signaling pathways. This could be carried out through various mechanisms:

(i) Sequestration or deactivation of transcription factors, taking over nuclear signaling pathways. Leishmania's metalloprotease GP63 has been identified within parasite exosomes [281], and localizes in the macrophage perinuclear area where it may contribute to changes in host transcription. It cleaves the p65-RelA subunit of NF-κB, and the resulting fragment p35 RelA migrates to the nucleus, where it induces chemokine gene expression [252]. GP63 may also enter the nucleus and cleave the cJun component within the AP1 transcription factor, causing the deactivation of the host MAPK signaling that can modulate essential cellular processes, including apoptosis, stress response, and survival [282][283][284]. Surprisingly, the GP63 metalloprotease was absent among the Leishmania proteins in my nuclear proteome analysis. This mechanism has been observed in Shigella flexneri, which can inhibit NF-κB binding to specific promoters [285], and in Chlamydia trachomatis, which directly cleaves the p64/RelA protein to effectively prevent NF-κB activation [286].

(ii) Post-translational modifications of host nuclear proteins via secreted enzymes. The identification of Leishmania casein kinase 1.2 (CK1.2) among the proteins I found within the nuclei of infected macrophages provides insight into a potential mechanism through which the parasite might exert immune-suppressive effects on host cells. Indeed, CK1.2 has a known exokinase activity, enabling it to modify host proteins through phosphorylation, including IFNAR1, a receptor implicated in IFN-α/β signaling [287]. This discovery encourages further investigations into the precise implications of CK1.2 actions on host immune responses, offering a promising avenue for understanding the intricate interactions between Leishmania and its host.

Second, Leishmania nucleomodulins could directly target host nuclear proteins via several mechanisms: (i) Association with host nuclear proteins. The discovery of 24 Leishmania proteins within the set of LSD1 interactor proteins in my co-IP analysis prompted us to contemplate this type of direct manipulation mechanism. This type of association has been previously described in the case of Toxoplasma gondii, which secretes GRA16, interacting with host nuclear proteins, such as the deubiquitinase HAUSP and the phosphatase PP2A [288]. A deeper exploration of these interactions is crucial to understand their impact on LSD1 functions in Leishmania-infected macrophages.

(ii) Modification of host nuclear proteins. Previous reports have highlighted the influence of the GP63 metalloprotease on host nuclear proteins, including the degradation of nucleoporins that play a role in nuclear transport processes [109]. Notably, GP63 was not detected in my analysis. Conversely, multiple Leishmania nucleoporins emerged. This observation suggests a direct potential for these proteins to influence nuclear transport processes. Moreover, it raises the intriguing possibility that they might even play a role in facilitating the transportation of Leishmania proteins into the nuclei of the host cells.

In addition, various apicomplexans and kinetoplastids, including Leishmania, release kinases and phosphatases into the host cell. It is possible that some of these secreted effectors could modify chromatin-related processes or directly interact with histones. For example, Mycobacterium tuberculosis secretes a protein phosphatase that dephosphorylates histones [289].

(iii) Molecular mimicry of nuclear proteins. One intriguing observation from the analysis of the nuclear proteome in infected macrophages is the significant presence of all Leishmania histones. Additionally, Histone H2A, H2B, and H4, were also found as LSD1 interactors in the co-IP study. The possibility of Leishmania histone proteins being transported into the host nucleus presents an exciting concept: the changes in expression observed during infection could potentially originate from hybrid nucleosomes containing both host and parasite histones as previously suggested by Dacher et al [240].

(iv) Association with host DNA: Leishmania nucleomodulins could potentially be directly interacting with the host DNA. This mechanism has been described in the case of Theileria spp. These parasites release proteins resembling eukaryotic AT hook domain proteins, which play a pivotal role in DNA-and RNA-dependent processes, thereby influencing cytoskeletal protein transcription and morphology [290].

After confirming the presence of these proteins within the macrophage nucleus, further investigations are necessary to unveil the specific mechanisms that these proteins could employ to exert influence over epigenetic patterns.

In conclusion, the manipulation of the host epigenome by pathogen-derived effector molecules is being increasingly recognized as a pivotal fundamental element in microbial pathogenesis. This phenomenon holds the potential for the development of therapeutic strategies, while also reducing concerns related to drug resistance. How pathogen effector proteins access the nucleus, especially in the absence of conventional nuclear localization signals, remains to be elucidated and may involve exosomal targeting of parasite proteins to the host cell delivery system (i.e. the ER), or hitchhiking via interaction with host nuclear proteins.

ABSTRACT

Intracellular pathogens exploit host cell functions to favor their own survival. In recent years, the subversion of epigenetic regulation has emerged as a key microbial strategy to modify host cell gene expression and evade antimicrobial immune responses. Using the protozoan parasite Leishmania as a model system, we have recently demonstrated that infection causes histone H3 hypomethylation, which is associated with the establishment of an anti-inflammatory phenotype, suggesting that host cell demethylases may play a role in the intracellular survival of these parasites. In this study, we employed a combination of pharmacological, RNA sequencing and interaction studies to investigate the role of the macrophage lysine demethylase LSD1 (KDM1a) in Leishmania intracellular infection in vitro. Treatment of infected macrophages with two validated LSD1-specific inhibitors resulted in a significant reduction in parasite burden. We confirmed the impact of these inhibitors on LSD1 activity within macrophage nuclear extracts using an in vitro demethylase assay and established their LSD1 target engagement in situ by cellular thermal shift assay. RNA-seq analysis of infected and inhibitor-treated macrophages linked parasite killing to a partial reversion of infectiondependent expression changes, restoring the macrophage anti-microbial response and limiting cholesterol biosynthesis. While we ruled out any impact of Leishmania on LSD1 expression or localization, we uncovered significant alterations in LSD1 complex formation within infected macrophages, involving unique interactions with host proteins as well as interactions with Leishmania proteins that appear to be secreted into the macrophage nucleus. Our study sheds important new light on the epigenetic mechanisms of macrophage immuno-metabolic subversion by intracellular Leishmania and identifies LSD1 as a potential candidate for hostdirected, anti-leishmanial therapy.

INTRODUCTION

Many viral, bacterial and eukaryotic pathogens infect mammalian cells and have co-evolved strategies to modulate the gene expression profiles of their host cells and promote their intracellular survival [START_REF] Benoit | Macrophage polarization in bacterial infections[END_REF][START_REF] Herbein | The macrophage in HIV-1 infection: from activation to deactivation?[END_REF][START_REF] Herbert | Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology[END_REF][START_REF] Kamhawi | Leishmania: A Maestro in Epigenetic Manipulation of Macrophage Inflammasomes[END_REF][START_REF] Noel | Alternatively activated macrophages during parasite infections[END_REF][START_REF] Pearce | The immunobiology of schistosomiasis[END_REF][START_REF] Raes | Alternatively activated macrophages in protozoan infections[END_REF]. This co-evolution is well illustrated for the protozoan parasites of the genus Leishmania that proliferate inside the sand fly insect vector as motile promastigotes and as non-motile amastigotes inside fully acidified phagolysosomes of mammalian macrophages. Leishmania subverts the immunological and metabolic functions of these important and highly toxic immune cells by interfering with cell signaling [START_REF] Gregory | Subversion of host cell signalling by the protozoan parasite Leishmania[END_REF], metabolism [START_REF] Rabhi | Transcriptomic signature of Leishmania infected mice macrophages: a metabolic point of view[END_REF] or epigenetic control [START_REF] Afrin | Leishmania-Host Interactions-An Epigenetic Paradigm[END_REF][START_REF] Kamhawi | Leishmania: A Maestro in Epigenetic Manipulation of Macrophage Inflammasomes[END_REF]. The host consequences of Leishmania immune subversion are often devastating causing severe immune pathologies termed 'leishmaniases' that range from self-healing cutaneous to lethal visceral forms [START_REF] Conceicao-Silva | Leishmania Spp-Host Interaction: There Is Always an Onset, but Is There an End?[END_REF][START_REF] Mazzarella | Inhibiting LSD1 suppresses coronavirus-induced inflammation but spares innate antiviral activity[END_REF].

Despite the severe consequences to human health, the mechanisms underlying the subversion of macrophage functions by intracellular Leishmania remain poorly understood. Epigenetic control of host immuno-metabolic processes in infected macrophages and dendritic cells has recently emerged as a new parasite strategy for host cell subversion [START_REF] Kamhawi | Leishmania: A Maestro in Epigenetic Manipulation of Macrophage Inflammasomes[END_REF][START_REF] Lecoeur | Going ballistic: Leishmania nuclear subversion of host cell plasticity[END_REF]. First, Leishmania has been shown to manipulate host cell miRNAs to limit pro-inflammatory gene expression [START_REF] Ganguly | Leishmania survives by exporting miR-146a from infected to resident cells to subjugate inflammation[END_REF][START_REF] Lemaire | MicroRNA expression profile in human macrophages in response to Leishmania major infection[END_REF][START_REF] Mukherjee | Antimony-Resistant Leishmania donovani Exploits miR-466i To Deactivate Host MyD88 for Regulating IL-10/IL-12 Levels during Early Hours of Infection[END_REF][START_REF] Muxel | Toll-Like Receptor and miRNA-let-7e Expression Alter the Inflammatory Response in Leishmania amazonensis-Infected Macrophages[END_REF][START_REF] Nandan | Leishmania regulates host macrophage miRNAs expression by engaging transcription factor c-Myc[END_REF][START_REF] Tiwari | Identification and Characterization of miRNAs in Response to Leishmania donovani Infection: Delineation of Their Roles in Macrophage Dysfunction[END_REF]. Second, intracellular Leishmania infection has been correlated to changes in the macrophage DNA methylation pattern, influencing host cell signalling pathways, oxidative phosphorylation, and cell adhesion [START_REF] Marr | Leishmania donovani infection causes distinct epigenetic DNA methylation changes in host macrophages[END_REF]. Finally, we and others have shown that Leishmania infections alters host cell histone modification, such as acetylation or methylation, whose levels are dynamically regulated by writer enzymes (e.g. acetyltransferases, methyltransferases) and eraser enzymes (i.e. deacetylases, demethylases). Our recent work has established a first link between a macrophage epigenetic eraser activity and Leishmania infection: applying a ChIPqPCR approach we provided first evidence that L. amazonensis infection causes hypomethylation at pro-inflammatory gene promoters at activating histone H3K4 and repressing H3K9 marks, which correlated with a corresponding decrease in transcript output [START_REF] Kamhawi | Leishmania: A Maestro in Epigenetic Manipulation of Macrophage Inflammasomes[END_REF][START_REF] Lecoeur | Leishmania amazonensis Subverts the Transcription Factor Landscape in Dendritic Cells to Avoid Inflammasome Activation and Stall Maturation[END_REF]. These H3 modifications are both removed by the Lysine-Specific Demethylase 1 (LSD1, a.k.a. KDM1a) [START_REF] Shi | Histone demethylation mediated by the nuclear amine oxidase homolog LSD1[END_REF], which in accordance to a given lysine mark can increase or decrease gene expression.

LSD1 is composed of three domains: the catalytic AO domain, the Swi3/Rcs8/Moira (SWIRM) domain and the Tower domain [START_REF] Burg | KDM1 class flavin-dependent protein lysine demethylases[END_REF]. Aside its enzymatic function, it carries important scaffolding functions via the Tower domain that serves as binding site for the CoREST transcription repressor complex (essential for LSD1 to associate with the nucleosome and exert its demethylase activity) and the SWIRM domain that functions as a platform for protein-protein interactions [START_REF] Burg | KDM1 class flavin-dependent protein lysine demethylases[END_REF]Laurent and Shi, 2016). LSD1 is involved in diverse biological processes including differentiation [START_REF] Whyte | Enhancer decommissioning by LSD1 during embryonic stem cell differentiation[END_REF], autophagy [START_REF] Ambrosio | Histone methyl-transferases and demethylases in the autophagy regulatory network: the emerging role of KDM1A/LSD1 demethylase[END_REF] or metabolic processes [START_REF] Sakamoto | Lysine Demethylase LSD1 Coordinates Glycolytic and Mitochondrial Metabolism in Hepatocellular Carcinoma Cells[END_REF]. Conceivably, deregulation of LSD1 functions cause severe human diseases, including cancer [START_REF] Majello | Expanding the Role of the Histone Lysine-Specific Demethylase LSD1 in Cancer[END_REF] and, as a consequence, represents one of the prime therapeutic targets, with various compounds undergoing clinical testing for treating small lung cancer cells (SCLC) or acute myeloid leukaemia (Fang et al., 2019). Surprisingly, despite the availability of LSD1-specific inhibitors and the known role of role of LSD1 to control macrophage polarization, its potential as a host-directed target for anti-microbial intervention has not been explored yet, except for viral infections [START_REF] Mazzarella | Inhibiting LSD1 suppresses coronavirus-induced inflammation but spares innate antiviral activity[END_REF][START_REF] Zwergel | Trends of LSD1 inhibitors in viral infections[END_REF]. We previously proposed an experimental framework for the discovery of host-directed inhibitors targeting macrophage histone-modifying enzymes [START_REF] Lamotte | Discovery of novel hit compounds with broad activity against visceral and cutaneous Leishmania species by comparative phenotypic screening[END_REF][START_REF] Lamotte | The enemy within: Targeting host-parasite interaction for antileishmanial drug discovery[END_REF] for which large compound libraries are available given current efforts to cure non-communicable human diseases such as cancer, obesity, auto-immunity by restoring a healthy epigenetic profile (Hogg et al., 2020;Nebbioso et al., 2018).

Here, we established a first proof-of-principle analysis for host-directed therapy by exploring the possibility to target LSD1 for anti-leishmanial drug discovery. Screening a diverse epigenetic inhibitor library against intracellular and extracellular L. amazonensis parasites identified a series of anti-parasitic compounds designed to target mammalian LSD1 (https://cordis.europa.eu/project/id/602080/fr ). We demonstrate that LSD1 inhibition partially reverts infection-induced expression changes that are likely incompatible with robust parasite survival and growth, and provide first evidence that L. amazonensis subverts the LSD1 scaffolding functions to establish a macrophage phenotype permissive for intracellular infection.

MATERIAL AND METHODS

Ethics statement

Six-week-old female C57BL/6 and Swiss nu/nu mice were purchased from Janvier (Saint Germain-sur-l'Arbresle, France). All animals were housed in A3 animal facilities according to the guidelines of Institut Pasteur and the "Comité d'Ethique pour l'Expérimentation Animale" (CEEA) and protocols were approved by the "Ministère de l'Enseignement Supérieur, Direction Générale pour la Recherche et l'Innovation" under number 2013-0047. EP is authorized to perform experiments on vertebrate animals (license 75-1265) issued by the ''Direction Départementale de la Protection des Populations de Paris'' and is responsible for all the experiments conducted personally or under his supervision as governed by the laws and regulations relating to the protection of animals.

Bone marrow-derived macrophages and cell line cultures

Bone marrow cell suspensions were recovered from tibias and femurs of female C57Bl/6JRj mice in Dulbecco's phosphate buffered solution (PBS), and cultured in complete medium containing 4 g/L glucose, 1 mM pyruvate and 3.97 mM L-Alanyl-L-Glutamine and supplemented with 10% heat-inactivated fetal calf serum (FCS), streptomycin (50 µg/mL), and penicillin (50 IU/mL) (de La [START_REF] De La Llave | A combined luciferase imaging and reverse transcription polymerase chain reaction assay for the study of Leishmania amastigote burden and correlated mouse tissue transcript fluctuations[END_REF]. Briefly, one million cells per ml of complete medium supplemented with 50 ng/ml of mouse recombinant colony-stimulating factor 1 (rm-CSF1, ImmunoTools) were incubated in bacteriological Petri dishes (Greiner bio-one 664161) at 37°C in a 7.5% CO2 atmosphere. After 6 days of culture, the medium was removed and adherent cells were incubated with pre-warmed PBS pH 7.4 containing 25 mM EDTA for 30 min at 37°C. Bone marrow-derived macrophages (BMDMs) were detached by gentle flushing, collected, and resuspended in complete medium supplemented with 30 ng/ml of rmCSF-1.

BMDMs were seeded in 100 mm tissue culture dishes (Petri BD Falcon, 353003) for nuclear or total protein extraction, and RNA isolation, and in 384-well tissue culture-treated flatoptically clear microplates (CellCarrier™ plate, PerkinElmer) for High Content Assay (HCA).

Parasite isolation and promastigote differentiation

mCherry-transgenic, tissue-derived amastigotes of Leishmania amazonensis strain LV79 (WHO reference number MPRO/BR/72/M1841) and Leishmania donovani strain 1S2D (WHO reference number MHOM/SD/62/1S-CL2D) were isolated from infected footpads of Swiss nude mice [START_REF] Lecoeur | Leishmania amazonensis Subverts the Transcription Factor Landscape in Dendritic Cells to Avoid Inflammasome Activation and Stall Maturation[END_REF] and infected spleen of golden Syrian hamsters, respectively.

Promastigotes were differentiated from animal-derived amastigotes and maintained at 27°C in M199 medium supplemented with 10% FCS, 20 mM HEPES pH 6.9, 4 mM NaHCO3, 2 mM glutamine, 20 μM folic acid, 100 μM adenine, 13.7 µM hemin, 8 μM 6-biopterin, 100 U ml -1 of penicillin, and 100 μg ml -1 of streptomycin. Promastigotes were then maintained in culture by serial dilution in fresh medium once they reached stationary phase and used in Log growth phase for Dye Reduction Assay (DRA).

Chemical compounds

All small chemical compounds used in screening assays were part of the EU FP7 A-ParaDDisE project (https://cordis.europa.eu/project/id/602080/fr) and represent potential or validated epigenetic inhibitors. Inhibitors of lysine specific demethylase 1, including MC2652, MC4030 and Tranylcypromine (TCP) were synthesized by the laboratory of Antonello Mai (Facolta di Farmacia e Medicina, Sapienza Universita, Roma, Italy). All compounds were solubilized in DMSO at a 10 mM concentration and stored as aliquots at -20°C in 1.5 mL Eppendorf tubes.

Compounds targeting LSD1 (LSD1i) were used at different concentrations depending on the assay as indicated in Results.

Cell infection and treatment

After seeding at day 6 of differentiation, BMDMs were left for 5 hours at 37°C to attach to their new culture support. BMDMs were infected with lesion-derived L. amazonensis amastigotes at a multiplicity of infection (MOI) of 4 and then cultured at 34°C. LSD1i were added to uninfected and Leishmania-Infected Macrophages (LIMs) 24 hours after infection, at a final concentration of 10 µM (HCA, epifluorescence microscopy) or 15 µM (WB, CETSA).

High Content Assay

The High Content Assay (HCA) was used to assess the anti-leishmanial activity against intramacrophagic amastigotes of L. amazonensis [START_REF] Lamotte | Discovery of novel hit compounds with broad activity against visceral and cutaneous Leishmania species by comparative phenotypic screening[END_REF]. 425 compounds (A-ParaDDisE libraryhttps://cordis.europa.eu/project/id/602080/fr) were added one day after macrophage infection at 10 μM final concentration (1% DMSO final concentration). Controls included a vehicle control (DMSO), a leishmanicidal control (amphotericin B), and a macrophage cytotoxic control (cycloheximide). Each compound was tested in quadruplicates.

Briefly, after 3 days of co-incubation with compounds, BMDM nuclei and parasitophorous vacuoles (PVs) were stained with Hoechst 33342 (Invitrogen Molecular Probes, 10 µM) and LysoTracker Green (Life Technologies, DND-26, 1 µM), respectively. Acquisition of images was performed on live cell cultures using the OPERA QEHS device with a 10x air objective (NA 0.4) using the green (488 nm), the blue (405 nm) and the red (561 nm) channels for the detection of PVs, nuclei and amastigotes, respectively [START_REF] Lamotte | Discovery of novel hit compounds with broad activity against visceral and cutaneous Leishmania species by comparative phenotypic screening[END_REF]. Images were transferred to the Columbus Conductor™ Database (Perkin Elmer Technologies) and analysed using the integrated Image analysis building blocks [START_REF] Lamotte | Discovery of novel hit compounds with broad activity against visceral and cutaneous Leishmania species by comparative phenotypic screening[END_REF]. Quantitative values obtained for host cell and parasite numbers were exported to Excel and SigmaPlot for further analysis and graphical representations.

Nuclear and cytoplasmic protein extraction

For western blotting, LSD1 Activity Assay and LSD1 co-immunoprecipitation (co-IP) assay, nuclear and cytoplasmic extracts were obtained after 2 days of infection. The medium was removed from each dish, and cells were gently washed with PBS at room temperature (RT).

PBS was then replaced by 1 ml of Lysis Buffer (LBI or LBII, depending on the subsequent procedure, Table S2).

After 10 min of incubation on ice, cells were detached with a cell scraper and collected in 2 ml Eppendorf tubes. The cell homogenate was passed 5 times through a 27 G needle to isolate nuclei. The quality of isolated nuclei was assessed by the EVOS Cell Imaging System and by Western blotting (Figure S6). Samples were centrifuged for 10 min at 4°C and i) 250 g for activity assay, western blot, or ii) 500 g for LSD1 co-IP assay. Supernatants containing cytoplasmic proteins were removed and kept for further analysis, whereas 150 µl of Extraction Buffer (EBI or EBII, depending on the subsequent procedure, supplementary table 2) were added to pellets. Tubes were vortexed and incubated for 10 min at 4°C. Sonication was performed using the Diagenode's Bioruptor® Standard for 5 min at high power (30" on/30" off sonication cycles). Extracts were cleared by centrifugation at 15,000g for 10 min at 4°C.

Cellular Thermal Shift Assay (CETSA)

Uninfected and infected BMDMs were treated or not at 1 day after infection with 10 µM LSD1i (MC2652, MC4030, and TCP) or 0.1% DMSO as control. After one day of treatment, cells were washed with the pre-warmed medium before adding 1.5 ml of cold Extraction Buffer (1x PBS -1x PIC -1 mM PMSF). Then, cells were collected with a cell scraper in two 1.5 ml Eppendorf tubes and subjected to three cycles of freezing in liquid nitrogen and thawing in a thermomixer at 25°C with a 400 rpm agitation step. Lysates were submitted to 3 sonication cycles (30" on/30" off) at high power, then pooled in one Eppendorf tube per condition and centrifuged at 20,000 g for 20 min at 4°C. Supernatants containing soluble proteins were transferred to 1.5 ml tubes, then distributed into twelve 0.2 ml PCR tubes and heated to different temperatures (from 37°C to 70°C, ∆T°=+3°C) in a Thermocycler (Applied ProFlex ™ 96 Well PCR System) for 3 min followed by cooling at 4°C. Afterwards, protein solutions were transferred to 1. Normalization was carried out with the Amersham ImageQuant TL analysis software considering the band intensity of the sample heated to 37°C as 100%. Melting curves were generated with GraphPad Prism software package.

Western blotting

Experiments were performed on 5-10 g proteins. Proteins were resolved by SDS-PAGE (4-12% Bis-Tris NuPAGE gels) in MOPS buffer and electroblotted onto polyvinylidene difluoride (PVDF) membranes, that were blocked with 2% Amersham ECL Prime blocking agent in 1x PBS containing 0.25% Tween 20. Membranes were then probed overnight at 4°C with the following primary antibodies: rabbit anti-LSD1 (C69G12, Cell Signaling Technology), rabbit anti-MAO-A (10539-1-AP, Proteintech), mouse anti-GAPDH (60004-1-Ig, Proteintech), rabbit anti-RCOR1 (27686-1-AP), mouse anti-YY1 (66281-1-Ig, Proteintech), rabbit anti-Histone-H3 (17168-1-AP, Proteintech).

Following incubation, either with Amersham ECL Plex Cy3 or Cy5 conjugated antibodies or with the appropriate peroxydase conjugate secondary antibodies (Thermo Scientific), fluorescence or luminescence (SuperSignal West Pico reagent, ThermoFisher Scientific) were measured in an Amersham ImageQuant 800 Imaging System (Cytiva). Relative protein expression was calculated by densitometric analysis using the Amersham ImageQuant analysis software. For every band, the ratio between the values obtained for the target protein and the YY1 (nuclear extracts) and the GAPDH (cytoplasmic extracts) normalization controls were calculated. Fold changes were calculated using the control sample as calibrator, control values of uninfected and unstimulated samples being set to 1. Modulations of the LSD1 protein content were statistically determined by the nonparametric Wilcoxon rank-sum test using the GraphPad Prism 7.03 software.

Determination of LSD1 localization by epifluorescence microscopy analysis

For intracellular LSD1 localisation, 2x10 5 macrophages were seeded in 500 µL of complete medium per well of 24-well plates containing sterile glass coverslips, infected or not with L. amazonensis amastigotes with a MOI of 4 for one day, and treated with 10 µM LSD1i or DMSO. After 3 days of treatment (4 days of infection), cells were fixed in a 4% PFA solution for 30 min at RT. After a PBS wash (1 ml/well, 10 min, RT), quenching solution (50 mM ammonium chloride) was added to capture free aldehyde groups (1 ml/well, 15 min, RT).

Viability assay for Leishmania promastigotes and amastigotes

Anti-leishmanial activity of compounds on free parasites was evaluated using an adapted resazurin-based dye reduction (DRA) assay with cell-cycling promastigotes from logarithmic growth phase maintained in M199 supplemented medium.

Fixed concentrations or serial dilutions of the inhibitors were done in triplicate or quadruplicate, at 27°C for promastigotes and 34°C for amastigotes in 96-well plates or 384-well plates. DMSO vehicle and AmB were used as controls. Two days later, resazurin was added (10 μL per well for a final concentration of 5 µg/ml) and resorufin-derived fluorescence intensity was measured 24 h later using a Tecan Safire 2 reader (excitation 558 ± 4.5 nm; emission 585 ± 10 nm).

Following background subtraction (complete parasite culture medium with resazurin in absence of parasites), data were expressed as percentages of growth compared to DMSO-treated controls (POC).

RNA extraction

Large RNA above 200 nucleotides were isolated from macrophage samples using the NucleoSpin miRNA kit (Macherey-Nagel) according to the manufacturer's instructions.

Evaluation of RNA quality was carried out by optical density measurement using a Nanodrop device (Kisker, http://www.kisker-biotech.com). RNAs were isolated from 3 independent biological replicates of uninfected and Leishmania-infected macrophages treated or not with LSD1i MC4030.

RNA-Seq analysis

Library preparation and sequencing was performed at the Biomics platform of Institut Pasteur.

Briefly, DNAse-treated RNA extracts were processed for library preparation using the Truseq Stranded mRNA sample preparation kit (Illumina, San Diego, California) according to the manufacturer's instructions. An initial poly (A)+ RNA isolation step (included in the Illumina protocol) was performed with 1 µg of large RNA to isolate the mRNA fraction and remove ribosomal RNA. The mRNA-enriched fraction was fragmented by divalent ions at high temperatures. The fragmented samples were randomly primed for reverse transcription followed by second-strand synthesis to create double-stranded cDNA fragments. No end repair step was necessary. An adenine was added to the 3'-end and specific Illumina adapters were ligated. Ligation products were submitted to PCR amplification. The quality of the obtained libraries was controlled using a Bioanalyzer DNA1000 Chips (Agilent, # 5067-1504) and quantified by spectrofluorimetric analysis (Quant-iT™ High-Sensitivity DNA Assay Kit, #Q33120, Invitrogen).

Sequencing was performed on the Illumina Hiseq2500 platform to generate single-end 65 bp reads bearing strand specificity. Reads were cleaned using cutadapt version 1.11 and only sequences at least 25 nt in length were considered for further analysis. STAR version 2.5.0a [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF], with default parameters, was used for alignment on the reference genome (GRCm38 from Ensembl database 94). Genes were counted using featureCounts version 1.4.6-p3 (Liao et al., 2014) from the Subreads package (parameters: -t gene -s 0). Transcriptomic data are made publicly available at the NCBI's Gene Expression Omnibus repository (Superseries ongoing submission).

LSD1 target genes

A list of LSD1 target genes was compiled from bibliographic analyses and presented in Table S3. Target genes were evidenced after LSD1 pharmacological inhibition [START_REF] Augert | Targeting NOTCH activation in small cell lung cancer through LSD1 inhibition[END_REF][START_REF] Barth | LSD1 inhibition by tranylcypromine derivatives interferes with GFI1mediated repression of PU.1 target genes and induces differentiation in AML[END_REF][START_REF] Egolf | LSD1 Inhibition Promotes Epithelial Differentiation through Derepression of Fate-Determining Transcription Factors[END_REF][START_REF] Hoshino | Genome-wide ChIP-seq data with a transcriptome analysis reveals the groups of genes regulated by histone demethylase LSD1 inhibition in esophageal squamous cell carcinoma cells[END_REF][START_REF] Maiques-Diaz | Enhancer Activation by Pharmacologic Displacement of LSD1 from GFI1 Induces Differentiation in Acute Myeloid Leukemia[END_REF], LSD1 Knocking Out [START_REF] Barth | LSD1 inhibition by tranylcypromine derivatives interferes with GFI1mediated repression of PU.1 target genes and induces differentiation in AML[END_REF][START_REF] Jin | Loss of LSD1 (lysine-specific demethylase 1) suppresses growth and alters gene expression of human colon cancer cells in a p53-and DNMT1(DNA methyltransferase 1)-independent manner[END_REF], and siRNA LSD1 knockdown [START_REF] Chen | Identification of downstream metastasisassociated target genes regulated by LSD1 in colon cancer cells[END_REF][START_REF] Jin | LSD1 collaborates with EZH2 to regulate expression of interferon-stimulated genes[END_REF] using transcriptomic [START_REF] Augert | Targeting NOTCH activation in small cell lung cancer through LSD1 inhibition[END_REF][START_REF] Barth | LSD1 inhibition by tranylcypromine derivatives interferes with GFI1mediated repression of PU.1 target genes and induces differentiation in AML[END_REF][START_REF] Chen | Identification of downstream metastasisassociated target genes regulated by LSD1 in colon cancer cells[END_REF][START_REF] Egolf | LSD1 Inhibition Promotes Epithelial Differentiation through Derepression of Fate-Determining Transcription Factors[END_REF][START_REF] Hoshino | Genome-wide ChIP-seq data with a transcriptome analysis reveals the groups of genes regulated by histone demethylase LSD1 inhibition in esophageal squamous cell carcinoma cells[END_REF][START_REF] Jin | Loss of LSD1 (lysine-specific demethylase 1) suppresses growth and alters gene expression of human colon cancer cells in a p53-and DNMT1(DNA methyltransferase 1)-independent manner[END_REF], proteomic [START_REF] Jin | LSD1 collaborates with EZH2 to regulate expression of interferon-stimulated genes[END_REF] and Chip Seq [START_REF] Augert | Targeting NOTCH activation in small cell lung cancer through LSD1 inhibition[END_REF][START_REF] Chen | Identification of downstream metastasisassociated target genes regulated by LSD1 in colon cancer cells[END_REF][START_REF] Egolf | LSD1 Inhibition Promotes Epithelial Differentiation through Derepression of Fate-Determining Transcription Factors[END_REF][START_REF] Jin | Loss of LSD1 (lysine-specific demethylase 1) suppresses growth and alters gene expression of human colon cancer cells in a p53-and DNMT1(DNA methyltransferase 1)-independent manner[END_REF][START_REF] Jin | LSD1 collaborates with EZH2 to regulate expression of interferon-stimulated genes[END_REF][START_REF] Maiques-Diaz | Enhancer Activation by Pharmacologic Displacement of LSD1 from GFI1 Induces Differentiation in Acute Myeloid Leukemia[END_REF] analyses.

Gene ontology analysis

Ensembl gene identifiers were translated into ENTREZ gene identifiers prior to the enrichment analysis. ENTREZ genes linked to several Ensembl IDs were associated with the one having the highest expression level. Functional gene-set enrichment analysis was performed using the Fisher statistical test for the over-representation of differentially expressed genes (gene-level adjusted P-value lower than 5% For the of analysis epigenetic regulatory factors, the differential expression values, expressed as log2 fold changes (log2 FC), were computed for each gene, and were visually depicted as a network using the Cytoscape software package (version 3.10.0). Each node within this network projects the log2FC information. Protein-protein interactions were extracted from the STRING Database (version 5.0) (https://string-db.org/). These interactions were filtered based on confidence scores, with a minimum interaction score of 0.700, thus generating an interaction network grounded in known or predicted associations among these genes. Gene Ontology (GO) terms we also incorporated for each network node, facilitating functional annotations pertaining to biological processes, molecular functions, and cellular components.

LC-MS/MS Analysis

Online chromatography was performed with an RSLCnano system (Ultimate 3000, Thermo Scientific) coupled to an Orbitrap Eclipse mass spectrometer (Thermo Scientific

Proteomics data processing

For identification, the data were searched against the Mus musculus (UP000000589_10090)

and the Leishmania amazonensis MHOMBR71973M2269 in the TriTrypDB (release 60) databases using Sequest HT through proteome discoverer (version 2.4). Enzyme specificity was set to trypsin and a maximum of two miss cleavages sites were allowed. Oxidized methionine, Met-loss, Met-loss-Acetyl and N-terminal acetylation were set as variable modifications.

Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.6

Da for MS/MS peaks. The resulting files were further processed using myProMS v3.10.0 (https://github.com/bioinfo-pf-curie/myproms) [START_REF] Poullet | myProMS, a web server for management and validation of mass spectrometry-based proteomic data[END_REF]. FDR calculation used

Percolator (The et al., 2016) and was set to 1% at the peptide level for the whole study. The label free quantification was performed by peptide Extracted Ion Chromatograms (XICs), reextracted across all conditions and computed with MassChroQ version 2.2.21 [START_REF] Valot | MassChroQ: a versatile tool for mass spectrometry quantification[END_REF]. For protein quantification, XICs from proteotypic and non-proteotypic peptides were used by assigning to the best protein, peptides shared by multiple match groups, and missed cleavages, charge states and sources were allowed. Label-free quantification (LFQ) was performed following the algorithm as described [START_REF] Cox | Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ[END_REF].

The final LFQ intensities were used as protein abundance and further analyzed using Prostar v1.32.1 following the guidelines outlined in the software manual [START_REF] Tardif | Statistical Analysis of Quantitative Peptidomics and Peptide-Level Proteomics Data with Prostar[END_REF]. Briefly, we addressed missing data in all conditions by first removing Partially Observed Values (POV)

and Missing in Entire Condition (MEC). Subsequently, we applied mean centering to normalize the filtered data. For the imputation of POV and MEC values, we employed the Structured Least Square Adaptive (SLSA) regression and the 2.5% quantile algorithm. Finally, we conducted a differential analysis using the Welch test.

RESULTS

L. amazonensis subverts the macrophage epigenetic landscape during infection.

We apply a multidisciplinary approach to (i) identify potential epigenetic pathways affected by Leishmania infection, (ii) reveal their relevance for intracellular parasite survival, and (iii)

assess the mechanisms underlying their subversion. We first conducted an RNA-Seq analysis of Bone Marrow-Derived Macrophages (BMDMs) infected or not by Leishmania amazonensis virulent amastigotes (day 2 PI) (Figure 1A), which revealed significant expression changes for 5742 genes (Figure S1A, adjusted p value < 0.05) affecting the macrophage innate immune response-including antigen presentation and toll receptor signaling cascade-as well as metabolic processes such as cholesterol biosynthesis (Figure S2). To gain insight into epigenetic subversion strategies, we more specifically investigated the impact of infection on the expression of macrophage Epigenetic Regulatory Factors (EpiRFs), including histone / DNA modifying enzymes, associated transcription factors, and members of epigenetic complexes. From 787 EpiRFs obtained from manual curation of publicly available databases including EpiFactors (http://epifactors.autosome.org., Table S1), 315 showed significant changes in transcript abundance during infection, with increased expression observed for 154 (e.g. Dnmt, FC = +1.39; Dot1l, FC = +2.78), and decreased expression affecting 161 (e.g. Sirt3, FC = -1.46; Ehmt,: FC = -1.38) (Figure 1B). Overall, 39.7 % of EpiRFs were modulated by the parasite, revealing epigenetic regulation as a key target for parasite subversion of host cell immune-metabolomic functions.

Pharmacological interference with macrophage epigenetic regulation reduces burden of intracellular L. amazonensis.

We next used a pharmacological approach to assess the relevance of this epigenetic subversion strategy for intracellular L. amazonensis survival. We assessed 425 epigenetic inhibitors provided by the FP7 A-PARADDISE project, which were designed to target various classes of mammalian epigenetic regulators, including sirtuins, Histone Acetyl Transferases (HATs), Histone Deacetylases (HDACs), Histone Methyl Transferases (HMT), Histone DeMethylases (HDMs) and DNA Methyl Transferases (DNMTs) (see https://cordis.europa.eu/project/id/602080). We screened this library by monitoring the intracellular amastigote survival in L. amazonensis-infected BMDMs using a high content assay, and extracellular parasite survival using a resazurin-based viability assay applied on promastigotes in culture [START_REF] Lamotte | Discovery of novel hit compounds with broad activity against visceral and cutaneous Leishmania species by comparative phenotypic screening[END_REF]. This dual strategy allowed us to distinguish anti-leishmanial compounds that affect parasite viability directly (killing both extracellular and intracellular parasites) from compounds that affect parasite viability indirectly via hostdependent mechanisms (killing exclusively intracellular parasites) (Lower right quadrant, figure 2A).

Of particular interest were the 18 compounds that caused significant decrease in intracellular but not extracellular parasite survival. Such putative host-dependent mode of action has been observed for different classes of targets including HMTs, HDMTs and DNMTs, suggesting that interference with macrophage epigenetic regulation both at DNA and histone levels can interfere with intracellular parasite development or trigger parasite death. Most effective were a series of inhibitors directed against the Lysine-Specific Demethylase 1 (LSD1, a.k.a.

KDM1A). This enzyme demethylates histone H3 at K4 and K9 residues, which has been linked

to macrophage polarization and the transcription of pro-inflammatory cytokines [START_REF] Janzer | Lysine-specific demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1) synergistically repress proinflammatory cytokines and classical complement pathway components[END_REF][START_REF] Tan | Lysine-Specific Histone Demethylase 1A Regulates Macrophage Polarization and Checkpoint Molecules in the Tumor Microenvironment of Triple-Negative Breast Cancer[END_REF]. The anti-leishmanial effect of these LSD1 inhibitors resonates well with our previous data that correlated L. amazonensis infection with macrophage H3K4 and H3K9 hypomethylation, suggesting that this demethylase may be key for parasite-mediated, epigenetic host cell subversion.

We next focused our attention on two derivatives of the canonical flavoenzyme monoamine oxidase A inhibitor Tranylcypromine (TCP), i.e. the compounds MC2652 and MC4030. These inhibitors were synthesized by replacing the 3-phenyl ring of TCP with distinct heterocyclic thiophene moieties, and display an increased LSD1 inhibitory effect in cancer cell lines [START_REF] Fioravanti | Heterocycle-containing tranylcypromine derivatives endowed with high anti-LSD1 activity[END_REF]. While TCP did not affect intracellular parasite survival (data not shown), both MC2652 and MC4030 strongly reduced burden of both intracellular L. amazonensis and L. donovani as judged by epifluorescence microscopy (Figures 2B andS3).

We next documented the effects of these TCP derivatives on murine macrophage LSD1, first evaluating their inhibitory potential on the enzymatic activity of LSD1 in BMDM nuclear extracts. Both inhibitors demonstrated a more effective inhibition of H3K4me2 demethylation compared to TCP (Figure 2C). To determine in cellula whether this inhibition could be attributed to target engagement -i.e. direct binding to LSD1, we performed a Cellular Thermal Shift Assay (CETSA). The binding of the LSD1i to the demethylase was confirmed by the LSD1 thermal stabilization in the presence of the inhibitors (Figure 2D and 2E and Figure S4).

Importantly, this interaction was specific to LSD1, as no thermal shift was observed for MAO-A or GAPDH in either uninfected (Figure 2D, and 2E) or L. amazonensis-infected BMDMs (Figure 2D1 and 2D2,and Figure S4A and S4B).

L. amazonensis affects LSD1 complex formation.

Surprisingly, even though our screening data suggest an important role of LSD1 in supporting intracellular Leishmania survival, infection itself neither modulated LSD1 protein levels during the first three days of infection (Western blot analysis, Figure 3A), nor affect LSD1 nuclear localization (immunofluorescence analysis, Figure 3B).

Since LSD1 serves as an important scaffolding protein and its activity relies on its interaction with key partners in regulatory complexes [START_REF] Burg | KDM1 class flavin-dependent protein lysine demethylases[END_REF], we next analyzed expression changes of 90 known LSD1-associated proteins during L. amazonensis infection mining our RNA-Seq data (Figure 3C). While no significant transcriptional modulation was observed for LSD1 itself, 35 of the LSD1-associated proteins (35.4%) were modulated (Figure 3C), [START_REF] Antoine | The biogenesis and properties of the parasitophorous vacuoles that harbour Leishmania in murine macrophages[END_REF]. Changes in transcript abundance of known LSD1 interaction partners subverted the composition of the main LSD1 complex (Table 1).

Using CETSA we observed an important shift in LSD1 thermal stability in infected compared to uninfected BMDMs, providing first, indirect evidence that LSD1 complexes undergo infection-dependent changes (Figures 4A and2E). We next performed a coimmunoprecipitation (co-IP) assay using nuclear extracts from uninfected and infected BMDMs to directly assess changes in LSD1 complex formation at day 2 post-infection. We employed two previously described, custom-made antibodies (termed Ab1 and Ab2, Biogenes) [START_REF] Duteil | Lsd1 Ablation Triggers Metabolic Reprogramming of Brown Adipose Tissue[END_REF]. Notably, the antibody targeting the C-terminus of LSD1 (Ab1) exhibited a higher efficacy in precipitating LSD1 interactors (Figure S5) and was therefore considered for further assessment.

In comparison to co-IPs performed with non-specific IgGs, we identified a total of 489 and 330 proteins as LSD1-specific interaction partners in uninfected compared to infected BMDMs. Of these, 259 were common to both conditions (Figure S5), including well-known LSD1 interactors, such as RCOR1, ARID1A, POLR2A (Figure 4B). Notably, 230 LSD1 interactors were exclusively identified in uninfected BMDMs, while 71 were unique to infected BMDMs.

Surprisingly, it was found that 24 out of these 71 interaction partners corresponded to Leishmania proteins (Table 2). Whether this is the result of specific protein transfer or parasite contaminating material remains to be established. STRING analysis performed on the remaining 47 macrophage proteins identified as unique LSD1 partners in infected BMDMs (Figure 4C) revealed notably three interaction networks that were enriched in GO terms linked to (i) ribosomal biogenesis, (ii) mRNA processing, binding, and splicing, and (iii) nucleosomal DNA binding and linker histones H1/H5 (Figure 4C). Collectively, these findings demonstrate that Leishmania infection has profound consequences on the formation of LSD1 complexes, which affects the protein's thermal stability and potentially down-stream regulated targets relevant for epigenetic, post-transcriptional and translational regulatory processes.

LSD1 inhibition counteracts parasite-driven expression changes in LIMs.

In order to elucidate host cell pathways that may govern the host-directed, anti-leishmanial effect of compound MC4030, we analyzed the impact of drug treatment on uninfected and infected BMDMs. As noted above, L. amazonensis infection alone had a profound effect on the macrophage transcriptome (see Figures 1B andS1), affecting many cellular pathways, including protein kinase activity, sterol biosynthesis, and responses to lipopolysaccharide, hypoxia, and oxidative stress (Figure S2). Among the 5742 modulated transcripts, 1101 (19.17%) were previously reported to be regulated by LSD1 (Figure 5A1), which further supports our hypothesis that Leishmania targets LSD1 to establish permissive conditions for intracellular macrophage infection.

MC4030 treatment of uninfected BMDMs resulted in expression changes of 1097 genes, 287 of which are regulated by LSD1 (26.1%) (Figure 5A2). These results further support the specificity of MC4030 to target macrophage LSD1 and sustains a host-directed mechanism for its anti-leishmanial activity. Finally, MC4030 treatment of infected BMDMs affected the abundance of 456 transcripts, 117 (25.7%) of which are regulated by LSD1 (Figure 5A3).

MC4030 treatment seems to counteract the infection-induced phenotype as judged by PCA analysis of the three experimental groups, which shows a shorter distance between MC4030treated infected and uninfected BMDMs compared to the distance between infected and uninfected BMDMs in absence of the inhibitor (Figure 5B). This is further confirmed by the double ratio plot shown in Figure 5C, providing direct evidence that 95% of expression changes caused by the treatment do counteract infection-dependent expression changes observed in infected BMDMs. For example, many genes implicated in the macrophage response to lipopolysaccharide and hypoxia showed reduced expression during infection, which was restored after drug treatment (Figure 5D1 and D2). Conversely, the increased expression of genes implicated in signal transduction and steroid biosynthesis observed in infected BMDMs was significantly reduced upon treatment (Figure 5D3 and D4). Importantly, this reversion was not just the consequence of parasite elimination but a direct effect of LSD1 inhibition given that the treatment had only a slight effect on parasite burden at the time point chosen for the RNA-Seq analysis (i.e. 48h of treatment).

In conclusion, our data provide evidence that MC4030 kills intracellular Leishmania by restoring a macrophage expression profile that interferes with the parasite survival by rescuing the host cell immune response and limiting the parasite's access to nutrients essential for its proliferation.

DISCUSSION

Leishmania infection of macrophages is associated with significant changes in gene expression that contribute to the creation of a favorable environment for parasite survival and replication within host cells. Here we provide pharmacological evidence that Leishmania exploits the host demethylase LSD1, raising questions on how Leishmania engages with LSD1, how this engagement changes the host cell phenotype, and how interfering with this engagement leads to parasite killing.

Leishmania can engage with LSD1 to modulate one or both of its biological functions, i.e. its enzymatic activity and / or its scaffolding properties. The absence of LSD1 modulation with respect to transcript abundance, protein abundance or subcellular localization levels suggested that LSD1 subversion relies on other mechanisms independent of its expression, such as complex formation. This hypothesis was confirmed by CETSA analyses that provided first evidence that the parasite increases LSD1 thermal stability. This increase might reflect changes in the nature of LSD1 host complexes, or new interactions with one or more of the Leishmania proteins we co-purified with nuclear LSD1, which opens the exiting possibility that Leishmania similar to pathogenic bacteria (Bierne and Cossart, 2012)-may have co-evolved 'nucleomodulins" similar that can directly engage with nuclear host cells components to subvert their functions.

Infection-dependent LSD1 interactions directly inform on possible mechanisms of immune subversion. For example, LSD1 specifically interacts with the heterogeneous nuclear ribonucleoprotein K (HnRNPK), a non-histone target of LSD1 (Natarajan et al., 2022) that been previously implicated in the stabilization of the mRNA of CXCL10, a chemokine associated with resistance to Leishmania infection [START_REF] Antonia | Pathogen Evasion of Chemokine Response Through Suppression of CXCL10[END_REF]. Likewise, other infectionspecific LSD1 interactions may favor parasite survival, such as the splicing factor proline-and glutamine-rich (SFPQ), the ATP-dependent RNA helicase A (DHX9), and Matrin 3 (MATR3), and the non-POU domain-containing octamer-binding protein (NONO). These molecules are respectively involved in i) viral RNA amplification [START_REF] Labeau | Characterization and functional interrogation of the SARS-CoV-2 RNA interactome[END_REF], ii) nuclear retention of RNAs that are defective or immature (Zhang and Carmichael, 2001), iii) DNA nonhomologous end joining (NHEJ) recombination required for double-strand break repair and V(D)J recombination [START_REF] Salton | Involvement of Matrin 3 and SFPQ/NONO in the DNA damage response[END_REF] and iv) modulation of transcription and translation processes [START_REF] Li | The hnRNPK/A1/R/U Complex Regulates Gene Transcription and Translation and is a Favorable Prognostic Biomarker for Human Colorectal Adenocarcinoma[END_REF]. LSD1 interaction partners within infected macrophages were also enriched in ribosomal proteins, whose methylation state may be affected by LSD1 [START_REF] Hamidi | Identification of Rpl29 as a major substrate of the lysine methyltransferase Set7/9[END_REF]. Altogether, these infection-specific changes in LSD1 interactions may significantly remodel the transcriptomic landscape of the host macrophage for the benefit of intracellular Leishmania survival via inhibition of immune-related gene expression (e.g. down regulation of pro-inflammatory response) or mobilizing of nutrients required for parasite growth (e.g. increase of the biosynthetic cholesterol pathway). Indeed, reversion of these expression changes in presence of the LSD1 inhibitor likely resorted the host cells innate defense mechanisms [START_REF] Tan | Lysine-Specific Histone Demethylase 1A Regulates Macrophage Polarization and Checkpoint Molecules in the Tumor Microenvironment of Triple-Negative Breast Cancer[END_REF] and limited cholesterol availability and its master regulator, SREBP2 [START_REF] Horton | Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2[END_REF], both of which translated into a host-directed, anti-leishmanial activity, confirming that these pathways might be essential for parasite survival [START_REF] Mukherjee | Leishmania donovani activates SREBP2 to modulate macrophage membrane cholesterol and mitochondrial oxidants for establishment of infection[END_REF].

In conclusion, our results shed new light on a Leishmania-specific epigenetic subversion strategy that targets the LSD1 scaffolding function of the host cell to change transcriptional complexes and establish an infection-permissive phenotype. This strategy was demonstrated in acute myeloid leukemia where the interaction between LSD1 and RCOR1 with the SNAGdomain transcription repressor GFI1 can be dispersed by TCP-derived inhibitors allowing myeloid differentiation (Maiques-Diaz, 2018). The identification of two LSD1-specific inhibitors with host-directed, anti-leishmanial activity pharmacologically validates host cell epigenetic regulation and downstream-regulated pathways as a fertile ground for the discovery of macrophage drug targets that impact parasite survival, which may be broadly applicable to other intracellular pathogens known to target host cell epigenetic regulation for their survival, such as Mycobacterium tuberculosis [START_REF] Pennini | Mycobacterium tuberculosis 19-kDa lipoprotein inhibits IFN-gamma-induced chromatin remodeling of MHC2TA by TLR2 and MAPK signaling[END_REF], Listeria monocytogenes (Hamon and Cossart, 2008) or Toxoplasma gondii [START_REF] Schneider | Toxoplasma gondii triggers phosphorylation and nuclear translocation of dendritic cell STAT1 while simultaneously blocking IFNgamma-induced STAT1 transcriptional activity[END_REF]. Nonspecific binding events were revealed by performing co-IP experiments with rabbit IgGs (grey dots). Only statistically significant interactions (cut-off p < 0.01) were retained (Prostar software tool).

FIGURE LEGENDS

(C) Gene ontology enrichment analysis of STRING networks identified between LSD1 interaction partners in LIMs using the Cytoscape V3.10.0 software package.

FIGURE 1 .

 1 FIGURE 1. GLOBAL PREVALENCE OF CUTANEOUS LEISHMANIASIS IN 2020 ................................................ FIGURE 2. PATIENTS WITH CUTANEOUS LEISHMANIASIS. .............................................................................. FIGURE 3. GLOBAL PREVALENCE OF VISCERAL LEISHMANIASIS IN 2020. .................................................... FIGURE 4. PATIENTS WITH VISCERAL AND POST KALA-AZAR DERMAL LEISHMANIASIS. ............................. FIGURE 5. PROMASTIGOTE AND AMASTIGOTE FORMS AND THEIR KEY CELLULAR STRUCTURES ............. FIGURE 6. LEISHMANIA LIFE CYCLE INSIDE THE SANDFLY VECTOR AND THE VERTEBRATE HOST ............. FIGURE 7. LUTZOMYIA LONGIGALPIS AND PHLEBOTOMUS PAPATASI ........................................................ FIGURE 8. CHEMICAL STRUCTURE OF KEY DRUGS USED AS THERAPEUTIC OPTIONS AGAINST LEISHMANIASIS...................................................................................................................................... FIGURE 9. HOST REGULATORY PATHWAYS TARGETED BY PARASITIC INFECTIONS ................................... FIGURE 10. TARGETING HOST CELL EPIGENOME AS A NOVEL APPROACH FOR ANTILEISHMANIAL DRUG DISCOVERY ............................................................................................................................................ FIGURE 11. OVERVIEW OF CHROMOSOME STRUCTURE AND POTENTIAL EPIGENETIC MODIFICATIONS.30 FIGURE 12. HISTONE MODIFYING ENZYMES ................................................................................................ FIGURE 13. READERS OF HISTONE H3 TAIL PTMS. ................................................................................... FIGURE 14. MAIN LOCATIONS OF LYSINE METHYLATION ON HISTONES H3 AND H4 ........................... FIGURE 15. CHEMICAL MECHANISM OF LYSINE DEMETHYLATION BY THE LSD FAMILY .......................... FIGURE 16. COMPARISON OF THE DOMAIN STRUCTURES OF HUMAN LSD1 AND LSD2 ...................... FIGURE 17. LSD1 DUAL FUNCTIONS AS TRANSCRIPTIONAL REPRESSOR AND ACTIVATOR.....................FIGURE 18. LSD1 INTERACTIONS WITH NONHISTONE PROTEINS ............................................................. FIGURE 19. LSD1 POST-TRANSLATIONAL MODIFICATIONS AND THEIR IMPACT ON BIOCHEMICAL AND BIOLOGICAL PROCESSES ....................................................................................................................... FIGURE 20. SCHEMATIC OVERVIEW OF THE EXPERIMENTAL DESIGN. ........................................................ FIGURE 21. STRUCTURE OF RESAZURIN SUBSTRATE AND THE PINK FLUORESCENT RESORUFIN PRODUCT ................................................................................................................................................................ FIGURE 22. PLATE DESIGN FOR THE RESAZURIN TEST ................................................................................. FIGURE 23. EXPERIMENTAL PROCEDURE OF CELLULAR THERMAL SHIFT ASSAY ..................................... FIGURE 24. EXPERIMENTAL PROCEDURE FOR NUCLEAR PROTEIN EXTRACTION ....................................... FIGURE 25. ANTIBODY-BEADS COMPLEX FORMATION AND CROSS-LINKING ......................................... FIGURE 26. EXPERIMENTAL PROCEDURE FOR NUCLEAR PROTEIN SAMPLE PRE-CLEARING AND IMMUNOPRECIPITATION. ..................................................................................................................... FIGURE 27. OVERVIEW OF THE CO-IP WORKFLOW AND PROTEIN DETECTION ....................................... FIGURE 28. EXPERIMENTAL PROCEDURE FOR CHROMATIN EXTRACTION ................................................. FIGURE 29. EXPERIMENTAL PROCEDURE FOR CHROMATIN IMMUNOPRECIPITATION. ............................. FIGURE 30. WORKFLOW FOR THE DSDNA HS ASSAY KIT USING THE QUBIT® 2.0 FLUOROMETER... FIGURE 31. EXAMPLE OF AN AMPLIFICATION PLOT OF QPCR TO DETERMINE THE NUMBER OF CYCLES FOR LIBRARY AMPLIFICATION .............................................................................................................. FIGURE 32. WORKFLOW FOR PCR PURIFICATION WITH AMPURE XP BEADS ........................................ FIGURE 33. DUAL SCREENING ASSAY OF AN EPIGENETIC INHIBITOR LIBRARY .......................................... FIGURE 34. CHEMICAL STRUCTURE OF HOST-DIRECTED TCP-BASED LSD1 INHIBITORS (LSD1I) ........ FIGURE 35. ANALYSIS OF TRANSCRIPTIONAL MODULATION INDUCED IN BMDMS INFECTED BY L. AMAZONENSIS IN THE PRESENCE OR NOT OF MC4030 .................................................................. FIGURE 36. ANALYSIS OF RNASEQ DATA BY PRINCIPAL COMPONENT ANALYSIS ...................................

  FIGURE 37. COMPARATIVE ANALYSIS OF DEGS IN L. AMAZONENSIS-INFECTED BMDMS TREATED WITH MC4030............................................................................................................................................... FIGURE 38. REVERSION OF LEISHMANIA-INDUCED MODULATED PATHWAYS BY MC4030 ................... FIGURE 39. IMPACT OF LSD1I ON PARASITE LOAD IN L. AMAZONENSIS-INFECTED BMDMS AND RAW 267.4..................................................................................................................................................... FIGURE 40. IMPACT OF LSD1I ON PARASITE LOAD IN L. DONOVANI-INFECTED BMDMS. ................ FIGURE 41. IMPACT OF LSD1I ON PARASITE LOAD ON LIVING L. AMAZONENSIS-INFECTED BMDMS. .............................................................................................................................................................. FIGURE 42. IMPACT OF LSD1I ON PARASITE LOAD IN LIVING L. DONOVANI-INFECTED BMDMS. .... FIGURE 43. LSD1I IC50 VALUES CALCULATED FROM RESAZURIN ASSAYS ............................................ FIGURE 44. STANDARD CURVE OF DEMETHYLATED LYSINE 4 OF HISTONE H3 ..................................... FIGURE 45. IN VITRO ANALYSIS OF LSD1 ACTIVITY IN THE PRESENCE OF LSD1I AND TCP ................ FIGURE 46. THERMAL SHIFT ASSAY PRINCIPLE ......................................................................................... FIGURE 47. LINEARITY OF LSD1 BAND SIGNAL BY WB ANALYSIS WITH FLUORESCENCE DETECTION . FIGURE 48. CETSA ANALYSIS TO ASSESS SELECTIVE LSD1 TARGET ENGAGEMENT IN PROTEIN LYSATES OF UNINFECTED BMDMS ................................................................................................................. FIGURE 49. CETSA ANALYSIS TO ASSESS SELECTIVE LSD1 TARGET ENGAGEMENT IN PROTEIN LYSATES FROM INFECTED BMDMS ................................................................................................................. FIGURE 50. LSD1 IMMUNOFLUORESCENCE MICROSCOPY ON FIXED UNINFECTED AND INFECTED BMDMS ............................................................................................................................................. FIGURE 51. NUCLEI ISOLATED FROM BOTH UNINFECTED AND INFECTED BMDMS EXHIBITING REMNANTS OF CYTOPLASMIC CONTENTS ........................................................................................ FIGURE 52. UNINFECTED, INFECTED BMDMS AND ISOLATED NUCLEI .................................................. FIGURE 53. WESTERN BLOT ANALYSIS OF CANDIDATE NUCLEAR REFERENCE PROTEINS. ...................... FIGURE 54. LSD1, YY1 AND GAPDH WESTERN BLOTS ........................................................................ FIGURE 55. REPRESENTATIVE WESTERN BLOTS OF GAPDH AND YY1.................................................. FIGURE 56. EXAMPLE OF COOMASSIE STAINED SDS-PAGE GEL WITH NUCLEAR AND CYTOPLASMIC EXTRACTS ............................................................................................................................................. FIGURE 57. QUANTIFICATION OF LSD1 PROTEIN IN NUCLEAR EXTRACTS ............................................. FIGURE 58. CETSA ANALYSIS FOR LSD1 TARGET ENGAGEMENT IN PROTEIN LYSATES FROM UNINFECTED AND INFECTED BMDMS ............................................................................................. FIGURE 59. NUCLEI ISOLATION AFTER PFA FIXATION .............................................................................. FIGURE 60. NUCLEI ISOLATION PROCEDURE FOR CO-IP ASSAY ............................................................. FIGURE 61. SYPRO RUBY STAINED SDS-PAGE GEL AND WB FROM EXPERIMENT 6 ......................... FIGURE 62. SYPRO RUBY STAINED SDS-PAGE GEL AND WB FROM EXPERIMENT 7 ......................... FIGURE 63. VENN DIAGRAMS DEPICTING THE OVERLAP BETWEEN IMMUNOPRECIPITATED PROTEINS WITH SPECIFIC Α-LSD1 ANTIBODIES AND THE IGG CONTROL ...................................................... FIGURE 64. SYPRO RUBY STAINED SDS-PAGE GEL AND WB FROM EXPERIMENT 10....................... FIGURE 65. VENN DIAGRAMS COMPARING THE LSD1 INTERACTING PROTEINS PRECIPITATED WITH Α-LSD1 SPECIFIC ANTIBODIES .............................................................................................................. FIGURE 66. VENN DIAGRAMS COMPARING THE LSD1 INTERACTING PROTEINS BETWEEN UNINFECTED AND INFECTED CONDITIONS .............................................................................................................. FIGURE 67. VOLCANO PLOTS DISPLAYING SPECIFIC LSD1 INTERACTORS IN UNINFECTED AND INFECTED CONDITIONS ........................................................................................................................................ FIGURE 68. PROTEIN CLUSTERS OF MACROPHAGE LSD1 PROTEIN INTERACTORS UNIQUE IN INFECTED BMDMS .............................................................................................................................................

FIGURE 85 .

 85 FIGURE 69. COMPARISON BETWEEN STANDARD CHIP-SEQ AND CHIPMENTATION............................ FIGURE 70. WESTERN BLOT TO ANALYZE ANTIBODY SPECIFICITY AND PROTEIN INTEGRITY ................. FIGURE 71. DNA SIZE DURING SONICATION TIME COURSE .................................................................... FIGURE 72. CHIP-WESTERN BLOT ANALYSIS DEMONSTRATING THE CAPACITY OF AB1 TO IMMUNOPRECIPITATE LSD1 FROM CHROMATIN SAMPLES OF UNINFECTED BMDMS ............... FIGURE 73. WESTERN BLOT TO ANALYZE THE ANTIBODY SPECIFICITY AND PROTEIN INTEGRITY IN CHROMATIN BIOLOGICAL REPLICATES .............................................................................................. FIGURE 74. AGAROSE GEL ELECTROPHORESIS OF PURIFIED FRAGMENTED DNA .................................. FIGURE 75. WESTERN BLOT ANALYSIS TO DETECT LSD1 IMMUNOPRECIPITATED CHROMATIN OF UNINFECTED AND INFECTED SAMPLES FROM REPLICATE 3 ............................................................ FIGURE 76. DIAGRAM ILLUSTRATING THE TAGMENTATION PROCEDURE ON BEADS ............................. FIGURE 77. SCHEMATIC VIEW OF DNA PURIFICATION AND LIBRARY PREPARATION FROM IMMUNOPRECIPITATED SAMPLES. ..................................................................................................... FIGURE 78. QPCR AMPLIFICATION PLOT USING VARIOUS AMOUNTS OF CHROMATIN INPUT SAMPLES FROM UNINFECTED AND INFECTED BMDMS .................................................................................. FIGURE 79. QUALITY ANALYSIS OF PURIFIED INPUT LIBRARIES ................................................................ FIGURE 80. QPCR AMPLIFICATION PLOT OF IMMUNOPRECIPITATED SAMPLES ..................................... FIGURE 81. QUALITY ANALYSIS OF PURIFIED IMMUNOPRECIPITATED LIBRARIES .................................... FIGURE 82. COOMASSIE-STAINED SDS-PAGE GEL OF MACROPHAGE NUCLEAR EXTRACTS. .............. FIGURE 83. ASSESSMENT OF NUCLEAR EXTRACTS QUALITY FOR MASS SPECTROMETRY ANALYSIS. COOMASSIE-STAINED SDS-PAGE GEL OF THE NUCLEAR EXTRACTS ........................................... FIGURE 84. VOLCANO PLOT OF NUCLEAR PROTEINS IN INFECTED VS. UNINFECTED BMDMS ............ FIGURE 85. GO ANALYSIS IN CELLULAR COMPONENT OF NUCLEAR PROTEINS THAT SHOW INCREASED OR DECREASED ABUNDANCE FOLLOWING LEISHMANIA INFECTION .............................................. FIGURE 86. SCATTERPLOT REPRESENTING THE CLUSTERS OF PARASITE PROTEINS IDENTIFIED IN THE NUCLEAR PROTEOME ANALYSIS.........................................................................................................FIGURE 87. TARGETING MACROPHAGE LSD1 AS A NOVEL STRATEGY FOR ANTI-LEISHMANIAL HOST-DIRECTED DRUG DISCOVERY ..............................................................................................................

Figure 4 .

 4 Figure 4. Patients with visceral (A) and post kala-azar dermal (B) leishmaniasis [12].

  amastigotes and shows an elongated body, measuring 15-25 µm in length and 1-5 µm in width. It exhibits a long flagellum that extends externally from the anterior end, spanning the length of the body and providing the propulsive force required for movement in the sandfly alimentary tract.

Figure 5 .

 5 Figure 5. Promastigote (A) and amastigote (B) forms and their key cellular structures [24].

Figure 8 .

 8 Figure 8. Chemical structure of key drugs used as therapeutic options against leishmaniasis.

Figure 9 .

 9 Figure 9. Host regulatory pathways targeted by parasitic infections. Red connectors indicate inhibition, while green arrows represent activation [108].

Figure 10 .

 10 Figure 10. Targeting host cell epigenome as a novel approach for antileishmanial drug discovery. Created with BioRender.com.

Figure 11 .

 11 Figure 11. Overview of chromosome structure and potential epigenetic modifications. Histone octamers bind to DNA, compacting it into nucleosomes, which then form chromatin fibers and chromosomes. Chromatin is subjected to epigenetic regulations, including DNA methylation (me, yellow bubbles) and post-translational modifications of histones, including acetylation (ac), methylation (me), phosphorylation (P), and ubiquitination (ub). Long noncoding RNAs (lncRNAs) can also play a significant role in epigenetic regulation via interaction with epigenetic modifiers, or influencing transcription through their interaction with DNA [116].

  include bromodomain (BD), double PHD finger (DPF) and Yeats domain. Bromodomain proteins are the principal readers of acetylated lysine marks and are categorized into eight families based on their sequence and structural similarity [135]. Histone methylation readers include ATRX-DNMT3-DNMT3L (ADD), ankyrin, bromo-adjacent homology (BAH), chromobarrel, chromodomain (CD), double chromodomain (DCD), malignant brain tumor (MBT), plant homeodomain (PHD), Pro-Trp-Trp-Pro (PWWP), Tudor, tandem Tudor (TTD), WD40, and the zinc finger CW (zf-CW) domains (Figure 13) [136].

Figure 13 .

 13 Figure 13. Readers of Histone H3 tail PTMs. Recognition of the methylated (me) lysine (pink), methylated (me) arginine (purple), acetylated (ac) lysine (orange), and phosphorylated (ph) serine and threonine (blue) residues [137].

Figure 14 .

 14 Figure 14. Main locations of lysine methylation on Histones H3 and H4. Methyltransferases (writers) and demethylases (erasers) are also displayed, representing with one, double or triple circle their preferences for specific methylation states [141].

Figure 15 .

 15 Figure 15. Chemical mechanism of lysine demethylation by the LSD family [143].

Figure 16 .

 16 Figure 16. Comparison of the domain structures of human LSD1 and LSD2 (A). Ribbon structure of LSD1 in complex with CoREST and Histone H3 (B). Ribbon structure of LSD2 in complex with NPAC and Histone H3 (C) [149].

Figure 17 .

 17 Figure 17. LSD1 dual functions as transcriptional repressor (A) and activator (B) [164].

   by demethylating K391 residue, promoting tumor angiogenesis. Overall, LSD1 influences various cellular processes in different tissue environments, including cell proliferation, differentiation, stemness via βcatenin signaling, chemoresistance in cancer, epithelial-mesenchymal transition (EMT), DNA methylation leading to metastasis, cell motility, angiogenesis, and senescence (Figure18)[143,[165][166][167][168][169].

Figure 18 .

 18 Figure 18. LSD1 interactions with nonhistone proteins [143].

INTRODUCTION 45 Figure 19 .

 4519 Figure 19. LSD1 post-translational modifications and their impact on biochemical and biological processes [143].

Figure 20 .

 20 Figure 20. Schematic overview of the experimental design. Created with BioRender.com.

Figure 21 .

 21 Figure 21. Structure of resazurin substrate and the pink fluorescent resorufin product resulting from reduction in viable cells.

Figure 22 .

 22 Figure 22. Plate design for the resazurin test. The cytotoxic activity was tested in triplicate for the DMSO vehicle control, LSD1i, and TCP, resulting in the preparation of two plates for each condition. Each plate included its viability control: amphotericin B or miltefosine for the parasites or cycloheximide for BMDMs and RAW264.7 cells, along with a medium-only control or untreated cells as control.

Figure 23 .

 23 Figure 23. Experimental procedure of Cellular Thermal Shift Assay. Created with BioRender.com.

Figure 24 .

 24 Figure 24. Experimental procedure for nuclear protein extraction. Created with BioRender.com.

  Tris-HCl pH 8.0 -0.5 mM EGTA -100 mM NaCl -0.1% Sodium Deoxycholate Sonication was performed using the Diagenode's Bioruptor® Standard for 5

  C15410206). For immunoprecipitation, in the three different conditions, 3 µg of antibody (Ab) were incubated (Ab1, α-LSD1 20752; Ab2, α-LSD1 3544; and negative control). Then, three washes with 200 µl PBS-T, pH 7.4 were performed. Afterwards, Dynabeads-Ab complexes were washed with 250 µl of Conjugation Buffer and subsequently cross-linked by incubating for 30 min at RT with a 5 mM solution of Bissulfosuccinimidyl suberate (BS 3 ). The reaction was quenched by adding 12.5 µl of 1M Tris pH 7.4 for 15 min at RT. Cross-linked Dynabeads-Ab complexes were washed with 200 µl of Beads buffer three times (Figure 25).

Figure 25 .

 25 Figure 25. Antibody-Beads complex formation and cross-linking.

Figure 26 .

 26 Figure 26. Experimental procedure for nuclear protein sample pre-clearing and immunoprecipitation. Created with BioRender.com.

Figure 27 .

 27 Figure 27. Overview of the Co-IP workflow and protein detection/identification. Created with BioRender.com.

Figure 28 .

 28 Figure 28. Experimental procedure for chromatin extraction. Created with BioRender.com.

Figure 29 .

 29 Figure 29. Experimental procedure for chromatin immunoprecipitation. Created with BioRender.com.

  DNA was carried using the iQuant™ dsDNA HS Assay Kit and the Qubit 2.0 Fluorometer. The assay is highly selective for doublestranded DNA (dsDNA) over single-stranded DNA (ssDNA), RNA, proteins, and free nucleotides. The assay kit is designed to be accurate for DNA sample initial concentrations ranging from 0.005 to 120 ng/µl (depending on the sample volume), resulting in a detection range of 0.1 to 120 ng.The Qubit Working Solution was prepared by combining 1 µl of Qubit Reagent with 199 µl of Qubit Buffer per sample, considering the two standards included in the kit. For the standards, 10 µl of each Standard Solution (provided in the kit) were mixed with 190 µl of the Working Solution. As for the samples, between 1-2 µl of each sample was mixed with 198-199 µl of the Working Solution (resulting in a final volume of 200 µl in each case). After vortexing the tubes, they were incubated at room temperature for 2 min before being read in the Qubit fluorometer (Figure30).

Figure 30 .

 30 Figure 30. Workflow for the dsDNA HS Assay Kit using the Qubit® 2.0 Fluorometer [197].

Figure 31 .

 31 Figure 31. Example of an amplification plot of qPCR to determine the number of cycles (N) for library amplification. Dotted line represents half of the curve saturation, and arrows intersecting the X-axis represent the number of cycles at which half of the saturation is achieved. Rn, Normalized reporter [198].

  every 1.0 µl of the sample, 1.6 µl of AMPure XP beads were added and incubated for 5 min at RT. Bead-bound DNA fragments were then collected at the bottom of the tube using a magnet to separate them from contaminants. Next, the bead-bound DNA fragments were washed twice with 70% ethanol to remove contaminants. Finally, purified DNA fragments were eluted with 30 µl of Elution Buffer from the ChIP DNA Clean & Concentrator from Zymo Research and transferred to a new tube (Figure32).

Figure 32 .

 32 Figure 32. Workflow for PCR purification with AMPure XP beads [199].

Figure 33 .

 33 Figure 33. Dual screening assay of an epigenetic inhibitor library. Different target classes of epigenetic inhibitors from the A-PARADDISE project (A). Dot plot displaying the dual screening results of the epigenetic inhibitor library against extracellular promastigotes (X-axis) and intracellular amastigotes (Y-axis) at a concentration of 10 µM (B). POC, percent of survival.

Figure 34 .

 34 Figure 34. Chemical structure of host-directed TCP-based LSD1 inhibitors (LSD1i) that decrease intracellular amastigote load.

Figure 35 .

 35 Figure 35. Analysis of transcriptional modulation induced in BMDMs infected by L. amazonensis in the presence or not of MC4030. Analysis of transcriptional modulations induced in BMDMs infected by L. amazonensis in the presence or not of MC4030. Volcano plot of the expression changes between infected (I) and uninfected (UI) BMDMs (A), MC4030-treated and untreated uninfected BMDMs (B), and MC4030treated and untreated infected BMDMs (C). Only DEG with adjusted p values<0.05 are shown.Green and red dots represent LSD1 direct and indirect target genes that were down and up modulated, respectively. Grey dots correspond to modulated genes that have not been identified as LSD1 targets (Table27).

Figure 36 .

 36 Figure 36. Analysis of RNAseq data by principal component analysis (PCA). UI, uninfected; I, infected; UId, uninfected MC4030-treated; Id, infected MC4030-treated. The numbers indicate the biological replicate.

Figure 37 .Chapter 1 .

 371 Figure 37. Comparative analysis of DEGs in L. amazonensis-infected BMDMs treated with MC4030. Each DEG is represented as a circle, where the border reflects the expression modulation associated with infection, and the interior represents the modulation associated with treatment using the inhibitor on infected macrophages. Upregulation is indicated by red color, while downregulation is shown in green, both in the border and the interior of the circle. The thickness of the circle's border and the size of the circle correspond to the adjusted p-values for the DEGs associated with infection and drug treatment, respectively. Block A contains 293 upregulated DEGs during infection, downregulated after treatment. Block B shows 84 downregulated DEGs during infection, upregulated after treatment. Block C represents 8 upregulated DEGs and 1 downregulated gene during infection, with a synergic effect after treatment. Block D and E display 33 and 37 upregulated and downregulated DEGs uniquely after treatment.

Figure 38 .

 38 Figure 38. Reversion of Leishmania-induced modulated pathways by MC4030. Pathways that are increased during infection and downmodulated by the drug (A) and vice versa (B).

Figure 39 .

 39 Figure 39. Impact of LSD1i on parasite load in L. amazonensis-infected BMDMs and RAW 267.4. After 24h of infection, cells were treated or not with the indicated compounds, fixed at day 4 PI (3 days in contact with compounds), and stained with Giemsa. Uninfected BMDMs (A). L. amazonensis-infected BMDMs (B). Uninfected RAW 264.7 macrophages (C). L. amazonensis-infected RAW 264.7 macrophages (D). Cumulative histogram representing the percentage of cells containing different parasite loads as indicated by the color code (E). Note the increase in uninfected (cured) cells (green color) in samples treated with LSDi. Microscopic analysis and counting were performed on at least 200 cells for each condition. Red arrows indicate L. amazonensis intramacrophagic amastigotes. Scale bars, 10 µm.

Figure 40 .

 40 Figure 40. Impact of LSD1i on parasite load in L. donovani-infected BMDMs. Overlay of fluorescence and brightfield microscopy images showing immuno-stained amastigotes (red color) and Hoechst-stained macrophage nuclei (blue color) on fixed cells (A). Cumulative histogram representing the percentage of cells containing different parasite loads as indicated by the color code (B). Cells were treated or not 24 hours after the infection with the indicated compounds, fixed at days 4 PI (3 days in contact with compounds) and immuno-stained. Uninfected (cured) cells are represented by the green color, while infected cells with different parasite loads are shown in different orange shades. Microscopic analysis and counting were performed on at least 200 cells for each condition. Scale bars, 10 µm.

Figure 41 .

 41 Figure 41. Impact of LSD1i on parasite load on living L. amazonensis-infected BMDMs. At day 1 PI, macrophage cultures were incubated with DMSO or LSD1i. After 3 days, living cell cultures were incubated with Hoechst 33342 and YO-PRO®-1 and image acquisition was performed with the EVOS™ M5000 Imaging System. Overlay of fluorescence and brightfield images showing mCherry-expressing amastigotes (red color), Hoechst-stained macrophage nuclei (blue color), and YO-PRO®-1-stained nuclei of dead macrophages (green color). Scale bars, 20 µm (A). The violin plots represent the level of red fluorescence per cell for each sample. 100-160 cells per condition were analyzed using ImageJ. Uninfected, white; infected, blue; MC2652-treated, green; MC4030-treated, orange color. Data significance is indicated by the p-value, *: p<0.0001 (B).

Figure 42 .

 42 Figure 42. Impact of LSD1i on parasite load in living L. donovani-infected BMDMs. At day 1 PI, macrophage cultures were incubated with DMSO or LSD1i. After 3 days, image acquisition of living cell cultures was performed with the EVOS™ M5000 Imaging System. Overlay of fluorescence and brightfield images showing mCherry-expressing amastigotes (red color). Scale bars, 20 µm (A). The violin plots represent the level of red fluorescence per cell for each sample. 100-180 cells per condition were analyzed using ImageJ. Infected, gray; MC2652-treated, pink; MC4030-treated, green color. Data significance is indicated by the p-value, *: p<0.0001 (B).

Figure 43 .

 43 Figure 43. LSD1i IC50 values (µM) calculated from resazurin assays. IC50 values for various batches of MC2652 (green bars) and MC4030 (yellow bars) were calculated using free lesion-derived L. amazonensis amastigotes (Ama) and promastigotes (Pro) in C-medium and Ld1spro medium. The year of the batch is indicated by the number above the compound name. Numbers above the histograms indicate the Mean IC50 in µM. The bars represent the standard deviation of 3 technical replicates.

Chapter 1 .Figure 44 .

 144 Figure 44. Standard curve of demethylated lysine 4 of Histone H3 (H3K4me0).

Figure 45 .

 45 Figure 45. In vitro analysis of LSD1 activity in the presence of LSD1i and TCP. Nuclear extracts from uninfected, untreated BMDMs (1 µg) were treated with two different concentrations of LSD1i (2019 batch) and TCP, with a reaction time of 60 minutes.

Figure 46 .

 46 Figure 46. Thermal Shift Assay Principle. LSD1, blue ribbon; LSD1i, yellow bubble; DMSO-treated, black line; LSD1i treated, green line; Tm, melting temperature; ∆Tm, thermal shift. Created with BioRender.com.

Figure 47 .

 47 Figure 47. Linearity of LSD1 band signal by WB analysis with fluorescence detection. The fluorescent intensity of the LSD1 band was measured using different volumes of protein samples(11,[START_REF] Alvar | Leishmaniasis worldwide and global estimates of its incidence[END_REF] 5, and 2 µl) obtained from uninfected BMDMs treated with DMSO, LSD1i, or TCP. The data was analyzed using regression analysis in Excel to determine if the relationship between the measured variables was linear. The calculated R 2 values demonstrated that the data points followed a linear relationship.

Figure 48 .

 48 Figure 48. CETSA analysis to assess selective LSD1 target engagement in protein lysates of uninfected BMDMs. Representative Western blots of (1) LSD1, (2) MAO-A and (3) GAPDH in the presence of DMSO (negative control) and LSD1i (MC2652 and MC4030) (A). Melting curves of (1) LSD1, (2) MAO-A and (3) GAPDH. BMDMs were treated for 24 hours with DMSO (gray line and area), MC2652 (green line), MC4030 (yellow line) and TCP (black line) (B). Results are presented as a percentage of the fluorescent signal detected at the lowest temperature in each melting curve (37°C). Values correspond to the mean and SEM of 2-3 biological replicates are represented by the error bars.

Figure 49 .

 49 Figure 49. CETSA analysis to assess selective LSD1 target engagement in protein lysates from infected BMDMs. Representative Western blots of (1) LSD1, (2) MAO-A and (3) GAPDH in the presence of DMSO (negative control) and LSD1i (MC2652 and MC4030) (A). Melting curves of (1) LSD1, (2) MAO-A and (3) GAPDH. BMDMs were treated for 24 hours with DMSO (gray line and area), or 15 µM MC2652 (green line), MC4030 (yellow line) and TCP (black line) (B). Results are presented as a percentage of the fluorescent signal detected at the lowest temperature in each melting curve (37°C). Values correspond to the mean and SEM of 2-3 biological replicates are represented by the error bars.

Chapter 2 .Figure 50 .

 250 Figure 50. LSD1 Immunofluorescence microscopy on fixed uninfected and infected BMDMs. Four days PI, cells were probed with primary α-LSD1 antibody (green), mCherry parasites (red) and Hoechst (blue). Representative images for each dataset are shown. Scale bars, 10 µm.

Figure 51 .

 51 Figure 51. Nuclei isolated from both uninfected and infected BMDMs exhibiting remnants of cytoplasmic contents. Scale bars, 20 µm.

Chapter 2 .

 2 Effect of Leishmania infection on macrophage LSD1 126 [228].

Figure 52 .

 52 Figure 52. Uninfected (A), infected (B) BMDMs and isolated nuclei (C & D). Obtained at day two PI with an optimized protocol. Scale bars, 20 µm.

Figure 53

 53 Figure53presents the WB results of the tested antibodies. The first blot depicts the corresponding band for POLR2B, which appears clean. However, the challenge with this protein lies in its molecular weight close to LSD1 (approximately 110 kDa), which would hinder the detection of both proteins in a single blot. The same issue arises with SF3A1. SNRPB and SPI1 displayed nonspecific bands, and SPI1 exhibited a band with very weak intensity. Finally, the mouse monoclonal antibody for the transcription factor YY1 resulted in several non-specific bands, while the rabbit polyclonal antibody specific gave a specific and strong signal and thus was chosen as the most suitable option for normalization.

Figure 53 . 2 . 2 . 2

 53222 Figure 53. Western blot analysis of candidate nuclear reference proteins. The expected signals are as follows: POL2RB, 135 kDa; SNRPB, 25-30 kDa; SPI1 30-35 kDa; SF3A1,120 kDa; YY1, 60-65 kDa. Molecular weight markers are indicated (kDa) on the left side of each membrane. 5 µg of nuclear protein extracts from uninfected BMDMs were loaded in all the WB.

Figure 54 .

 54 Figure 54. LSD1, YY1 and GAPDH Western Blots. Samples correspond to 5 µg of nuclear extracts from uninfected (UI) and infected (Inf) BMDMs (Mφ) or total protein extracts from L. amazonensis (L. am) promastigotes (Pro) and amastigotes (Ama).

Figure 55 .

 55 Figure 55. Representative Western blots of GAPDH and YY1. Samples correspond to 5 µg of nuclear and cytoplasmic extracts from uninfected (UI) and infected (Inf) BMDMs, after 24 and 72h PI.

Figure 56 .

 56 Figure 56. Example of Coomassie stained SDS-PAGE gel with nuclear and cytoplasmic extracts. Samples correspond to 5 µg of nuclear and cytoplasmic extracts from uninfected (UI) and infected (Inf) BMDMs after 24 and 72h PI, as well as total protein extracts from L. amazonensis (L. am) promastigotes (Pro) and amastigotes (Ama).

Figure 57 .

 57 Figure 57. Quantification of LSD1 protein in nuclear extracts. Representative Western Blots of LSD1 and YY1 (loading control) using 5 µg of nuclear protein extracts from uninfected (UI) and infected (Inf) BMDMs at 24, 48 and 72 hours after infection (A). Normalized quantities were calculated with the YY1 signal. Quantitative values were obtained from WB performed with nuclear extracts obtained between 24 and 72 hours after infection for at least two biological replicates (B).

Figure 58 .

 58 Figure 58. CETSA analysis for LSD1 target engagement in protein lysates from uninfected (UI) and infected (I) BMDMs. Representative western blots of (1) LSD1, (2) MAO-A and (3) GAPDH, in the presence of DMSO (A). Melting curves of (1) LSD1, (2) MAO-A and (3) GAPDH (B). BMDMs were infected or not and treated at 24 h PI for 24 h with DMSO. Results are presented as percentage of the fluorescent signal detected at the lowest temperature in each melting curve (37°C). Values correspond to the mean with the standard error of the mean (SEM) of 2-3 biological replicates represented by the bars.

Figure 59 .

 59 Figure 59. Nuclei isolation after PFA fixation. Left panel shows uninfected (UI) sample and right panel shows infected (Inf) sample.

Figure 60 .

 60 Figure 60. Nuclei isolation procedure for Co-IP assay. Uninfected (left) and infected (right) BMDMs after a few minutes of contact with the LBII (A). Isolated nuclei from uninfected (left) and infected (right) BMDMs after passages in a 27 gauge needle (B). Scale bars, 20 µm.

Chapter 2 .Figure 61 .

 261 Figure 61. SYPRO ruby stained SDS-PAGE gel (A) and WB (B) from experiment 6. CE, Cytoplasmic Extract; Input, nuclear proteins from uninfected BMDMs; Ab1, homemade α-LSD1 antibody (20752); Ab2, homemade α-LSD1 antibody (3544); C-, Rabbit IgGs. Molecular weight markers (M) are indicated (kDa) on the left side of the gel (A) or the membrane (B).

Figure 62 .

 62 Figure 62. SYPRO ruby stained SDS-PAGE gel (A) and WB (B) from experiment 7. UI, Uninfected; I, Infected; Input, nuclear proteins from BMDMs; Ab1, homemade α-LSD1 antibody (20752); Ab2, homemade α-LSD1 antibody (3544); C-, Rabbit IgGs. Molecular weight markers (M) are indicated (kDa) on the left side of the gel (A) or the membrane (B).

Figure 63 .

 63 Figure 63. Venn diagrams depicting the overlap between immunoprecipitated proteins with specific α-LSD1 antibodies and the IgG control (C-). Immunoprecipitated proteins identified by mass spectrometry from uninfected (A) and infected (B) nuclear protein samples. At least 3 peptides for each protein were identified. Group A (in red) = C-, Rabbit IgG as negative control; Group B (in blue) = Ab1 (20752); Group C (in yellow) = Ab2 (3544).

Figure 64 .

 64 Figure 64. SYPRO ruby stained SDS-PAGE gel (A) and WB (B) from experiment 10. Before pre-clear, nuclear proteins from uninfected BMDMs; Ab1, homemade α-LSD1 antibody (20752); Ab2, homemade α-LSD1 antibody (3544); C-, Rabbit IgGs. Molecular weight markers (M) are indicated (kDa) on the left side of the gel (A) or the membrane (B).

Figure 65 .

 65 Figure 65. Venn diagrams comparing the LSD1 interacting proteins precipitated with α-LSD1 specific antibodies Ab1 or Ab2. UI, uninfected condition; I, infected condition.

Figure 66 .

 66 Figure 66. Venn diagrams comparing the LSD1 interacting proteins between uninfected (UI) and infected (I) conditions. Left panel, Ab1; right panel, Ab2. Blue frame indicates the dataset used for the following analyses.

Figure 67 .

 67 Figure 67. Volcano plots displaying specific LSD1 interactors in uninfected (upper panel)and infected (lower panel) conditions. Nonspecific binding events were eliminated by comparing proteins in the negative control (rabbit IgGs) with proteins immunoprecipitated using the LSD1-specific antibody. Only statistically significant interacting proteins (cut-off p < 0.01) were retained. The analysis was performed using the Prostar software tool. UI, uninfected; I, infected; Ab1 α-LSD1 specific antibody (20752).

Figure 68 .

 68 Figure 68. Protein clusters of macrophage LSD1 protein interactors unique in infected BMDMs. CL, NetworkNeighbor AL; GO, gene ontology; IPR, InterPro Domains; KW, UniProt Keywords; MMU, reactome pathways; mmu, KEGG pathways; SM, SMART domains.

Figure 69 .

 69 Figure 69. Comparison between standard ChIP-seq and ChIPmentation [237].

Figure 70 .

 70 Figure 70. Western blot to analyze antibody specificity and protein integrity. Chromatin samples from uninfected BMDMs subjected to increasing number of sonication cycles were tested. Molecular weight markers (M) are indicated (kDa) on the left side of the blot.

Figure 71 .

 71 Figure 71. DNA size during sonication time course. Purified DNA from chromatin samples (after reverse cross-linking) of uninfected BMDMs subjected to increasing number of sonication samples were tested. Molecular weight markers (M) are indicated (base pairs, bp) on the left and right side of the agarose gel.

Figure 72 .

 72 Figure 72. ChIP-Western Blot analysis demonstrating the capacity of Ab1 to immunoprecipitate LSD1 from chromatin samples of uninfected BMDMs. Sheared chromatin samples obtained after 15 sonication cycles (30'' on/30 off'', high power) were submitted to immunoprecipitation (IP) either with α-LSD1 (Ab1, 20752) or Rabbit IgGs as negative control (C-). Input, chromatin sample before IP; Supernatant, chromatin sample recovered after IP. Molecular weight markers (M) are indicated (kDa) on the left side of the blot.

Figure 73 .

 73 Figure 73. Western blot to analyze the antibody specificity and protein integrity in chromatin biological replicates. Samples from 3 biological replicates (Rep), uninfected (UI) and infected (Inf) and from evLIMS isolated from two Rag2 knockout mice (Rag1 & Rag2). Molecular weight markers (M) are indicated (kDa) on the left side of the blot.

Figure 74 .

 74 Figure 74. Agarose gel electrophoresis of purified fragmented DNA. Chromatin samples from 3 biological replicates (Rep) of uninfected (UI) and infected (Inf) BMDMs, as well as from evLIMs of 2 Rag2-KO mice, were subjected to 15 sonication cycles. Subsequently, the samples underwent reverse cross-linking, and the DNA was purified to visualize fragment size. Molecular weight markers (M) are indicated (base pairs, bp) on the left and right side of the agarose gel.

Figure 75 .

 75 Figure 75. Western blot analysis to detect LSD1 immunoprecipitated chromatin of uninfected and infected samples from Replicate 3. LSD1 was immunoprecipitated (IP Eluate) from chromatin samples from of uninfected (UI) and infected (Inf) BMDMs, C-, rabbit IgGs as negative control; Input, chromatin sample before IP; Supernatant, chromatin sample recovered after IP. Molecular weight markers (M) are indicated (kDa) on the left side of the blot.

Figure 76 .

 76 Figure 76. Diagram illustrating the tagmentation procedure on beads. Tn5, Transposase; TF, Transcription Factor [198].

Figure 77 .

 77 Figure 77. Schematic view of DNA purification and library preparation from immunoprecipitated samples.

Figure 78 .

 78 Figure 78. qPCR Amplification plot using various amounts (6, 3, 0.6 ng) of chromatin input samples from uninfected (UI) and infected (Inf) BMDMs. The black horizontal line intersecting the Y-axis represents the midpoint of saturation, and the black vertical lines intersecting the X-axis represent the number of cycles at which half of the saturation is achieved. Rn, Normalized reporter; UI, uninfected; Inf, infected.

Figure 79 .

 79 Figure 79. Quality analysis of purified input libraries. Purified libraries from various amounts (6, 3 and 0.6 ng) of chromatin input samples from uninfected (UI) and infected (Inf) BMDMs were visualized in a virtual gel (A) and the corresponding individual electropherogram (B). The blue arrow (A) and the blue vertical lines (B) indicate the size of the library fragments. Molecular weight markers (Ladder) are indicated (base pairs, bp) on the left side of the virtual gel and are also indicated in the X-axes of the electropherograms. Fluorescence intensity is represented on the Y-axis (FU). Purple and green lines (A) and peaks at 1500 and 15 bp (B) represent high-and low-molecular weight markers, respectively.

Figure 80 .

 80 Figure 80. qPCR amplification plot of immunoprecipitated samples. The black horizontal line intersecting the Y-axis represents the midpoint of saturation, and the black vertical lines intersecting the X-axis represent the number of cycles at which half of the saturation is achieved. Rn, Normalized reporter; UI, uninfected; Inf, infected; C-, negative control (rabbit IgGs).

Figure 81 .

 81 Figure 81. Quality analysis of purified immunoprecipitated libraries. Purified libraries from immunoprecipitated samples (with specific α-LSD1 antibody and with the negative control) from uninfected (UI) and infected (Inf) BMDMs were visualized in a virtual gel (A) and the corresponding individual electropherogram (B). Blue arrow in the gel and blue vertical lines in the eletropherograms indicate the library fragments size. Molecular weight markers (Ladder) are indicated (base pairs, bp) on the left side of the virtual gel and are also indicates in the X-axis of the electropherograms. Fluorescence intensity is represented in the Y-axis (FU). Purple and green lines (A) and peaks at 1500 and 15 bp (B) represent high-and low-molecular weight markers, respectively.

Figure 82 .

 82 Figure 82. Coomassie-stained SDS-PAGE gel of macrophage nuclear extracts. Uninfected (UI) and infected (Inf) BMDMs nuclear extracts from two biological replicates (Rep., 5 µg per sample). Molecular weight markers (M) are indicated (kDa) on the left side of the gel.

Figure 83 .

 83 Figure 83. Assessment of nuclear extracts quality for mass spectrometry analysis. Coomassie-stained SDS-PAGE gel of the nuclear extracts (A). Western blots for LSD1 and GAPDH using nuclear and cytoplasmic extracts. Biological quadruplicates of uninfected (UI) and infected (Inf) BMDMs were used (Rep. 1-4) (B). Molecular weight markers (M) are indicated (kDa) on the left side of the gel. 5 µg of each sample were loaded for all conditions.

Figure 84 .

 84 Figure 84. Volcano plot of nuclear proteins in infected (B) vs. uninfected (A) BMDMs. Proteins present exclusively in uninfected samples are indicated as "Only in A," while proteins exclusively found in infected samples are denoted as "Only in B." The size of each dot corresponds to adjusted p-values.

Figure 85 .

 85 Figure 85. GO analysis in cellular component (CC) of nuclear proteins that show increased (up) or decreased (down) abundance following Leishmania infection (A). Subclusters from a STRING analysis comparing nuclear protein abundances in infected vs uninfected BMDMs (B). Green and red colors represent down-and up-regulation in the infected state, respectively. Dot size corresponds to the adjusted p-value.

Figure 86 .

 86 Figure 86. Scatterplot representing the clusters of parasite proteins identified in the nuclear proteome analysis. Bubble color indicates the p-value; bubble size indicates the frequency of the GO term in the GO Annotation database (bubbles of more general terms are larger).

Figure 87 .

 87 Figure 87. Targeting macrophage LSD1 as a novel strategy for anti-leishmanial hostdirected drug discovery. Leishmania parasites release effectors that enter macrophage nuclei through yet-unknown mechanisms, interacting with host nuclear proteins like LSD1, thereby influencing the macrophage epigenome. LSD1, a pivotal epigenetic regulator known for its role in demethylating H3K4 and H3K9, experiences functional alterations due to changes in its protein associations during infection. This perturbation aids parasite survival and establishes a macrophage phenotype permissive for Leishmania infection. Repurposed LSD1 inhibitors designed for cancer treatment partially restores normal macrophage functions, reducing parasite load (right panel). Treating uninfected macrophages with LSD1 inhibitors (LSD1i) alters gene expression (left panel), confirming their impact on the macrophage epigenome and supporting their potential in host-directed therapy. Created with BioRender.com.

  5 ml Eppendorf tubes and cleared at 20,000 g for 20 min at 4°C. Carefully, 75 µL of supernatant containing the soluble protein fraction was collected in a new LoBind® Eppendorf tube. The detection of the remaining soluble proteins was performed by Western Blot, where 11 µL of each cleared sample were mixed with 2 µL of NuPAGE® Reducing Agent and 5 µL of NuPAGE® LDS Sample Buffer. The samples for each CETSA melt curve were loaded on the same gel. The quantification was performed by measuring the fluorescence intensity emitted by the ECL Plex fluorescent secondary antibodies with the Amersham™ ImageQuant™ 800.

  Cells were rinsed twice for 5 min in PBS and incubated with the blocking and permeabilization solution (5% donkey normal serum, 5% goat normal serum, 0.3% Triton X-100 in PBS 1X) for 1 hour at RT. Primary antibody solution (α-LSD1 antibody, Cell Signaling, 1/100; Mouse immune serum for parasite detection, 1/1000; 1% Bovine Serum Albumin, 0.3% Triton X-100, PBS 1X) was added for an overnight incubation at 4°C. The next day, cells were rinsed 3 times for 5 min in PBS 1X and subsequently incubated for 1h30 at RT in the dark with the secondary antibody solution (10 µg/ml Donkey α-mouse, 10 µg/ml Goat α-rabbit, 1% BSA, 0.3% Triton X-100 in PBS 1X). Afterwards, cells were rinsed once in PBS 1X for 5 min, incubated for 15 min in a PBS 1X solution containing 5 M Hoechst 33342 and rinsed one last time with PBS 1X for 5 min.Finally, coverslips were rinsed in Milli-Q water, dried and mounted using SlowFade™ Gold Antifade Mountant on clean glass slides. The slides were stored overnight in the dark at 4°C and sealed with transparent varnish. The slides were observed on an Axiophot ZEISS epifluorescence microscope for counting of uninfected and infected cells, and intracellular parasites and for identifying LSD1 sub-cellular localization. Four fields per culture condition were acquired at 20X magnification allowing the analysis of at least 100 cells.Determination of parasite load in live macrophages by epifluorescence imagingParasite load was estimated by the level of the mCherry fluorescence of live hosting macrophages. Nuclei from macrophages were stained by 5 M Hoechst 33342 (ThermoFisher Scientific) for 15 min at room temperature. Image acquisition was done with the EVOS M5000 Imaging System on live cells after 4 days of infection and 3 days of treatment, using the DAPI light cube filter for detection of Hoechst-stained nuclei (Excitation: 357/44 nm; Emission: 447/60 nm) and the Texas red light cube for parasite mCherry detection (Excitation: 585/29 nm; Emission: 624/40 nm). Five fields were acquired with the 20x objective. Image analysis was performed using the ImageJ software package on at least 100 cells. Parasite loads were determined based on the Raw Integrated Density of the mCherry fluorescence of macrophages in the absence or presence of LSD1i. Statistical analyses applying the Wilcoxon-Mann-Whitney test and graphical representations were performed with the GraphPad Prism software package.

  IP was performed using nuclear protein samples from four biological replicates of uninfected and infected BMDMs. Dynabeads™ Protein G (Invitrogen) were resuspended, transferred to Eppendorf tubes (50 µl for sample pre-clearing and 25 µl for immunoprecipitation) and washed three times with 200 µl of PBS-T pH 7.4 (1x PBS, 0.02% Tween-20, 1x PIC).For sample pre-clearing, beads were incubated for 30 min at RT with 5 µg of negative control antibody (C-, IgG from healthy rabbits, Diagenode). For immunoprecipitation, 3 µg of antibody Ab1 or C-were incubated with the beads (Ab1, anti-LSD1 20752, kindly provided and validated by Eric Metzger, from Roland Schüle team at the University of Freiburg, Germany)[START_REF] Duteil | LSD1 promotes oxidative metabolism of white adipose tissue[END_REF]. Following three washes with 200 µl PBS-T, pH 7.4, Dynabeads-Ab complexes were washed with 250 µl of Conjugation Buffer (20 mM Sodium phosphate pH 7.4, 150 mM NaCl) and subsequently cross-linked by incubating for 30 min at RT with a 5 mM solution of Bissulfosuccinimidyl suberate (BS 3 diluted in Conjugation Buffer). The reaction was quenched by adding 12.5 µl of 1M Tris pH 7.4 for 15 min at RT. Cross-linked Dynabeads-Ab complexes were washed three times with 200 µl of Beads buffer (50 mM Tris pH 7.4, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM NaF, 0.1% NP-40). Immunoprecipitation was performed by suspending the cross-linked Dynabeads-Ab complexes with 300 µg of pre-cleared samples and incubating overnight at 4°C. The following day, supernatants were recovered and transferred to a new tube (for further analysis) and Dynabeads-Ab-Antigen (Ag) complexes were washed 3 times with 1 ml of Wash Buffer (50 mM Tris pH 7.4, 250 mM NaCl, 5 mM EDTA, 5 mM NaF, 0.1% NP-40) and four times with 1 ml of 100 mM NH4HCO3. Finally, Dynabeads-Ab-Ag complexes were resuspended in 100 µl NH4HCO3 and digested by adding 0.2 μg of trypsin-LysC (Promega) for 1 h at 37°C. Samples were then loaded into custom-made C18 StageTips packed by stacking three AttractSPE® disk (#SPE-Disks-Bio-C18-100.47.20 Affinisep) into a 200 µL micropipette tip for desalting. Peptides were eluted using a ratio of 40:60 CH3CN:H2O + 0.1% formic acid and vacuum concentrated to dryness with a SpeedVac device. Peptides were reconstituted in 10 µL of injection buffer in 0.3% trifluoroacetic acid (TFA) before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

  including 19 showing decreased mRNA abundance (e.g. Zfp217, FC = -1.87; Suv39h1, FC = +1.40), and 16 showing increased mRNA abundance (e.g. Mbd3, FC = +1.48; Gatad2a, FC = +1.

Figure 1 :

 1 Figure 1: Experimental pipeline (A) and differential expression analysis (B) of Epigenetic

Figure 2 :

 2 Figure 2: Identification and validation of LSD1 inhibitors with host-dependent, anti-

Figure 3 .

 3 Figure 3. Impact of L. amazonensis infection on LSD1 expression and localization and on

Figure 4 :

 4 Figure 4: Analyses of LSD1 interactions.

Figure 5 :

 5 Figure 5: Transcript profiling of uninfected (UI) BMDMs and LIMs at day 2 post-

  Figure 1, Gutiérrez-Sanchez M. et al.

Figure 3 ,

 3 Figure 2, Gutiérrez-Sanchez M. et al.

  .... FIGURE 29. EXPERIMENTAL PROCEDURE FOR CHROMATIN IMMUNOPRECIPITATION. ............................. FIGURE 30. WORKFLOW FOR THE DSDNA HS ASSAY KIT USING THE QUBIT® 2.0 FLUOROMETER... FIGURE 31. EXAMPLE OF AN AMPLIFICATION PLOT OF QPCR TO DETERMINE THE NUMBER OF CYCLES FOR LIBRARY AMPLIFICATION .............................................................................................................. FIGURE 32. WORKFLOW FOR PCR PURIFICATION WITH AMPURE XP BEADS ........................................ FIGURE 33. DUAL SCREENING ASSAY OF AN EPIGENETIC INHIBITOR LIBRARY .......................................... FIGURE 34. CHEMICAL STRUCTURE OF HOST-DIRECTED TCP-BASED LSD1 INHIBITORS (LSD1I) ........ FIGURE 35. ANALYSIS OF TRANSCRIPTIONAL MODULATION INDUCED IN BMDMS INFECTED BY L. FIGURE 36. ANALYSIS OF RNASEQ DATA BY PRINCIPAL COMPONENT ANALYSIS ................................... v FIGURE 37. COMPARATIVE ANALYSIS OF DEGS IN L. AMAZONENSIS-INFECTED BMDMS TREATED WITH MC4030............................................................................................................................................... FIGURE 38. REVERSION OF LEISHMANIA-INDUCED MODULATED PATHWAYS BY MC4030 ................... FIGURE 39. IMPACT OF LSD1I ON PARASITE LOAD IN L. AMAZONENSIS-INFECTED BMDMS AND RAW 267.4..................................................................................................................................................... FIGURE 40. IMPACT OF LSD1I ON PARASITE LOAD IN L. DONOVANI-INFECTED BMDMS. ................ FIGURE 41. IMPACT OF LSD1I ON PARASITE LOAD ON LIVING L. AMAZONENSIS-INFECTED BMDMS. .............................................................................................................................................................. FIGURE 42. IMPACT OF LSD1I ON PARASITE LOAD IN LIVING L. DONOVANI-INFECTED BMDMS. .... FIGURE 43. LSD1I IC50 VALUES CALCULATED FROM RESAZURIN ASSAYS ............................................ FIGURE 44. STANDARD CURVE OF DEMETHYLATED LYSINE 4 OF HISTONE H3 ..................................... FIGURE 45. IN VITRO ANALYSIS OF LSD1 ACTIVITY IN THE PRESENCE OF LSD1I AND TCP ................ FIGURE 46. THERMAL SHIFT ASSAY PRINCIPLE ......................................................................................... FIGURE 47. LINEARITY OF LSD1 BAND SIGNAL BY WB ANALYSIS WITH FLUORESCENCE DETECTION . FIGURE 48. CETSA ANALYSIS TO ASSESS SELECTIVE LSD1 TARGET ENGAGEMENT IN PROTEIN LYSATES OF UNINFECTED BMDMS ................................................................................................................. FIGURE 49. CETSA ANALYSIS TO ASSESS SELECTIVE LSD1 TARGET ENGAGEMENT IN PROTEIN LYSATES FROM INFECTED BMDMS ................................................................................................................. FIGURE 50. LSD1 IMMUNOFLUORESCENCE MICROSCOPY ON FIXED UNINFECTED AND INFECTED

AMAZONENSIS IN THE PRESENCE OR NOT OF MC4030 ..................................................................

  ..... FIGURE 68. PROTEIN CLUSTERS OF MACROPHAGE LSD1 PROTEIN INTERACTORS UNIQUE IN INFECTED FIGURE 69. COMPARISON BETWEEN STANDARD CHIP-SEQ AND CHIPMENTATION............................ FIGURE 70. WESTERN BLOT TO ANALYZE ANTIBODY SPECIFICITY AND PROTEIN INTEGRITY ................. FIGURE 71. DNA SIZE DURING SONICATION TIME COURSE .................................................................... FIGURE 72. CHIP-WESTERN BLOT ANALYSIS DEMONSTRATING THE CAPACITY OF AB1 TO IMMUNOPRECIPITATE LSD1 FROM CHROMATIN SAMPLES OF UNINFECTED BMDMS ............... FIGURE 73. WESTERN BLOT TO ANALYZE THE ANTIBODY SPECIFICITY AND PROTEIN INTEGRITY IN CHROMATIN BIOLOGICAL REPLICATES .............................................................................................. FIGURE 74. AGAROSE GEL ELECTROPHORESIS OF PURIFIED FRAGMENTED DNA .................................. FIGURE 75. WESTERN BLOT ANALYSIS TO DETECT LSD1 IMMUNOPRECIPITATED CHROMATIN OF UNINFECTED AND INFECTED SAMPLES FROM REPLICATE 3 ............................................................ FIGURE 76. DIAGRAM ILLUSTRATING THE TAGMENTATION PROCEDURE ON BEADS ............................. FIGURE 77. SCHEMATIC VIEW OF DNA PURIFICATION AND LIBRARY PREPARATION FROM IMMUNOPRECIPITATED SAMPLES. ..................................................................................................... FIGURE 78. QPCR AMPLIFICATION PLOT USING VARIOUS AMOUNTS OF CHROMATIN INPUT SAMPLES FROM UNINFECTED AND INFECTED BMDMS .................................................................................. FIGURE 79. QUALITY ANALYSIS OF PURIFIED INPUT LIBRARIES ................................................................ FIGURE 80. QPCR AMPLIFICATION PLOT OF IMMUNOPRECIPITATED SAMPLES ..................................... FIGURE 81. QUALITY ANALYSIS OF PURIFIED IMMUNOPRECIPITATED LIBRARIES .................................... FIGURE 82. COOMASSIE-STAINED SDS-PAGE GEL OF MACROPHAGE NUCLEAR EXTRACTS. .............. FIGURE 83. ASSESSMENT OF NUCLEAR EXTRACTS QUALITY FOR MASS SPECTROMETRY ANALYSIS. FIGURE 84. VOLCANO PLOT OF NUCLEAR PROTEINS IN INFECTED VS. UNINFECTED BMDMS ............

BMDMS ............................................................................................................................................. vi COOMASSIE-STAINED SDS-PAGE GEL OF THE NUCLEAR EXTRACTS ...........................................

TABLE 1 .

 1 SPECIES OF LEISHMANIA CAUSING LEISHMANIASIS IN HUMANS ................................................ TABLE 2. SUMMARY OF HISTONE METHYL MARKS. ..................................................................................... TABLE 3. SUMMARY OF CHARACTERISTICS OF HDAC ENZYMES. ............................................................. TABLE 4. SUMMARY OF JMJC DEMETHYLASE SUBFAMILIES........................................................................

TABLE 5 .

 5 HDACS INVOLVED IN MACROPHAGE FUNCTION AND POLARIZATION ..................................... TABLE 6. HMTS INVOLVED IN MACROPHAGE FUNCTION AND POLARIZATION. ......................................

TABLE 7 .

 7 COMPOSITION OF CULTURE MEDIUM FOR PROMASTIGOTES ..................................................... TABLE 8. COMPOSITION OF THE COMPLETE MEDIUM FOR BONE MARROW-DERIVED MACROPHAGES. ................................................................................................................................................................

TABLE 9 .

 9 DIFFERENT PLASTIC PLATES USED FOR THE SPECIFIED PROCEDURES. ........................................ TABLE 10. COMPOSITION OF BUFFERS FOR IMMUNOFLUORESCENCE. .....................................................

TABLE 11 .

 11 EXPERIMENTAL CONDITIONS FOR EACH CELL TYPE IN THE RESAZURIN REDUCTION ASSAY. .

TABLE 12 .

 12 COMPOSITION OF LYSIS BUFFERS AND EXTRACTION BUFFERS FOR NUCLEAR PROTEIN AND CHROMATIN EXTRACTION.

...................................................................................................................

TABLE 13 .

 13 FUNCTION OF THE COMMON COMPONENTS AMONG LYSIS AND EXTRACTION BUFFERS. ..

TABLE 14 .

 14 COMPOSITION OF BUFFERS FOR WESTERN BLOT. ....................................................................

TABLE 15 .

 15 PRIMARY ANTIBODIES FOR WESTERN BLOT ..............................................................................

TABLE 16 .

 16 SECONDARY ANTIBODIES FOR WESTERN BLOT. ........................................................................

TABLE 17 .

 17 COMPOSITION OF BUFFERS FOR COOMASSIE STAINING. ........................................................

TABLE 18 .

 18 COMPOSITION OF BUFFERS FOR CO-IMMUNOPRECIPITATION ASSAY. ...................................

TABLE 19 .

 19 BUFFERS FOR CHROMATIN IMMUNOPRECIPITATION. ..............................................................

TABLE 20 .

 20 SAMPLES USED FOR THE PREPARATION OF LIBRARIES AND THEIR CORRESPONDING UDIS.

TABLE 21 .

 21 PCR SET UP FOR LIBRARY PREPARATION PER SAMPLE. .............................................................

TABLE 22 .

 22 PRE-AMPLIFICATION PCR CYCLING CONDITIONS. .................................................................

TABLE 23 .

 23 QPCR CYCLING CONDITIONS. ...................................................................................................

TABLE 24 .

 24 POST-AMPLIFICATION PCR CYCLING CONDITIONS. ...............................................................

TABLE 25 .

 25 FUNCTION OF THE COMPONENTS OF THE PROTEIN EXTRACTION BUFFER FOR MASS SPECTROMETRY ANALYSIS.

...................................................................................................................

TABLE 26 .

 26 COMPOSITION OF THE FP7 A-PARADDISE EPIGENETIC INHIBITOR LIBRARY ....................

TABLE 27 .

 27 SOURCES CONSULTED TO OBTAIN THE LIST OF LSD1 INDIRECT OR DIRECT TARGET GENES.92

TABLE 28 .

 28 LSD1I CYTOTOXICITY IN BMDMS AND RAW 264.7 MACROPHAGES IN C-MEDIUM. .....

TABLE 29 .

 29 LSD1I ACTIVITY ON FREE LESION-DERIVED L. AMAZONENSIS AMASTIGOTES IN C-MEDIUM

AND LD1SPRO MEDIUM. ....................................................................................................................

TABLE 30 .

 30 LSD1I CYTOTOXICITY IN L. AMAZONENSIS PROMASTIGOTES IN C-MEDIUM AND LD1SPRO

MEDIUM. ..............................................................................................................................................

TABLE 31 .

 31 SUMMARY OF THE RESULTS OBTAINED IN THE LSD1 ACTIVITY ASSAY. ................................

TABLE 32 .

 32 LSD1, MAO-A AND GAPDH MELTING TEMPERATURES AND THERMAL SHIFTS FROM PROTEIN LYSATES OF UNINFECTED BMDMS BY CETSA ANALYSIS. .............................................

TABLE 33 .

 33 LSD1, MAO-A AND GAPDH MELTING TEMPERATURES AND THERMAL SHIFTS FROM PROTEIN LYSATES OF INFECTED BMDMS BY CETSA ANALYSIS. ..................................................

TABLE 34 .

 34

EXAMPLES OF NUCLEAR-LOCALIZED PROTEINS WITHOUT SIGNIFICANT DIFFERENCES IN TRANSCRIPT AMOUNT. .......................................................................................................................

TABLE 35 .

 35 LSD1, MAO-A AND GAPDH MELTING TEMPERATURES AND THERMAL SHIFTS FROMHuman leishmaniases are a group of diseases caused by around 20 species of Leishmania protozoan parasites. The parasites are transmitted to humans by the bite of an infected female phlebotomine sandfly. Leishmaniases are neglected tropical diseases influenced by various major risk factors. Socioeconomic conditions, such as poverty and inadequate housing with poor sanitation, create favorable environments for sandfly breeding and their access to humans. Malnutrition, particularly diets low in proteins, iron, vitamin A and zinc, increases the risk of infection progressing to a more severe disease. Population mobility plays a role in epidemics, as non-immune individuals moving into high transmission areas can contribute to the spread of the parasite. Recently, environmental and climate changes, including urbanization,

	INTRODUCTION

deforestation and human incursion into forested areas, have been linked to the increased incidence of leishmaniases. Climate change exacerbates the problem by altering temperature and rainfall patterns, influencing the size and geographic distribution of sandfly populations. Additionally, droughts, famines, wars and floods lead to population migrations, potentially introducing the parasite into new areas.

Table

  1

Table 1 .

 1 Species of Leishmania causing leishmaniasis in humans[START_REF] Steverding | The history of leishmaniasis[END_REF].

	Subgenus	Species	OW/NW	Clinical disease	Distribution
		L. aethiopica	OW	LCL, DCL	East Africa
		L. amazonensis	NW	LCL, DCL, MCL	South America
					Central Africa, South
		L. donovani	OW	VL, PKDL	Asia, Middle East, India,
					China
					Mediterranean
		L. infantum	OW, NW	VL, CL	countries, Southeast Europe, Middle East,
	Leishmania				Central Asia, Americas
					North and Central
		L. major	OW	CL	Africa, Middle East,
					Central Asia
		L. mexicana	NW	LCL, DCL	Americas
					North and Central
		L. tropica	OW	LCL, VL	Africa, Middle East,
					Central Asia, India
		L. venezuelensis	NW	LCL	South America
		L. waltoni	NW	DCL	Dominican Republic
		L. braziliensis	NW	LCL, MCL	South America
		L. guyanensis	NW	LCL, MCL	South America
		L. lainsoni	NW	LCL	South America
		L. lindenbergi	NW	LCL	South America
	Viannia	L. naiffi	NW	LCL	South America
		L. panamensis	NW	LCL, MCL	Central and South America
		L. peruviana	NW	LCL, MCL	South America
		L. shawi	NW	LCL	South America
	Mundinia	L. martiniquensis L. orientalis	NW, OW	LCL, VL	Martinique, Thailand

Abbreviations. NW: New World, OW: Old World, DCL: Diffuse Cutaneous Leishmaniasis, LCL: Localized Cutaneous Leishmaniasis, MCL: Mucocutaneous Leishmanisis, VL: Visceral Leishmaniasis, PKDL: Post Kala-azar Dermal Leishmaniasis.

Table 2 .

 2 Summary of histone methyl marks.

	Mark	Enriched at	KMTs
		Actively transcribed regions
	H3K4me1	Enhancers	SMYD1,2 MLL1-5 family, SET1 A/B,
	H3K4me2	5' body genes	ASH1L, ASH2L, PRDM9 and SET7,9
	H3K4me3	Promoters
	H3K36me3	Actively transcribed regions	NSD1,2,3, SMYD2 and SETD2,3
	H3K79me2,3 Actively transcribed regions	DOT1L
	H4K20me1	Actively transcribed regions	SET8/PR-SET7
			EHMT1 also known as GLP,
	H3K9me2,3	Heterochromatin	EHMT2/G9a, SETDB1, SETDB2, SUV39H1, SUV39H2, PRDM2 and
			PRDM8
	H3K27me2,3	Heterochromatin	Polycomb repressive complex 2 (PRC2)-EZH1/2, NSD2 and NSD3
	H4K20me2,3	Heterochromatin	SUV4-20H1 and SUV4-20H2

Table 3 .

 3 Summary of characteristics of HDAC enzymes.

	Class HDAC	Characteristics
	I.

Table 4 .

 4 Summary of JmjC demethylase subfamilies.

	Subfamily	Members	Substrate
	KDM2	KDM2A & KDM2B are members of F-box protein family.	H3K36me1/me2
	KDM3	KDM3A (JMJD1B), KDM3C (JMJD1C) (JMJD1A), KDM3B	H3K9me1/2
		KDM4A, KDM4B & KDM4C	H3K9me2/me3 & H3K36me2/me3
	KDM4	KDM4D	H3K9me2/me3
		KDM4E	H3K9me1 & H3K56me1
	KDM5	KDM5A-5D	H3K4me1/me2/me3
	KDM6	KDM6A (UTX) & KDM6B (JMJD3) KDM6C (UTY)	H3K27me3 H3K27
	KDM7	KDM7A (JMJD1D)
	Contain two
	domains:
	PHD and
	JmjC

Table 5 .

 5 HDACs involved in macrophage function and polarization

	HDAC	Described function	Reference
	HDAC2	C-Jun inhibition, PAI-1 repression, anti-inflammatory response	[172] [173]
	HDAC3	TGF-β repression, M2 phenotype phagocytosis	[174]
	HDAC5	TNF-α & MCP-1 induction, proinflammatory response	[175]
	HDAC6	Modulation of macrophages recruitment, proinflammatory IL-10 induction	[176] [177]
	HDAC8	IL-1β repression, anti-inflammatory response	[178]
	HDAC11	IL-10 repression	[177]

Regarding LSD1, most discoveries to date have been made in cancer models. LSD1 is an important target in cancer therapy due to its crucial role in macrophage differentiation. Notably, it can hinder THP1 differentiation into macrophages

[179]

and trigger the differentiation of stem-like leukemia cells into macrophages in MLL-rearranged leukemia cases

[180]

. In a breast cancer mouse model, LSD1 inhibition suppresses tumor epithelial-mesenchymal transition (EMT) and promotes an innate M1-like immune response, enhancing the effectiveness chemotherapy

[181]

. Moreover, LSD1 phosphorylation by PKCα is crucial for the demethylation of the transcription factor p65, enhancing its stability and sustaining the inflammatory response. This phosphorylation is necessary for recruiting p65 to the target gene promoter and activating the NF-кB pathway, demonstrating the essential role of LSD1 in prolonged INTRODUCTION 47 inflammation response [182].

Table 6 .

 6 HMTs involved in macrophage function and polarization.

	HMT	Specific Substrate	Described Function	Reference
	SUV39H2	H3K9	Sirt1 repression, proinflammatory response	[183]
	EHMT2/G9a	H3K9	Type I interferon repression	[184]
	SETDB1	H3K9	TNF-α repression	[185]
	MLL1	H3K9	NOS, TNF-α induction, proinflammatory response	[186] [187]
	MLL4	H3K4	GPI glycolipid induction, proinflammatory response	[188]
	ASH2	H3K4	TNF-α induction, proinflammatory response	[189]
	SMYD2	H3K36	IL-6 and TNF-α repression; TGF-β induction, M2 activation	[190]
	DOT1L	H3K79	IL-6 and IFN-β induction	[191]
	SET8	H3K20	TNF-α repression, anti-inflammatory response	[192]

Table 7 .

 7 Composition of culture medium for promastigotes (Ld1spro medium).

	Reagents	Final concentration	Provider
	M199	-	Gibco
	Heat inactivated FBS	10%	Gibco
	HEPES pH 6.9	20 mM	Sigma
	Adenine	100 µM	Sigma
	Penicillin / Streptomycin	100 unit-µg/ml	Corning
	L-glutamine	2 mM	Gibco
	Folic acid	10 µg/ml	Sigma
	Hemin	13.7 µM	Sigma
	NaHC03 -7.5%	4.2 mM	R.P. Normapur
	RPMI 1640 vitamins solutions	1 X	Sigma
	6-Biopterin	8 µM	Sigma l
	Adjustment of the pH to 7.4	32% Sodium hydroxide (Fisher scientific)

Table 8 .

 8 Composition of the Complete Medium for bone marrow-derived macrophages.

	Reagents	Final concentration	Provider
	DMEM	-	PAN-Biotech (P04-3588)
	FBS	10%	Gibco
	HEPES	10 mM	Gibco
	2-Mercaptoethanol	50 µM	Sigma
	Penicillin / Streptomycin	50 units-µg/ml	Corning

Table 9 .

 9 Different plastic plates used for the specified procedures. , adherent BMDMs were infected or not with L. amazonensis mouse or L. donovani hamster-derived amastigotes(MOI of 4 and 8, respectively), resulting in at least 80% of infected cells. It is worth noting that no washing step was performed since lesion-derived, free amastigotes are unable to survive extracellularly (in contrast to axenically differentiated amastigotes).

	Support	Procedure	Number of cells/ml of C-medium/well
	100 mm dishes	Nuclear protein & chromatin extraction	1x10 7 in 12 ml
	6-well plates	RNA extraction	1.5x10 6 in 3 ml
	12-well plates	EVOS imaging	5x10 5 in 1.5 ml
	24-well plates	Immunofluorescence (+glass coverslips)	2x10 5 in 1.5 ml
	96-well plates	Resazurin assay	5x10 4 in 100 µl
	16-well Lab-Tek™ Chamber Slide™	Giemsa analysis	5x10 4 BMDMs or 2x10 4 RAW264.7 cells in 100 µl

supplemented with 100 U/mL penicillin-streptomycin and 10% FBS. This cell line was used to compare Leishmania infection (MOI of 4 with L. amazonensis), antileishmanial effects and cytotoxicity for LSD1 inhibitors with BMDM cultures.

Table 10 .

 10 Composition of buffers for immunofluorescence.Image analysis was performed using the ImageJ software package on at least 100 cells. Parasite loads were determined based on the Raw Integrated Density of the mCherry fluorescence of macrophages in the absence or presence of LSD1i. Statistical analyses applying the Wilcoxon-Mann-Whitney test and graphical representations were performed with the GraphPad Prism software package.

	Fixation	-4% paraformaldehyde (PFA)
	Solution	-PBS 1x
	Quenching	-50 mM ammonium chloride
	Solution
	Blocking	-5% donkey normal serum	-5% goat normal serum
	Solution	-0.3% Triton X-100	-1x PBS
	Primary	-α-LSD1 (1/100) (C69G12, Cell Signaling)
	Antibody	-Hamster immune serum (IS998, 1/1000) -for parasite detection
	Solution	-1% Bovine Serum Albumin
		-0.3% Triton X-100	-1x PBS
	Secondary	-AlexaFluor 594 Goat α-guinea pig (10 µg/ml) (Jackson Immu-
	Antibody	noResearch)
	Solution	-AlexaFluor 488 Goat α-rabbit (10 µg/ml) (Jackson Immu-
		noResearch)
		-1% Bovine Serum Albumin
		-0.3% Triton X-100	-1x PBS

5.3 MULTIPARAMETRIC FLUORESCENCE ANALYSES OF LIVING CELLS

Epifluorescence microscopy was used to evaluate parasite load in living macrophages and the potential toxicity of LSD1i. Image acquisition was done with the EVOS M5000 Imaging System on live cells at 4 days PI and 3 days PT, using the DAPI light cube filter for detection of Hoechst-stained nuclei (Excitation: 357/44 nm; Emission: 447/60 nm) and the Texas red light cube for parasite mCherry detection (Excitation: 585/29 nm; Emission: 624/40 nm). Dead macrophages were detected by nuclear incorporation of YOPRO-1 by the GFP light cube (Excitation: 470/22 nm; Emission: 510/42nm).

Table 11 .

 11 Experimental conditions for each cell type in the resazurin reduction assay.

	Type

of cells Number of cells per well Viability control

				Negative
				control
	BMDMs	5x10 4	Cycloheximide	DMSO
	RAW 264.7	2x10 4	Miltefosine	DMSO
	Free-amastigotes	1x10 6	Amphotericin B	DMSO
	Promastigotes	2x10 5	Amphotericin B	DMSO

Table 12 .

 12 Composition of Lysis Buffers (LB) and Extraction Buffers (EB) for nuclear protein and chromatin extraction.

Table 13 .

 13 Function of the common components among Lysis and Extraction Buffers.

	Component	Function
	β-glycerophosphate	Potent protein phosphatase inhibitor that acts as a
	(sodium salt hydrate)	phosphate group donor.
	1,10-Phenanthroline Membrane-permeable metal chelator, known as a
			non-specific matrix metalloproteinase (MMP) in-
			hibitor.
	Sodium		Competitive inhibitor for protein phosphotyrosine
	Orthovanadate	phosphatases (PTPs)
	(Na3VO4)
	Phenylmethyl-	Inhibitor of serine proteases (serine hydrolase inac-
	sulfonyl	fluoride	tivator) such as trypsin, chymotrypsin, thrombin,
	(PMSF)		and papain.
	Sodium	pyrophos-	Functions as a sequestering, clarifying, buffering
	phate	decahydrate	agent and water softener.
	(TSPP)

Table 14 .

 14 Composition of buffers for Western blot.

	Electrophoresis	-1x NuPAGE MOPS SDS Running Buffer
	Running Buffer
	Transfer	-30 g Trizma base (Sigma-Aldrich)
	Buffer 10x	-144 g Glycine (Sigma-Aldrich)
		-QS 1000 ml distilled water
	Transfer Buffer 1x -1x Transfer buffer	-2.5% ethanol
	PBS-T	-1x PBS	-0.25% Tween-20
	Blocking Solution -2% Amersham ECL blocking agent (Cytiva) in PBS-T
	Detection Reagent -Amersham ECL Western Blotting Detection Reagent

Table 15 .

 15 Primary antibodies for Western Blot, all specific for mouse proteins.

	Antigen	Provider	Species	Reference	Dilution
	LSD1	Cell signaling Rabbit	C69G12	1:1000
	GAPDH	Proteintech	Mouse	60004-1-Ig	1:10000
	H3	Proteintech	Rabbit	17168-1-AP	1:1000
	HDAC1	Proteintech	Rabbit	10197-1-AP	1:5000
	LSD1	Proteintech	Mouse	67037-1-Ig	1:5000
	LSD2	Proteintech	Mouse	67886-1-Ig	1:1000
	MAO-A	Proteintech	Rabbit	10539-1-AP	1:500
	MAO-B	Proteintech	Rabbit	12602-1-AP	1:500
	POLR2B	Proteintech	Rabbit	20370-1-AP	1:500
	RCOR1	Proteintech	Rabbit	27686-1-AP	1:500
	SF3A1	Proteintech	Rabbit	15858-1-AP	1:2000
	SNRPB	Proteintech	Rabbit	16807-1-AP	1:500
	SPI1	Proteintech	Rabbit	55100-1-AP	1:500
	YY1	Proteintech	Rabbit	22156-1-AP	1:500
	YY1	Proteintech	Mouse	66281-1-Ig	1:1000

Table 16 .

 16 Secondary antibodies for Western blot.

	Specificity Reporter	Provider	Reference	Species	Dilution
	Rabbit	HRP	Thermo Scientific	32230	Goat	1/20000
	Mouse	HRP	Thermo Scientific	31462	Goat	1/10000
	Rabbit	Cy5 dye	Amersham Biosciences	PA45011	Goat	1/3500
	Mouse	Cy3 dye	Amersham Biosciences	PA43009	Goat	1/3500

Table 17 .

 17 Composition of buffers for Coomassie Staining.

	Coomassie R250	-0.05% Coomassie brilliant blue (Merck)
	stain slow	-50% Methanol	-10% Acetic Acid
	De-staining solution	-10% ethanol	-7% acetic acid

12.2 QUALITY CONTROL OF SHEARED CHROMATIN 12

  .2.1 DNA size check Reverse cross-linking was performed on a mix of 25 µl of input sample, 75 µl of ChIP elution buffer, 2 µl of Proteinase K (20 mg/ml) and 2 µl of RNAse A (2 mg/ml). Samples were incubated overnight in a thermomixer at 65°C and 1400 rpm. The following day, DNA was purified using the ChIP DNA Clean & Concentrator from Zymo Research, according to manufacturer's instructions.Afterwards, DNA concentrations were determined using a Nanodrop. They were calculated based on the absorbance at 260 nm knowing that an absorbance of 1.0 corresponds to 50 µg/mL of double-stranded DNA. The absorbances at 260, 280 and 230 nm were recorded and the 260/280 nm and 260/230 nm ratios were determined for quality control. DNA was considered usable if these ratios were between 1.8 to 2 and 2 to 2.2, respectively.

Finally, purified DNA samples were separated on 1% agarose gels in 1x Tris Acetate EDTA (TAE) Buffer containing a drop of ethidium bromide (EtBr) solution (0.7 mg/ml), in order to analyze the fragment size. A mix of 9 µl of sample and 1 µl of 10X BlueJuice Gel Loading Buffer or 5 µl of SmartLadder (Ref. MW-1700-02) were deposited per well and run at 120V for 30 min.

12.2.2 Antibody Test and protein integrity

A mix of 16.25 µl of input sample, 6.25 µl of NuPAGE® LDS Sample Buffer and 2.5 µl of NuPAGE® Reducing Agent was heated at 95°C for 5 min and LSD1 was revealed by Western Blot as described in Section 10 of this chapter.

Table 19 .

 19 Buffers for Chromatin Immunoprecipitation.

	Blocking	-1x PBS
	Solution	-0.5% Bovine Serum Albumin
	RIPA Wash	-50 mM Tris-HCl, pH 8.0	-1% NP-40
	Buffer	-150 mM NaCl	-0.1% Sodium deoxycholate
		-2 mM EDTA, pH 8.0	-0.1% SDS
	Low Salt
	Wash Buffer

Table 20 .

 20 Samples used for the preparation of libraries and their corresponding UDIs.

				i7 = reverse primer	i5 = forward primer
							Sequence as read in se-
							quencing
		Sample	Primer pair (UDI)	Sequence as in primer	Sequence as read in se-quencing	Sequence as in pri-mer	i5 bases for sample sheet Forward Strand Workflow NovaSeq 6000 v1.0, MiSeq, HiSeq 2000/2500	5 bases for sample sheet Reverse Strand Workflow NovaSeq 6000 v1.5, iSeq, Min-iSeq, NextSeq, HiSeq 3000/4000
		UI -6 ng	1	AACCGCGG CCGCGGTT AGCGCTAG AGCGCTAG	CTAGCGCT
		UI -3 ng	2	GGTTATAA TTATAACC	GATATCGA GATATCGA	TCGATATC
		UI -0.6 ng	3	CCAAGTCC GGACTTGG CGCAGACG CGCAGACG	CGTCTGCG
	Input
		I -6 ng	4	TTGGACTT AAGTCCAA TATGAGTA TATGAGTA	TACTCATA
		I -3 ng	5	CAGTGGAT ATCCACTG AGGTGCGT AGGTGCGT	ACGCACCT
		I -0.6 ng	6	TGACAAGC GCTTGTCA GAACATAC GAACATAC	GTATGTTC
	IP -	UI	7	CTAGCTTG CAAGCTAG ACATAGCG ACATAGCG	CGCTATGT
	LSD1	I	8	TCGATCCA TGGATCGA GTGCGATA GTGCGATA	TATCGCAC
	IP -	UI	9	CCTGAACT AGTTCAGG CCAACAGA CCAACAGA	TCTGTTGG
	C-	I	10	TTCAGGTC GACCTGAA TTGGTGAG TTGGTGAG	CTCACCAA

Input, chromatin before immunoprecipitation; IP, immunoprecipitated; C-, negative control (rabbit IgGs); UDI, Unique Dual Indexed; UI, uninfected; I, infected.

Table 21 .

 21 PCR set up for library preparation per sample.

	Reagents	Volume
	Purified DNA	20 µl
	10 µM UDI (i7 & i5 primers mix)	2.5 µl
	Nuclease-free water	2.5µl
	NEB Next High-Fidelity 2x PCR Master Mix	25 µl
	Total	50 µl

Table 22 .

 22 Pre-Amplification PCR Cycling Conditions.

	Steps	Temperature	Time	Cycles
	Gap fill-in	72°C	5 min	1
	Initial Denaturation	98°C	1 min	1
	Denaturation	98°C	10 sec
	Annealing	63°C	30 sec	4 cycles
	Extension	72°C	20 sec
	Hold	10°C	∞

Table 23 .

 23 qPCR Cycling Conditions.

	Steps	Temperature	Time	Cycles
	Initial Denaturation	98°C	1 min	1
	Denaturation Annealing Extension	98°C 63°C 72°C	10 sec 30 sec 20 sec (acquire data)	35 cycles

Table 24 .

 24 Post-Amplification PCR Cycling conditions.

	Steps	Temperature	Time	Cycles
	Initial Denaturation	98°C	1 min	1
	Denaturation	98°C	10 sec
	Annealing	63°C	30 sec	N cycles
	Extension

Table 25 .

 25 Function of the components of the protein extraction buffer for mass spectrometry analysis.

Table 26 .

 26 Composition of the FP7 A-PARADDISE Epigenetic Inhibitor Library

	Compounds origin		Number of tested compounds
	Wolfgang Sippl Laboratory, Martin-Luther-Universität Halle-Wittenberg, Germany	112
	Antonello Mai Laboratory, Facolta di Farmacia e Medicina, Sapienza Universita, Roma, Italy.	161
	Manfred Jung Laboratory, Institute of Pharmaceutical
	Sciences,	Albert-Ludwigs-Universität,	Freiburg,	48
	Germany.
	Arasu Ganesan Laboratory, School of Pharmacy, University of East Anglia, Norwich, UK.	10
	David Fairlie Laboratory, School of Chemistry and
	molecular Biosciences, University of Queensland,	36
	Australia.
	Johan Schultz Laboratory, Kancera AB, Solna, Sweden	108
	Through this screening campaign, a set of epigenetic inhibitors were identified
	that showed a significant reduction in the intracellular load of Leishmania

amastigotes without showing toxicity against the promastigote stage (Figure 33B, red box), indicating a potential host-directed mode of action. Interestingly, most of the hit compounds represent histone demethylase inhibitors (yellow dots).

Table 27 .

 27 Sources consulted to obtain the list of LSD1 indirect or direct target genes.

	Approach	References
	LSD1 pharmacological inhibition	[208]; [209]; [210]; [211]; [212]
	LSD1 knockout	[209]; [213]
	LSD1 siRNA	[214]; [215]
	Transcriptomics	[208]; [209]; [214]; [210]; [211]; [213]
	Proteomics	[215]
	ChIP	[208]; [214]; [210]; [213]; [215]; [212]

Table 28 .

 28 LSD1i cytotoxicity in BMDMs and RAW 264.7 macrophages in C-medium.

			BMDMs		RAW 264.7
	Batch LSD1i IC50 (µM) IC50 SD (µM) IC50 (µM) IC50 SD (µM)
	2019	MC2652 MC4030	>100 >100	n/a n/a	>100 >100	n/a n/a
	2022	MC2652 MC4030	>100 31.4	n/a 3.5
	2023	MC2652 MC4030	>100 58.2	n/a 3.3

Table 29 .

 29 LSD1i activity on free lesion-derived L. amazonensis amastigotes in C-medium and Ld1spro medium.

			C-medium	Ld1spro medium
	Batch	LSD1i IC50 (µM) IC50 SD (µM) IC50 (µM) IC50 SD (µM)
	2019	MC2652 MC4030	2.6 >100	0.2 n/a	>100 >100	n/a n/a
	2022	MC2652 MC4030	1.9 0.9	0.1 0.0	20.6 7.4	1.8 0.5
	2023	MC2652 MC4030	9.8 2.5	0.3 0.1	19.2 5.5	1.1 0.3

Table 30 .

 30 LSD1i cytotoxicity in L. amazonensis promastigotes in C-medium and Ld1spro medium.

			C-medium	Ld1spro medium
	Batch	LSD1i IC50 (µM) IC50 SD (µM) IC50 (µM) IC50 SD (µM)
	2019	MC2652 MC4030	4.9 >100	0.7 n/a	>100 >100	n/a n/a
	2022	MC2652 MC4030	3.2 1.8	0.2 0.0	12.5 5.0	1.6 1.1
	2023	MC2652 MC4030	3.7 2.1	0.2 0.1	23.0 8.1	1.2 0.6

Table 31 .

 31 Summary of the results obtained in the LSD1 activity assay.

			Demethylated	LSD1 activity	Inhibition
			Product (ng)	(ng/min/mg)	(%)
	LSD1i Concentration Mean	SD	Mean SD
	Ctrl	-	3.9	0.5	64.9	10.4	-
	MC2652	30 µM 100 µM	2.0 1.7	0.1 0.3	33.3 28.8	1.2 5.2	48.7 55.7
	MC4030	30 µM 100 µM	2.4 1.9	0.3 0.0	39.7 32.2	4.2 0.8	38.8 50.3
	TCP	30 µM 100 µM	3.7 2.7	0.6 0.8	60.9 45.8	9.9 13.9	6.1 29.4

Table 32 .

 32 LSD1, MAO-A and GAPDH melting temperatures (Tm) and thermal shifts (ΔTm) from protein lysates of uninfected BMDMs by CETSA analysis.

		LSD1		MOA-A	GAPDH
	UI-Sample Tm (°C) ΔTm (°C) Tm (°C) ΔTm (°C) Tm (°C) ΔTm (°C)
	DMSO	46.9	N/A	48.1	N/A	44.7	N/A
	MC2652	52.4	5.5	48.7	0.6	43.4	-1.3
	MC4030	50.2	3.3	49.5	1.4	43.7	-1.0
	TCP	51.0	4.1	50.1	2.1	41.3	-3.4
	Table 33. LSD1, MAO-A and GAPDH melting temperatures (Tm) and thermal shifts
	(ΔTm) from protein lysates of infected BMDMS by CETSA analysis.
		LSD1		MOA-A	GAPDH
	I-Sample	Tm (°C) ΔTm (°C) Tm (°C) ΔTm (°C) Tm (°C) ΔTm (°C)
	DMSO	50.2	N/A	51.1	N/A	44.2	N/A
	MC2652	56.7	6.5	50.4	-0.7	45.6	1.4
	MC4030	53.0	2.8	49.0	-2.1	45.6	1.4
	TCP	56.5	6.3	50.8	-0.3	42.9	-1.3

Table 34

 34

provides some examples of these nuclear proteins. I first tested a series of commercially available antibodies against these nuclear proteins on extracts Chapter 2. Effect of Leishmania infection on macrophage LSD1 127 from uninfected BMDMs, to stablish proper WB conditions.

Table 34 .

 34 Examples of nuclear-localized proteins without significant differences in transcript amount.

	Gene	Protein	Linear FC FDR
	POLR2B DNA-directed RNA polymerase II subunit	1.10	0.24
		RPB2
	SNRPB Small nuclear ribonucleoprotein-associated	-1.05	0.49
		protein B
	SPI1	Transcription factor PU.1	-1.08	0.33
	SF3A1	Splicing factor 3A subunit 1	1.03	0.66
	YY1	Transcriptional repressor protein YY1	-1.14	0.07

Table 35 .

 35 LSD1, MAO-A and GAPDH melting temperatures (Tm) and thermal shifts (ΔTm) from protein lysates of uninfected and infected BMDMs by CETSA analysis.

		LSD1		MOA-A	GAPDH
	Sample	Tm (°C) ΔTm (°C) Tm (°C) ΔTm (°C) Tm (°C) ΔTm (°C)
	UI-DMSO	46.9	N/A	48.1	N/A	44.7	N/A
	I-DMSO	50.2	3.3	51.1	3.0	44.2	-0.5

Table 36 .

 36 Summary of the LSD1 Co-IP experiments performed.

	Exp Sample Detergent	Protocol	Incubation times*	α-LSD1	C-	Pre-clearing
	1	UI BMDMs (150 µg)	-Digitonin -Triton	Parsig	-10 min, RT -30 min, RT	CS Dil 1:50	None	None
	2	UI & Inf BMDMs	DOC+LS	RIME	-O/N, 4°C -O/N, 4°C	CS Dil 1:50	Beads	None
	3	UI & Inf BMDMs (250 µg)	DOC+LS	RIME for NE Parsig for IP	-O/N, 4°C -O/N, 4°C	CS Dil Ptt (3.5µg) 1:50	Beads	Beads, 1 h, 4°C
	4	UI & Inf BMDMs	Digitonin	Parsig	-O/N, 4°C -O/N, 4°C	Ptt (4 µg)	Rabbit IgGs	Beads, 1 h, 4°C
	5	SN from Exp. 4	Digitonin	Parsig	-1 h, RT -Test 1h & O/N 4°C	Ptt (3 µg)	Rabbit IgGs	N/A
	6	UI BMDMs (300 µg)	Digitonin	Parsig	-30 min, RT -O/N, 4°C	Ab1 & Ab2 (3 µg)	Rabbit IgGs	Beads, 1 h, 4°C
	7	UI & Inf BMDMs (250 µg)	Triton	Parsig	-30 min, RT -O/N, 4°C	Ab1 & Ab2 (3 µg)	Rabbit IgGs	Beads, 1 h, 4°C
	8	UI & Inf BMDMs (250 µg)	Digitonin	Parsig	-30 min, RT -O/N, 4°C	Ab1 & Ab2 (3 µg)	Rabbit IgGs	Beads, 1 h, 4°C
								1° Beads, 30 min,
	9 SN from Exp. 6	Digitonin	Parsig	-30min, RT -O/N, 4°C	Ab1 & Ab2 (3 µg)	Rabbit IgGs	4°C 2° Beads+Rabbit
								IgGs, 30 min, 4°C
	10	SN from Exp. 8	Digitonin	Parsig	-30min RT -O/N, 4°C	Ab1 & Ab2 (3 µg)	Rabbit IgGs	Beads+Rabbit IgGs, 30 min, 4°C, twice
	Exp, Experiment; SN, Supernatant; DOC, sodium deoxycholate; LS, N-Laurylsarcosine; Parsig, Molecular Para-
	sitology and Signaling Unit, Pasteur Institut; RIME, Rapid immunoprecipitation mass spectrometry of endog-
	enous proteins; NE, Nuclear Extraction; O/N, overnight; CS, Cell Signaling; Ptt, Proteintech.
	Ab1 (20752) & Ab2 (3544) are homemade α-LSD1 antibodies.
	*First incubation time: Beads-Antibody complex. Second incubation time: Beads-Antibody-Sample.

Table 38 .

 38 Quantity and quality of purified DNA fragments from chromatin samples of uninfected BMDMs.

	Sonication cycles ng/µl 260/280 260/230
	0	11.1	1.43	1.03
	5	62.9	1.82	1.91
	10	62.4	1.78	1.79
	15	64.6	1.81	1.79
	20	50.1	1.80	1.79
	25	45.5	1.79	1.76
	5.1.3 Chromatin Immunoprecipitation
	This section investigates the ability of the α-LSD1 Ab1 (20752) to
	immunoprecipitate the protein of interest bound to chromatin after
	formaldehyde cross-linking. Sheared chromatin from 2.5 x 10 6 uninfected

260/280 260/230 Rep 1

		UI	42.0	1.84	2.29
		Inf	37.1	1.86	2.29
	Rep	UI	60.4	1.82	2.50
	2	Inf	47.6	1.83	2.24
	Rep	UI	36.8	1.83	2.24
	3	Inf	37.6	1.85	2.31
	Rag 1	49.4	1.80	2.18
	Rag 2	43.7	1.79	2.29

Table 40 .

 40 Quantity of purified tagmented DNA after reverse cross-linking.

		Input	ChIP-C-	ChIP-LSD1
	Sample	UI	Inf	UI	Inf	UI	Inf
	ng/µl	6.38	6.94	0.12	0.37	0.07	0.91
	Input, sheared chromatin before immunoprecipitation; ChIP, immunoprecipitated
	chromatin; UI, Uninfected BMDMs; Inf, Infected BMDMs; C-, rabbit IgGs. Total
	volume per sample= 20 µl.

Table 41 .

 41 Characteristics of the purified libraries from the preliminary ChIPmentation experiment

	Type of	Sample	Average	Concentration
	sample	library	size (bp)	(ng/µl)
		UI-6 ng	320	16.0
		UI-3 ng	315	13.8
	Input	UI-0.6 ng Inf-6 ng	330 297	9.2 14.9
		Inf-3 ng	295	14.7
		Inf-0.6 ng	322	4.1
	ChIP-LSD1	UI Inf	347 406	10.7 11.3
	ChIP-C-	UI Inf	320 367	11.5 10.6
	Input, chromatin sample before immunoprecipitation; ChIP,
	immunoprecipitated chromatin; C-, negative control (rabbit IgGs);
	UI, uninfected; Inf, infected BMDMs.

Table 42 .

 42 Examples of Leishmania proteins found in the nucleome of infected BMDMs.

	Gene ID	Transcript Product Description
	LAMA_000538700 ABC transporter putative
	LAMA_000006400	ABC-2 family transporter protein/ABC transporter, putative
	LAMA_000298900 AMP-binding enzyme, putative
	LAMA_000140900	BILBO1 N-terminal domain/EF-hand domain pair, putative
	LAMA_000224800 C2 domain containing protein, putative
	LAMA_000424900 cytoskeleton associated protein, putative
	LAMA_000106400	Dynamin family/Dynamin central region/Dynamin GTPase effector domain containing protein, putative
	LAMA_000432100 EF-hand domain pair, putative
	LAMA_000721300 Flagellum attachment zone protein 12
	LAMA_000145000 Flagellum attachment zone protein 3
	LAMA_000749500 Flagellum attachment zone protein 8
	LAMA_000365000 Flagellum targeting protein Kharon 1
	LAMA_000615100 Glyceraldehyde 3-phosphate dehydrogenase, putative
	LAMA_000120800	Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase/Hsp90 protein, putative
	LAMA_000111100 Histone H2A, putative
	LAMA_000154400 Histone H2B variant V
	LAMA_000295600 Histone H3 variant V
	LAMA_000780200 Histone H4
	LAMA_000021200 Hsp70 protein, putative
	LAMA_000514000 Hsp70 protein/MreB/Mbl protein, putative
	LAMA_000721100 KH-associated protein 1
	LAMA_000442600 Kinesin-C
	LAMA_000357800 Kinesin-D
	LAMA_000366000 Kinetoplast DNA-associated protein, putative
	LAMA_000364900 Kinetoplast-associated protein, putative
	LAMA_000296700 Lactate/malate dehydrogenase, putative
	LAMA_000574600 Leucine rich repeat, putative
		Leucine-rich	repeat/Leucine	rich	repeats	(2
	LAMA_000685500	copies)/Glycogen recognition site of AMP-activated
		protein kinase, putative
	LAMA_000195600 Lipase (class 3), putative
		Metallopeptidase family M24/FACT complex subunit
	LAMA_000128600	(SPT16/CDC68)/Histone	chaperone	Rttp106-like,
		putative

  ). Several types of gene-sets have been tested: (i) Hallmark

	(https://doi.org/10.1016%2Fj.cels.2015.12.004),		(ii)	reactome
	(https://doi.org/10.1093/nar/gkab1028),	(iii)	KEGG	pathways
	(https://doi.org/10.1093/nar/gkac963),	(iv)	GO	terms
	(https://doi.org/10.1093/genetics/iyad031)	and	(v)	Wikipathways
	(https://doi.org/10.1093/NAR/gkaa1024). Only gene-sets with a false discovery rate (FDR)

lower than 0.05 were considered significantly enriched in differentially expressed gene sets.

  ). Peptides were trapped on a 2 cm nanoviper Precolumn (i.d. 75 μm, C18 Acclaim PepMap TM 100, Thermo Scientific) at a flow rate of 3.0 µL/min in buffer A (2/98 MeCN/H2O in 0.1% formic acid) for 4 min to desalt and concentrate the samples. Separation was performed on a 50 cm nanoviper column (i.d. 75 μm, C18, Acclaim PepMap TM RSLC, 2 μm, 100Å, Thermo Scientific) regulated to a temperature of 50°C with a linear gradient from 2% to 25% buffer B (100% MeCN in 0.1% formic acid) at a flow rate of 300 nL/min over 91 min. MS1 data were collected in the Orbitrap (120,000 resolution; maximum injection time 60 ms; AGC 4 x 10 5 ). Charges states between 2 and 7 were required for MS2 analysis, and a 60 sec dynamic exclusion window was used. MS2 scan were performed in the ion trap in rapid mode with HCD fragmentation (isolation window 1.2 Da; NCE 30%; maximum injection time 35 ms;AGC 10 4 )

The amastigote form, mainly found in mononuclear phagocytes of the vertebrate host, is an intracellular and nonmotile form. It has an oval shape, measuring 3-5 µm in length and 1-3 µm in width. It possesses a short, embedded flagellum at the anterior end, which is probably involved in sensory functions. On the other hand, the promastigote form is present in the sandflies alimentary tract and is characterized by an extracellular and motile lifestyle. It is

AmB is a broad-spectrum antifungal drug that is effective against Leishmania parasites. Due to the loss of effectiveness of antimonial drugs in India, AmB has become the first-line treatment for VL. It works by binding to membrane sterols, particularly ergosterol, forming complexes that create ion channels and increase membrane permeability[START_REF] Gray | Amphotericin primarily kills yeast by simply binding ergosterol[END_REF]. However, it is associated with frequent

Multidrug therapy has been explored as a potential solution to combat the growing resistance of Leishmania parasites to single-drug therapy. Also, combining drugs offers other advantages, including increased activity through synergistic or additive effects, shorter treatment duration, lower doses, reduced toxicity and cost-effectiveness.

Unlike LSD1, which interacts through its Tower domain with the CoREST transcriptional repressor and enables binding to the nucleosome (see next

ACKNOWLEDGMENTS

With financial support from "la Région Île-de-France" (N°EX061034) and ITMO Cancer of Aviesan and INCa on funds administered by Inserm (N°21CQ016-00) for MS analysis, and from A-PARADDISE/602080 FP7 Project, FISR2019_00374 MeDyCa, and Progetto di Ateneo Sapienza 2021 (no. RM12117A61C811CE) for the preparation of LSD1 inhibitors.

OUTLOOK: CHARACTERIZING THE EFFECT OF LEISHMANIA INFECTION ON MACROPHAGE NUCLEAR PROTEIN COMPOSITION

Outlook SUPPLEMENTARY

S1. Heat map of mRNA expression values in BMDM samples.

BMDMs were infected with lesion-derived mCherry-transgenic amastigotes of L. amazonensis (LIMs) and compared to uninfected BMDMs (UI BMDMs) (A). One day after infection, MC4030 was added or not to the samples (B). RNA-Seq was performed one day after this treatment using three independent biological replicates. A Z-score normalization is performed on the normalized read counts across samples for each gene. Z-scores are computed on a geneby-gene (row-by-row) basis and used to plot heatmap (add the color legend with values).

S2. Gene Set Enrichment Analysis (GSEA) of transcriptionally modulated genes in LIMs.

Downregulated and upregulated processes are indicated in blue and red colors, respectively. S3: Analysis of the impact of LSD1i on parasite load in Leishmania-infected BMDMs.

BMDMs were infected with mCherry-transgenic amastigotes of L. amazonensis isolated from infected mouse footpads (A) or L. donovani isolated from infected hamster spleens (B). After one day of infection, MC2652 and MC4030 were added at a concentration of 10 M for 3 days. Supplementary Table1 Supplementary Table2

Supplementary Table 3