Keywords: Tuberculose, Mycobacterium tuberculosis, macrophages, criblage à haut débit, thérapie dirigée vers l'hôte, épigénétique, sirtuine, antibiotique, résistance host-targeting strategies Tuberculosis, Mycobacterium tuberculosis, macrophages, high throughput screening, host-directed therapy, epigenetics, sirtuin, antibiotic, resistance ABBREVIATIONS 1, 25-D3: 1, 25-dihydroxyvitamin D3 Apoptosis-associated speck-like protein containing a caspase recruitment Immune-mediated inflammatory disorders Latent tuberculosis infection Multi-drug resistant tuberculosis Mitochondrial-derived reactive oxygen species

Titre : Améliorer le traitement de la tuberculose grâce à des stratégies dirigées vers l'hôte.

Résumé : La tuberculose (TB) est l'une des dix premières causes de mortalité dans le monde et représente une menace majeure pour la santé publique. Cette maladie peut être guérie grâce à un traitement de 6 mois associant jusqu'à quatre antibiotiques. Cependant, il est alarmant de constater que des souches multirésistantes (MDR) de Mycobacterium tuberculosis (MTB) ne cessent d'apparaître et de se propager. Leur résistance aux antibiotiques de première ligne rende les traitements beaucoup plus difficiles. Les options thérapeutiques pour guérir la TB MDR sont limitées, coûteuses et associées à un taux de réussite plus faible. De plus, elles sont basées sur l'utilisation de médicaments de deuxième et troisième intention plus toxiques et sur une durée plus longue. Malgré des efforts de recherche considérables, le vaccin quant à lui, ne suffit pas à enrayer cette épidémie. Récemment, les approches dirigées vers l'hôte sont apparues comme une stratégie innovante et prometteuse pour éradiquer la tuberculose. Ces thérapies, contrairement aux antibiotiques qui ciblent spécifiquement les bactéries, visent à améliorer les défenses immunitaires de l'hôte. Leur combinaison avec les antibiotiques existants ou futurs peut se révéler d'une utilité majeure pour améliorer le traitement de la TB. MTB a su développer différentes stratégies pour contourner l'immunité innée et adaptative de l'hôte qu'elle colonise. Pour exemple, MTB module l'expression des gènes de l'hôte en ciblant l'épigénome ce qui lui permet de se développer dans les cellules immunitaires. Mon projet de thèse vise à déterminer si l'interférence pharmacologique de ces modifications de l'épigénome induites par MTB diminue la survie bactérienne intracellulaire chez l'hôte. Dans cette optique, nous avons mis au point une technique de criblage de composés épigénétiques qui modifient l'expression des gènes de la cellule. Nous avons testé l'efficacité des composés sur des macrophages infectés par une bactérie MTB exprimant la GFP grâce à l'utilisation d'un microscope confocal automatisé à haut débit. Ce criblage nous a permis de découvrir une molécule qui réduit considérablement l'infection bactérienne dans ces cellules en empêchant MTB de se multiplier. Cette molécule est un inhibiteur putatif de la Sirtuine 2 (SIRT2). La SIRT2 est une désacétylase dépendante du NAD+ qui régule de nombreux processus cellulaires et joue un rôle dans le contrôle des infections bactériennes telle que la listériose. Nous avons démontré que ce composé n'était pas toxique pour les cellules humaines. De plus, il n'affecte pas la croissance bactérienne en milieu liquide, suggérant un mécanisme d'action dépendant de l'hôte. Aussi, cette molécule potentialise l'activité de plusieurs antibiotiques antituberculeux sur des souches de MTB sensibles et résistantes aux médicaments, aussi bien in vitro dans des macrophages humains qu'in vivo chez la souris. Ces travaux de thèse pourraient permettre la création d'un nouveau traitement contre la tuberculose.

LISTE DES ÉLÉMENTS SOUS DROITS

Liste de tous les éléments retirés de la version complète de la thèse faute d'en détenir les droits Page 10: Figure 1. Global representation of countries with at least 100,000 incident cases of TB reported in 2019. The first eight countries (Bangladesh, China, India, Indonesia, Nigeria, Pakistan, Philippines and South Africa) with the highest cases of TB, accounts for nearly 65% of the total cases worldwide. Picture from (World Health Organization, 2020).

Page 32: Figure 2. Cell cycle of MTB infection. MTB is transmitted from a sick individual to a new host via fine MTB-containing droplets. Once in the lower part of the lung, MTB is phagocytosed by individual macrophages. Infected macrophages recruit other macrophages, forming a macrostructure so-called granuloma, composed of macrophages and other immune cells. Inside the granuloma, MTB replication is slow-down (or even stopped depending of the site of infection) and MTB can persist there for decades. However, in some cases, granuloma can undergo into necrosis, with the release and the spreading of MTB in the extracellular environment. Figure from [START_REF] Cambier | Host Evasion and Exploitation Schemes of Mycobacterium tuberculosis[END_REF].

Page 36: Figure 3. Host-MTB interaction influences the granuloma fate. At the local infection site, MTB interacts with different immune cell types. This host-MTB interaction influences the balance of pro-inflammatory and anti-inflammatory mediators, controlling the level of inflammation and modulating the granuloma fate. Picture from [START_REF] Cadena | Heterogeneity in tuberculosis[END_REF].

Page 65: Figure 4. Host-directed therapies against MTB. MTB interferes with different host defenses pathways. HDTs are used to overcome MTB resistance to microbial killing. Each box indicates drugs or classes of drugs with proven effects on antimicrobial immunity. HDAC: histone deacetylase; IFNγ: interferon-γ; mTOR, mammalian target of rapamycin; PDE: phosphodiesterase; TB: tuberculosis; TNF: tumour necrosis factor [START_REF] Kaufmann | Host-directed therapies for bacterial and viral infections[END_REF].

Page 76: Figure 5. Schematic representation of the three different epigenetic mechanisms: DNA methylation, histone modification and non-coding RNAs. DNA methylation occurs on CpG islands, on the carbon of the cytosine ring. Histone modification, and more specifically histone acetylation, induces a remodeling of the chromatin into euchromatin, allowing gene expression in those regions. Histone deacetylation leads to heterochromatin, where the gene expression is silenced. Non-coding RNAs are composed of (i) Long non-coding RNAs (lncRNAs) that impede transcription as well as chromatin stability, and (ii) microRNAs (miRNAs) that post-transcriptionally regulate gene expression. Figure from [START_REF] Gartstein | Prenatal influences on temperament development: The role of environmental epigenetics[END_REF]. nodes or genitourinary tract, and are more common in immunocompromised individuals [START_REF] Pai | Tuberculosis[END_REF]. According to the World Health Organization (WHO) and its global report published in 2019, TB is one of the top ten causes of human death worldwide (World Health Organization, 2020), responsible for 1.4 million death per annum, with 10 million of new TB cases every year. The majority of people that have developed TB in 2019 are from South-East Asia (44%), Africa (25%) and Western Pacific (18%). In these areas, some have a TB incidence higher than 500 new cases per 100,000 people per year (Figure 1) (World Health Organization, 2020).

The causative agent of human TB is mainly Mycobacterium tuberculosis (MTB). Transmission of the disease usually occurs upon inhalation of fine MTB-containing airborne droplets expelled by a symptomatic patient (generally by coughing). While large size droplets (>5 μm) are trapped in the upper respiratory system by mucus and ciliary action [START_REF] Turner | Cough and the transmission of tuberculosis[END_REF], smaller droplets enter the lower lung directly to the alveoli, where MTB is then phagocytized by tissue-resident alveolar macrophages [START_REF] Ryndak | Mycobacterium tuberculosis Primary Infection and Dissemination: A Critical Role for Alveolar Epithelial Cells[END_REF]. This primary infection results in different outcomes: active TB disease, latent TB infection (with potential reactivation), or eradication of the infection.

Clinical outcomes of tuberculosis

It is commonly stated that 5 to 10% of individuals infected with MTB develop active pulmonary TB within during their life [START_REF] Pai | Tuberculosis[END_REF]. However, these percentages need to be nuanced « élément sous droit, diffusion non autorisée » "copyrighted material, unauthorized distribution" Figure 1. Global representation of countries with at least 100,000 incident cases of TB reported in 2019 (World Health Organization, 2020).

depending on many factors such as age, living country, comorbidities or close contact with TB patients [START_REF] Trauer | Risk of active tuberculosis in the five years following infection ⋯ 15%?[END_REF]. Furthermore, several longitudinal epidemiological studies have shown that the majority of TB occurs within the first months after infection and rarely after two years [START_REF] Behr | Is Mycobacterium tuberculosis infection life long?[END_REF]. In active TB, the patient usually shows symptoms such as cough, fever, weight loss, night sweats and haemoptysis in advanced disease [START_REF] Loddenkemper | Clinical aspects of adult tuberculosis[END_REF]. At this stage of the active pulmonary TB, MTB can spread from a sick patient to a healthy individual. In some rare cases, dissemination also happens via the hematogenous and the lymphatic system to other organs, causing extrapulmonary TB.

In other cases, primary infection does not lead to active TB and remains nearly asymptomatic.

In immunocompetent individuals, the pathogen is usually eliminated by the host immune responses. However, if not fully eradicated from the host, MTB can persist in a latent state for decades, known as latent tuberculosis infection (LTBI). This latter is defined as a state of persistent immune response to stimulation by MTB antigens without evidence of clinical symptoms (S. H. [START_REF] Lee | Tuberculosis infection and latent tuberculosis[END_REF]. LTBI may progress into infectious active TB [START_REF] Getahun | Latent Mycobacterium tuberculosis Infection[END_REF].

Many factors can influence the development of an active TB or a reactivation of LTBI: virulence of the MTB strain, immunocompetence of the host, vaccination status, environmental stresses and social factors. Worryingly, about a quarter of the world's population is estimated to be latently infected by MTB (A. [START_REF] Cohen | The global prevalence of latent tuberculosis: A systematic review and meta-analysis[END_REF], representing a major reservoir for potential active TB cases. This assumption is based on meta-analyses of two immunoreactivity diagnostic tests: the tuberculin skin test (TST) and interferon-γ release assays (IGRAs).

However, TST or IGRAs could still be positive even if the infection has been successfully eliminated [START_REF] Esmail | The ongoing challenge of latent tuberculosis[END_REF]. It also should be stated that due to the short period between infection and active TB disease, some studies prefer to mention newly acquired infection rather than reactivation of an old one [START_REF] Behr | Revisiting the timetable of tuberculosis[END_REF][START_REF] Cardona | Reactivation or reinfection in adult tuberculosis: Is that the question[END_REF].

Risk factors

Risk factors play a key role in the spreading of TB, from exposure to active disease. Among them, some are considered "exogenous" such as the infectiousness of the strain, close contact and social behaviors like smoking or overuse of alcohol. Other host related factors are rather considered as "endogenous".

Human immunodeficiency virus (HIV): TB is the leading cause of death among people living with HIV. In 2019, TB caused 1.4 million deaths and 208,000 were diagnosed HIV-positive. In the African region, the burden of HIV-associated TB is the highest, with 86% of TB patients that are HIV-positive (World Health Organization, 2020). HIV coinfection is known as the most associated risk factor for developing active TB disease. Compared to healthy individual, a person with an early HIV-1 infection has 2 to 5 times more chance to develop active TB [START_REF] Bell | Pathogenesis of HIV-1 and mycobacterium tuberculosis co-infection[END_REF]. This ratio goes up to 20 times for people with an advanced HIV-1 disease [START_REF] Bell | Pathogenesis of HIV-1 and mycobacterium tuberculosis co-infection[END_REF]. HIV coinfection increases the susceptibility to primary infection and the risk of reactivation for LTBI. Moreover, it potentiates the immunosuppression of the host mainly by a deprivation of CD4 + T cells and a diminution of cytokines production (Bruchfeld et al., 2015). HIV is also linked to an inhibition of phagocytosis in macrophages [START_REF] Mazzolini | Inhibition of phagocytosis in HIV-1-infected macrophages relies on Nef-dependent alteration of focal delivery of recycling compartments[END_REF]. Furthermore, the use of antiretroviral therapy (ART) may lead to a TB immune reconstitution inflammatory response syndrome which is an increased inflammatory pathology related to TB after ART treatment [START_REF] Bell | Paradoxical reactions and immune reconstitution inflammatory syndrome in tuberculosis[END_REF].

Young age and gender: In 2019, children under 15 years of age represented 12% of the TB cases. However, clinical and radiographic manifestations of the disease are less specific in children compared to adults, making a medical diagnosis more challenging (World Health Organization, 2020). Following primary infection, TB-related mortality is higher during infancy (< 4 years) (Narasimhan et al., 2013). Young children are also at higher risk of contracting and developing TB infection due to the age-related immature immune system, as well as being involved in the TB spreading [START_REF] Alcaïs | Tuberculosis in children and adults: Two distinct genetic diseases[END_REF].

Men and women over the age of 15 account for 56% and 32% of the patients that have developed TB in 2019, respectively. The male:female (M:F) ratio of incident TB cases goes from 1.3 in the Eastern Mediterranean region to 2.1 in the European and Western Pacific regions.

In children, the global M:F ratio is close to 1 (World Health Organization, 2020). A metaanalysis reveals that TB prevalence is significantly higher among men than women in low-and middle-income countries [START_REF] Horton | Sex Differences in Tuberculosis Burden and Notifications in Low-and Middle-Income Countries: A Systematic Review and Meta-analysis[END_REF]. Many hypotheses have been put forward to explain this gender difference, notably the behavior and the physiology.

Men's behavior, such as smoking and elevated alcohol consumption in low-income countries, exposes them more often to TB burden [START_REF] Nhamoyebonde | Biological differences between the sexes and susceptibility to tuberculosis[END_REF]. Case notification rates are also higher for men and the ratio of prevalent-to-notified cases of TB (an indication of how long patients take to be diagnosed) is 1.5 times higher among men than women [START_REF] Horton | Sex Differences in Tuberculosis Burden and Notifications in Low-and Middle-Income Countries: A Systematic Review and Meta-analysis[END_REF].This study suggests that men are disadvantaged in accessing TB care and are less likely to achieve a timely diagnosis. However, this explanation is still under debate, as other studies suggest a bias for under-reporting and under-diagnosis of TB amongst women [START_REF] Srivastava | Tuberculosis in women: A reflection of gender inequity[END_REF].

Physiological differences may also explain the different TB outcomes between genders.

Indeed, hormones are known to have diverse effects on many immune cell types, including dendritic cells (DC), macrophages, and natural killer cells (NK). For instance, estradiol enhances macrophage activation [START_REF] Neyrolles | Sexual inequality in tuberculosis[END_REF]. Estrogen stimulates secretion of interferon-gamma (INF-γ), Tumor Necrosis Factor α (TNF-α) and interleukin 12 (IL-12) (a pro-inflammatory cytokine involved in immune defense against MTB) and inhibits production of IL-10 (an anti-inflammatory cytokine) [START_REF] Nhamoyebonde | Biological differences between the sexes and susceptibility to tuberculosis[END_REF].

Diabetes: Many studies suggest that diabetes is a risk factor for TB, as patients with diabetes have a three-time higher risk of developing active TB. Moreover, they have a worse outcome regarding the treatment efficacy, mycobacterial clearance by the immune system, progression from LTBI to active TB, relapse and death (nearly two-fold compared to people without diabetes) [START_REF] Gautam | Diabetes among tuberculosis patients and its impact on tuberculosis treatment in South Asia: a systematic review and meta-analysis[END_REF]. In diabetes-TB cases, activation of immunity is reduced or delayed in alveoli with lower proportion of activated alveolar macrophages, higher antiinflammatory IL-10 cytokines and lower IFN-γ pro-inflammatory cytokines (Restrepo, 2016).

Finally, monocytes from diabetic individual have a significant reduced binding and phagocytosis for MTB [START_REF] Restrepo | Understanding the Host Immune Response Against Mycobacterium tuberculosis Infection[END_REF]. A similar conclusion has been drawn from experiments using alveolar macrophages from diabetic mice [START_REF] Martinez | Diabetes and immunity to tuberculosis[END_REF].

Socioeconomic factors and malnutrition: Socioeconomic deprivation refers to the relative disadvantage in terms of economic and social resources necessities for an individual [START_REF] Apolinário | Tuberculosis inequalities & socio-economic deprivation in Portugal[END_REF]. It is defined by numerous factors such as social integration, lack of education, low income, overcrowding or unemployment. Those factors are partially responsible for health inequities, with mistreatment outcomes and an increased risk of developing disease such as TB [START_REF] Duarte | Tuberculosis, social determinants and co-morbidities (including HIV)[END_REF]. Close contacts, like in overcrowded households, homeless populations or prisoners increase the risk of infection by MTB [START_REF] Story | Tuberculosis in London: The importance of homelessness. problem drug use and prison[END_REF]. Another well-established risk factor for TB is nutritional deficiencies. The relation between TB and malnutrition is bi-directional: TB itself reduces appetite and changes in metabolic processes. Undernutrition status potentiates TB through immunodeficiency, but also increases the risk of LTBI reactivation and active TB which consequently increases the risk of death [START_REF] Feleke | Nutritional status of tuberculosis patients, a comparative cross-sectional study[END_REF]. This results from an impairment of the host immune response, mostly through the impairment of T-lymphocytes and macrophages functions with a diminution of cytokines production; but also regarding the lack of vitamins and minerals, such as vitamins A, B12, D and calcium, as a key mediator of the innate immune system [START_REF] Chandrasekaran | Malnutrition: Modulator of immune responses in tuberculosis[END_REF].

Genetic polymorphisms: Some case-control studies point out a link between host genetic factors and susceptibility to TB. For example, Mendelian susceptibility to mycobacterial diseases, a familial predisposition to develop a disease with weakly virulent mycobacteria, is related to polymorphism on nine characterized genes (IFNGR1-2, STAT1, IL12RB1, IL12B, IRF8, ISG15, TYK2, IKBG and CYBB) (Harishankar et al., 2018).

Other examples of genes polymorphisms and its links with TB are described below:

-Polymorphisms of human leukocyte antigens (HLA) of Class I and Class II alter the binding affinity of antigens and impair the antigenic presentation to CD4+ and CD8+ Tcells. HLA-DRB1 polymorphisms are the most associated ones with TB susceptibility and disease development in East Asian populations [START_REF] Tong | Polymorphisms in HLA-DRB1 Gene and the Risk of Tuberculosis: A Meta-analysis of 31 Studies[END_REF].

-TLRs are pattern recognition receptors critical for pathogens detection and activation of the host innate and adaptive immune responses. TLRs are found on many cell types, with several playing a role in controlling MTB infection by the immune system, including TLRs 1, 2, 6, 8 and 9 (Azad et al., 2012). As a result, polymorphisms in those TLRs modulate the outcome of TB, such as the increase or decrease risk of developing TB amongst certain ethnic populations (Y. [START_REF] Zhang | Toll-Like Receptor -1, -2, and -6 Polymorphisms and Pulmonary Tuberculosis Susceptibility: A Systematic Review and Meta-Analysis[END_REF]. Moreover, presence of the X chromosome has a putative impact on tuberculosis immunity, with the X-linked Toll-like receptor 8 (TLR8) gene polymorphisms implicated in susceptibility to pulmonary TB in male children (Dalgic et al., 2011).

-Natural Resistance-Associated Macrophage Protein 1 (NRAMP1), encoded by Solute Carrier Family 11A Member 1 gene (SCL11A1), is a transporter that influences MTB survival within the host, by regulating iron and cations homeostasis in macrophages [START_REF] Meilang | Polymorphisms in the SLC11A1 gene and tuberculosis risk: A meta-analysis update[END_REF]. NRAMP1/ SCL11A1 gene is heterogeneous across populations and the D543N single nucleotide polymorphism of SLC11A1 (NRAMP1) is significantly associated with susceptibility to infection by MTB Beijing strains in Indonesian TB patients [START_REF] Azad | Innate immune gene polymorphisms in tuberculosis[END_REF].

-The vitamin D-binding protein (VDBP) polymorphisms have been shown to impede the inflammatory profile upon MTB infection [START_REF] Coussens | Ethnic Variation in Inflammatory Profile in Tuberculosis[END_REF]. VDBP polymorphism is also related to ethnic variation and associated with susceptibility to TB [START_REF] Hawthorne | Vitamin D binding protein (DBP) levels during tuberculosis treatment are affected by DBP genotype / haplotype but not by total vitamin D levels[END_REF].

Genome-wide association studies (GWAS) has also been used to search for specific genetic variations that may be associated with susceptibility to TB. In 2018, a study described the homozygosity for TYK2 P1104A allele as being a rare monogenic cause of TB [START_REF] Boisson-Dupuis | Tuberculosis and impaired IL-23-dependent IFN-γ immunity in humans homozygous for a common TYK2 missense variant[END_REF]. However, it should be kept in mind that TB is a polygenic disease which is influenced by many genes.

Bacterial load, lineage and virulence of the strains: MTB strains are divided into lineages 1 to 4 and lineages 7 and 8. These lineages are geographically spread: lineage 1 (Indo-Oceanic or East African-Indian) is disseminated around the Indian Ocean, lineage 2 (East-Asian; including Beijing family strains) and lineage 4 (Euro-American) are worldwide spread, lineage 3 (Central Asian Strain) dominates the East Africa, Central-and South-Asia, while lineage 7 is mainly present in Ethiopia and lineage 8 in African Great Lakes region [START_REF] Brites | The nature and evolution of genomic diversity in the mycobacterium tuberculosis Complex[END_REF]Ngabonziza et al., 2020).

MTB lineage is a contributing factor to clinical outcomes of TB and well spread among populations. Some lineages might be more associated and adapted to certain populations, leading to different outcomes depending of the host [START_REF] Gagneux | Variable host-pathogen compatibility in Mycobacterium tuberculosis[END_REF]. Higher virulence is usually associated with higher bacterial concentrations [START_REF] Tram | Virulence of Mycobacterium tuberculosis Clinical Isolates Is Associated With Sputum Pre-treatment Bacterial Load, Lineage, Survival in Macrophages, and Cytokine Response[END_REF]. Isolates with high virulence phenotype are likely to belong to East Asian/Beijing lineage [START_REF] Ribeiro | Mycobacterium tuberculosis strains of the modern sublineage of the beijing family are more likely to display increased virulence than strains of the ancient sublineage[END_REF].

Furthermore, strains from lineages 2 and 4 are considered as the most transmissible strains, with a higher virulence and a different host immune response (notably by a reduced proinflammatory response upon infection) [START_REF] Coscolla | Consequences of genomic diversity in mycobacterium tuberculosis[END_REF][START_REF] Wiens | Global variation in bacterial strains that cause tuberculosis disease: A systematic review and metaanalysis[END_REF].

Some studies have also highlighted that lineages 1 and 2 are more often associated with extrapulmonary TB [START_REF] Caws | The influence of host and bacterial genotype on the development of disseminated disease with Mycobacterium tuberculosis[END_REF][START_REF] Click | Relationship Between Mycobacterium tuberculosis Phylogenetic Lineage and Clinical Site of Tuberculosis[END_REF].

Immunosuppressive conditions: Patients with immune-mediated inflammatory disorders (IMID) like rheumatoid arthritis also have a higher risk of developing active TB. These diseases are generally treated with anti-TNF-α to reduce inflammation. However, TNF-α has a role in MTB containment by the immune system. Moreover, an anti-TNF-α treatment increases the risk of MTB reactivation. Therefore, IMID is not only detrimental for TB outcomes due to dysregulations of the immune system, but also with the therapeutic approach used [START_REF] Petruccioli | Mycobacterium tuberculosis Immune Response in Patients With Immune-Mediated Inflammatory Disease[END_REF]. These immunosuppressive conditions are also found in transplanted patients due to their treatment [START_REF] Aguado | Tuberculosis and Transplantation[END_REF]. Patients with IMID therefore represent a high-risk group for developing TB.

Coronavirus Disease 2019 (COVID-19):

The COVID-19 is a disease caused by the SARS-CoV-2 virus which became a worldwide pandemic in 2020. Regarding its association with TB, a first meta-analysis based on six Chinese studies indicates that people with TB are not more likely to get COVID-19. However, it has been shown that those individuals present a higher risk of developing serious complications from COVID-19 [START_REF] Gao | Association between tuberculosis and COVID-19 severity and mortality: A rapid systematic review and meta-analysis[END_REF]. Other modelling analysis studies suggest that this disease could cause up to 6.3 million TB cases and 1.3 million additional deaths between 2020 and 2025 [START_REF] Cilloni | The potential impact of the COVID-19 pandemic on the tuberculosis epidemic a modelling analysis[END_REF].These numbers are related to the increasing household contacts and the reduction of healthcare access due to lockdown.

However, more studies are needed to fully understand the impact of COVID-19 on TB.

Alcohol: A meta-analysis has demonstrated that alcohol use, alcohol dosage and alcoholrelated problems are strongly associated with an increased risk of MTB infection (Imtiaz et al., 2017). According to this study, alcohol consumption is a well-known contributor to the TB burden, with the most severe impacts estimated for the African region. Furthermore, alcohol is often associated with an increase in poor treatment outcomes including treatment failure and death [START_REF] Ragan | The impact of alcohol use on tuberculosis treatment outcomes: A systematic review and meta-analysis[END_REF].

Tobacco smoke and indoor air pollution: Tobacco smoking is a risk factor for LTBI, progression to active TB, MTB treatment failure and higher rate of TB-related death [START_REF] Amere | Contribution of smoking to tuberculosis incidence and mortality in high-tuberculosis-burden countries[END_REF].

A recent study performed in Delhi shows significant associations with TB burden and exposure to indoor pollution (Jayachandran et al., 2007a). Indoor air pollution is composed of toxic particles (carbon monoxide, nitrogen oxide, polyaromatic hydrocarbons) from use of solid fuels, smokes, firewood and biomass combustion. Moreover, smoking and inhaling pollutants alters the cellular and immune responses by exacerbating pathological immune responses or by attenuating the normal defensive function of the immune system [START_REF] Torres-Juarez | LL-37 immunomodulatory activity during Mycobacterium tuberculosis infection in macrophages[END_REF]Qiu et al., 2017). Therefore, impaired mucociliary clearance function or alveolar macrophage processing directly affects the ability of MTB to access the alveoli and cause damage.

Many risk factors influence the progression of TB. To reduce poor outcomes associated with certain risk factors, one of the main objectives is to prevent and diagnose TB as early as possible to limit transmission and conversion to active TB.

II. Diagnosis and prophylaxis

In 2014, the WHO has adopted the "End TB Strategy". The goal of this project is to strengthen the fight against TB, with the aim to drastically reduce TB burden worldwide by 2035. By this date, the objectives are a 90% reduction in the TB incidence rate, a 95% reduction in the annual number of TB deaths and 0% TB-affected households. This program is mainly focused on TB prevention, with increased funding for care and research to better diagnose this disease and develop new TB treatments and vaccines.

Diagnosis

In 2019, 7.1 million people were newly TB-diagnosed, far from the estimated 10 million of new cases in previous years. This gap is mainly due to the under-reporting and under-diagnosis related to health care accessibility in some countries such as India, Nigeria and Indonesia (World Health Organization, 2020). Yet diagnosis is an important part of the fight against TB.

Rapid diagnosis and treatment are key factors to prevent deaths and limit further transmission of the disease. The choice of a diagnostic tool depends on the TB status: LTBI or active TB disease.

Latent tuberculosis infection

In a case of TB suspicion (subclinical TB with low symptoms or LTBI), two diagnostic tools are available: the tuberculin skin test and the interferon gamma release assay.

Tuberculin skin test or TST: TST is carried out by intradermally injecting a tuberculin purified protein derivative (PPD). In a TB positive individual, a cell-mediated immunity to PPD is present and a hypersensitivity reaction to the TST occurs after 48 to 72 hours. An induration reaction larger than 15 mm is considered positive. While easy to perform at a large scale, this test presents some limitations. A positive result can be due to a former mycobacterial infection. Repeated vaccination with Bacille Calmette-Guérin (BCG -the vaccine for TB) and exposure to non-tuberculous mycobacteria may also induce a false-positive TST [START_REF] Farhat | False-positive tuberculin skin tests: what is the absolute effect of BCG and non-tuberculous mycobacteria?[END_REF]. In contrast, patients with chronic kidney disease with high levels of parathormone and vitamin D may present a negative TST due to reduced cell-mediated immunity [START_REF] Deniz | Factors affecting TST level in patients undergoing dialysis: a multicenter study[END_REF]. More generally, TST tests show lower positive results notably among HIV patients and other immunocompromised individuals depending on the degree of immunosuppression [START_REF] Richeldi | Performance of tests for latent tuberculosis in different groups of immunocompromised patients[END_REF].

Interferon gamma release assays or IGRAs: IGRAs are in vitro blood test assays based on cellmediated immune response to two mycobacterial antigens: the early secretory antigenic target (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10). These tests detect the release of IFNγ by circulating T cells. Interestingly, IGRAs can distinguish a BCG-induced response and most environmental mycobacteria from an MTB infection as the antigens detected by the assay are more specific to MTB [START_REF] Pai | Gamma interferon release assays for detection of Mycobacterium tuberculosis infection[END_REF]. These tests have also reduced sensitivity regarding immunocompromised patients [START_REF] Richeldi | Performance of tests for latent tuberculosis in different groups of immunocompromised patients[END_REF].

In both cases, TST and IGRAs cannot be used to predict whether LTBI will progress to active TB and whether the positive test results are due to an acute or past disease, as memory lymphocyte can produce cytokines even if the infection is ancient. New tests are currently under development to address these issues. As an example, a skin test named C-Tb combining the two previous tests has been developed. In a similar way as TST, C-Tb is a hypersensitivity skin test, using recombinant ESAT-6 and CFP-10 proteins, like IGRAs. It is used in endemic countries without the problem of TST false BCG-positive results [START_REF] Aggerbeck | C-Tb skin test to diagnose Mycobacterium tuberculosis infection in children and HIV-infected adults: A phase 3 trial[END_REF].

Researchers have recently identified some mycobacterial antigens specifically and naturally expressed during LTBI, useful to discriminate an active TB from a latent TB [START_REF] Meier | A systematic review on novel mycobacterium tuberculosisantigens and their discriminatory potential for the diagnosis of latent and active tuberculosis[END_REF].

Measurement of cytokines or TB biomarkers predicting the progression to active TB disease is also under investigation [START_REF] Carranza | Diagnosis for Latent Tuberculosis Infection: New Alternatives[END_REF]. However, such tests require more expensive facilities that are not always available in low-income countries.

Active tuberculosis

In active TB, several technologies are used to diagnose the disease: imaging techniques (chest X-Ray or computed tomography PET-CT scan), sputum smear positivity, culture-based methods and molecular tests [START_REF] Pai | Tuberculosis[END_REF].

Chest radiography plays a role in diagnosis and management of TB. Images of a patient with TB usually show lymphadenopathy, parenchymal disease, pleural effusion or bronchial stenosis which can help the diagnosis of TB and evaluate its impact on the lungs [START_REF] Nachiappan | Pulmonary tuberculosis: Role of radiology in diagnosis and management[END_REF]. Although imaging is a fundamental part of the diagnosis, the presence of the disease still needs to be confirmed via some microbiological investigations.

Sputum smear analysis is the most widely used test to diagnose active TB disease in low-and middle-income countries. A mycobacterial culture is performed on both solid and liquid media followed by a direct visualization of mycobacteria using light microscopy. However, this smear analysis has some limitations among HIV-positive patients (Méndez-Samperio, 2017) and in children [START_REF] Reuter | Challenges and controversies in childhood tuberculosis[END_REF]. Some of these individuals are sputum-scarce which makes this technique of analysis difficult to execute. In younger children, gastric lavage and bronchoalveolar lavage represents one of the main procedures to obtain a culture of MTB [START_REF] Reuter | Challenges and controversies in childhood tuberculosis[END_REF]. For sputum-scare adults, sputum induction can be performed using ultrasonic nebulization of hypertonic saline [START_REF] Méndez-Samperio | Diagnosis of Tuberculosis in HIV Co-infected Individuals: Current Status, Challenges and Opportunities for the Future[END_REF]. However, the sputum obtained from these techniques can be paucibacillary which might lead to misdiagnosis [START_REF] Méndez-Samperio | Diagnosis of Tuberculosis in HIV Co-infected Individuals: Current Status, Challenges and Opportunities for the Future[END_REF][START_REF] Reuter | Challenges and controversies in childhood tuberculosis[END_REF]. To overcome this problem, the lateral flow urine lipoarabinomannan assay (LF-LAM) has emerged as a novel technique to detect active TB disease amongst HIV-positive and sputum smear-negative patients [START_REF] Sabur | Diagnosing tuberculosis in hospitalized HIV-infected individuals who cannot produce sputum: Is urine lipoarabinomannan testing the answer[END_REF]. It for RIF and INH resistance, have been designed to provide further information on additional antibiotic resistances such as the detection of the main mutations responsible for the resistance [START_REF] Georghiou | Analytical performance of the Xpert MTB/XDR® assay for tuberculosis and expanded resistance detection[END_REF][START_REF] Ok | Rapid Molecular Diagnosis of Tuberculosis and Its Resistance to Rifampicin and Isoniazid with Automated MDR/MTB ELITe MGB® Assay[END_REF]. Nowadays, drug susceptibility testing is a major part of TB diagnosis in order to prescribe the most accurate treatment to the patient.

DNA sequencing, also known as targeted next-generation sequencing (tNGS) has been proposed to detect resistance on multiple gene regions. These assays, like the portable Deeplex®-MycTB assay (GenoScreen, Lille, France), allow early detection of multidrug resistance and the prescription of more appropriate TB treatments accordingly [START_REF] Feuerriegel | Rapid genomic first-and second-line drug resistance prediction from clinical Mycobacterium tuberculosis specimens using Deeplex-MycTB[END_REF].

Vaccines

Discovered by Calmette and Guérin in 1921 from a live-attenuated strain of Mycobacterium bovis, the BCG vaccine is currently the major vaccine used worldwide to prevent TB. Since 1974, BCG vaccine has been used as the main part of the WHO's Expanded Program on Immunization to expand and strengthen vaccination throughout the world. Nowadays, studies

show that the effects of BCG vaccination vary widely regarding the age of the patient, the endemic situation of the country, the form of TB (extra-or pulmonary) or the time of protection [START_REF] Ahmed | A century of BCG: Impact on tuberculosis control and beyond[END_REF]. As an example, its effectiveness varies from 80%, notably against meningeal TB in children, to 0% in endemic areas like India [START_REF] Mangtani | Protection by BCG vaccine against tuberculosis: A systematic review of randomized controlled trials[END_REF].

Moreover, the vaccine-induced immunity diminishes over time and provides a maximum protection from 10 to 20 years [START_REF] Rodrigues | How does the level of BCG vaccine protection against tuberculosis fall over time?[END_REF]. Several hypotheses explain these differences between effectiveness and protection time. Over time and due to different culture methods, many BCG strains with different genetic backgrounds like BCG Pasteur, Danish, Glaxo or Japan, have emerged worldwide [START_REF] Bottai | The BCG strain pool: Diversity matters[END_REF]. Those genetic and phenotypic differences inevitably affect the efficacy of the BCG vaccine. Genetic or nutritional difference between population may also have an effect on BCG vaccination [START_REF] Boisson-Dupuis | Inherited and acquired immunodeficiencies underlying tuberculosis in childhood[END_REF]. Moreover, pre-exposition to other mycobacteria (NTM or environmental bacteria) provides a protective immunity against TB that is not improved by BCG vaccination [START_REF] Ahmed | A century of BCG: Impact on tuberculosis control and beyond[END_REF]. Those pre-expositions may decrease the BCG vaccine efficacy in TBendemic areas. In this context, developing a more effective vaccine against MTB that provides protection amongst all populations regarding their age and the different forms of TB, is crucial to overcome this global health emergency.

In August 2020, the WHO Global report on TB announced 14 vaccines in Phase I, II or III trials.

Briefly, the new TB vaccine candidates are classified into different categories: preventive preexposure vaccines for vaccination prior to infection by MTB; preventive post-exposure vaccines to prevent LTBI to become active TB; therapeutic vaccines combined with antibiotic treatment to clear the mycobacterial infection or to prevent re-infection. These vaccine candidates are either whole cell-derived vaccines or subunit vaccines [START_REF] Schrager | The status of tuberculosis vaccine development[END_REF].

Mycobacterial whole cell-derived vaccines: These vaccines are derived from MTB, BCG or NTM strains. As they are whole cell vaccines, they contain many different antigenic components that stimulate the immune response in a more diverse way. There are two types of whole cellderived vaccines: viable or inactivated.

-Viable whole-cell vaccines are based on genetic attenuation of a live strain, like VPM1002 -a recombinant BCG strain expressing a membrane-perforating listeriolysin.

This live vaccine with VPM1002 is currently in clinical trial phase III for prevention of TB infection in infants.

-Inactivated whole-cell vaccines are based on killed or fractionated whole mycobacteria, like MIP/Immuvac -a heat-killed Mycobacterium indicus pranii vaccine.

They are considered safer than live vaccines since they do not contain any infectious particles but they may require multiple doses.

Subunit vaccines: Subunit vaccines target selected antigens that trigger an immune response against MTB. These vaccines are more suitable to prevent active TB among infected or latently infected individuals, enhancing prior immunity mediated by T cells. Indeed, the use of several selected antigens may not be sufficient to induce effective and full immunity against the pathogen but strengthen a pre-existing immune response, induced by BCG for example [START_REF] Sable | Tuberculosis vaccine development: Progress in clinical evaluation[END_REF]. The subunit vaccines are divided into two categories:

-Adjuvanted protein subunit vaccines: they are based on the administration of antigenic proteins along with an adjuvant in order to potentiate the host immune response, like M72 /AS01E (M72 immunogenic fusion protein with AS01E as an adjuvant system).

-Recombinant viral-vectored vaccines: These vaccines are similar to protein subunit vaccines but no adjuvant addition is needed. The use of a viral vector allows a more robust immune response to the antigens. As an example, TB/FLU-04L is based on an attenuated replication-deficient influenza virus vector expressing antigens Ag85A and ESAT-6.

Other strategies are under investigation to increase the BCG vaccination efficacy. For example, BCG revaccination was evaluated in different studies but the results are variable [START_REF] Rodrigues | Effect of BCG revaccination on incidence of tuberculosis in school-aged children in Brazil: The BCG-REVAC cluster-randomised trial[END_REF][START_REF] Nemes | Prevention of M. tuberculosis Infection with H4:IC31 Vaccine or BCG Revaccination[END_REF]. The main limitation to revaccination seems to be related to the exposure to NTM or LTBI which can alter the immune response of the host and be inefficient for an enhanced protection. Moreover, several studies question the intradermal route of immunization for the BCG vaccine. A recent study performed in rhesus macaques

shows that intravenous administration of BCG vaccine clearly increases the protection against a TB infection with an enhanced immune response [START_REF] Darrah | Prevention of tuberculosis in macaques after intravenous BCG immunization[END_REF].

Some progress has been made with regard to the development of improved or new vaccines against TB. However, a number of studies still need to be conducted to produce a new licensed effective vaccine for the future. Developing efficient therapeutic treatments remains a crucial part of the fight towards the eradication of TB.

III. Treatments

Patients developing active TB can be effectively cured with an appropriate anti-TB treatment regimen. Usually, it relies on a 6-month multidrug therapy with the main objectives of killing mycobacteria, eliminating latent infection and preventing acquisition of drug resistance.

Drug-sensitive tuberculosis

In the 1980s, a four antibiotic treatment was endorsed to treat TB with:

-Rifampicin (RIF): RIF and more generally rifamycins (like rifapentine or rifabutine) stalls bacterial transcription by inhibiting the DNA-dependent RNA polymerase [START_REF] Wehrli | Rifampin: Mechanisms of action and resistance[END_REF]. and the nucleic acid biosynthesis by targeting dihydrofolate reductase [START_REF] Timmins | Mechanisms of action of isoniazid[END_REF][START_REF] Unissa | Overview on mechanisms of isoniazid action and resistance in Mycobacterium tuberculosis[END_REF].

Isoniazid (INH

-Pyrazinamide (PZA): PZA is a prodrug converted to pyrazinoic acid (POA) via the bacterial pyrazinamidase PncA once inside the bacteria. Several mechanisms of action have been proposed over the years. The most accepted one is that POA disrupts the membrane potential and affects the membrane transport function at acidic pH (Y. [START_REF] Zhang | Mode of action of pyrazinamide: Disruption of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid[END_REF]. However, the importance of the acidic pH for the antibacterial activity of POA is still under debate [START_REF] Peterson | Uncoupling environmental pH and intrabacterial acidification from pyrazinamide susceptibility in Mycobacterium tuberculosis[END_REF]. Other recent studies suggest that POA binds the ribosomal protein S1/RpsA, the guanosine pentaphosphate synthase (GpsI) or the aspartate decarboxylase PanD [START_REF] Gopal | Pharmacological and Molecular Mechanisms Behind the Sterilizing Activity of Pyrazinamide[END_REF]. PanD is responsible for the formation of β-alanine from L-aspartate, which is essential for vitamin B5 and coenzyme A biosynthesis in MTB. Liaison between PanD and POA triggers PanD degradation by MTB, thus blocking biosynthesis of the essential Coenzyme A (Q. Sun et al., 2020).

-Ethambutol (EMB): EMB affects the synthesis of the mycobacterial cell wall by blocking the polymerization of arabinogalactan via the inhibition of arabinosyl transferases, EmbA, B and C [START_REF] Belanger | The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol[END_REF]. It also targets the glutamate racemase (MurI), a crucial enzyme of peptidoglycan (PG) biosynthesis pathway of MTB [START_REF] Pawar | Ethambutol targets the glutamate racemase of Mycobacterium tuberculosis-an enzyme involved in peptidoglycan biosynthesis[END_REF].

This treatment is given during six months: an intensive phase of two months with the four antibiotics, followed by a continuation phase of four months using RIF and INH only. The success rate of this multidrug therapy is up to 85% for a drug-susceptible TB (World Health Organization, 2020).

In 2014, the WHO published the first guidelines on LTBI management (updated in 2020) [START_REF] Organizat | WHO consolidated guidelines on tuberculosis. Module 1: Prevention. Tuberculosis preventive treatment[END_REF]. The first recommendation is about the identification of LTBI amongst selected populations such as HIV-positive individuals, children with TB household contacts, people initiating anti-TNF treatment, receiving dialysis or populations with higher risk factors (health workers, immigrants from high burden countries, prisoners or homeless people). The risk of progression from LTBI to active TB is higher for these people and an early anti-TB treatment would manage to kill the bacteria before LBTI turns into active TB. Whether the patient has LTBI or active TB, such long treatment periods generate many side effects such as hepatotoxicity, skin rash, gastrointestinal intolerance, nephrotoxicity and peripheral neuropathy [START_REF] Pai | Tuberculosis[END_REF]. These side effects and the long duration of treatment usually lead to a decrease in patient compliance (patient's adherence to treatment and willingness to follow medical advice). Thus, another part of the TB treatment is the directly observed therapy (DOT) introduced to increase treatment adherence among patients [START_REF] Karumbi | Directly observed therapy for treating tuberculosis[END_REF]. DOT is based on the supervision of drug intake by health professionals or at home by family members or community health workers. However, this measure does not solve the problem of poor adherence to TB treatment because of its inconveniences for the patient. Other techniques to promote patient compliance have been deployed, such as video observation therapy and the use of smartphones for drug intake, proven to be more effective than DOT [START_REF] Story | Smartphone-enabled video-observed versus directly observed treatment for tuberculosis: a multicentre, analyst-blinded, randomised, controlled superiority trial[END_REF]. Patient compliance is a crucial step in preventing the spread of TB and avoiding the development of drug-resistant strains, which are much more difficult to treat.

Drug-resistant tuberculosis

Resistant TB is caused by MTB that is resistant to one of the four first-line antibiotics.

Multidrug-resistant TB (MDR-TB) occurs when MTB is resistant to at least RIF and INH. In 2019, an estimated 3.3% of new cases and 18% of previously treated cases were MDR or RIF resistance (RR-TB) respectively (World Health Organization, 2020). The global cases were in India (27%), in China (14%) and in Russia (8%) (World Health Organization, 2020). Alarmingly, extensively drug resistant TB (XDR-TB) have emerged. XDR-TB are MDR-TB with additional resistance to fluoroquinolone (FQ) and at least one of the injectable drugs such as kanamycin, capreomycin and amikacin [START_REF] Seung | Multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis[END_REF]. Many cases have been reported in India or in sub-Saharan Africa [START_REF] Pietersen | Long-term outcomes of patients with extensively drug-resistant tuberculosis in South Africa: A cohort study[END_REF][START_REF] Prasad | Adverse drug reactions in tuberculosis and management[END_REF]. Even more worrying, several cases of totally resistant MTB, meaning untreatable TB, are already notified in India [START_REF] Udwadia | Totally drug-resistant tuberculosis in India[END_REF]. Treatment of MDR/XDR-TB is longer and can go up to 20 months [START_REF] Mirzayev | World health organization recommendations on the treatment of drug-resistant tuberculosis, 2020 update[END_REF]. The difficulty of treating these types of strains represent a major public health challenge to control the global TB epidemic. MTB acquires antibiotic resistances through mutations in chromosomal genes, for example during suboptimal or inappropriate treatment [START_REF] Seung | Multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis[END_REF]. However, patients with MDR-TB can be directly infected with an already resistant MTB strain -which is called transmitted resistance [START_REF] Leung | Transmission of multidrug-resistant and extensively drug-resistant tuberculosis in a metropolitan city[END_REF]. The mechanisms of drug resistance in MTB are modifications/alterations or overexpression of the drug targets, like the RNA polymerase for RIF, or an impairment of pro-drug activation, related to KatG mutation for INH [START_REF] Gygli | Antimicrobial resistance in Mycobacterium tuberculosis: Mechanistic and evolutionary perspectives[END_REF]. Antibiotics are also subject to efflux. Indeed, efflux pump are upregulated during antibiotic treatment to export antimicrobial compounds from the bacterial cell. This efflux lead to the maintenance of a sublethal intracellular antibiotic concentration that contributes to the selection of more resistant mutants. Therefore, the overexpression of efflux pump genes contributes to resistant phenotype observed in MTB [START_REF] Laws | Efflux pumps in Mycobacterium tuberculosis and their inhibition to tackle antimicrobial resistance[END_REF]. As a result, other antibiotics (named second-line antibiotics) need to be used to treat those resistant TB.

Groups

For MDR-TB, second line antibiotics are associated with different regimens. For some of these antibiotics, they were originally used to treat other infectious diseases but were proven to be active against MDR-TB. Among them:

-Aminoglycosides: amikacin (and streptomycin) are injectable drugs used to treat MDR-TB. They inhibit protein synthesis by binding to the 16S rRNA in the 30S small ribosomal subunit [START_REF] Recht | Basis for prokaryotic specificity of action of aminoglycoside antibiotics[END_REF].

-Fluoroquinolones (FQ): levofloxacin and moxifloxacin are the most valuable second line antibiotics against MDR-TB. They inhibit DNA gyrase and topoisomerase IV which are essential for bacterial DNA replication [START_REF] Pranger | The Role of Fluoroquinolones in the Treatment of Tuberculosis in 2019[END_REF].

-Carbapenems: meropenem, ertapenem and imipenem belong to a subclass of β-Lactams. Their action is based on the inhibition of the PG synthesis. They are not the main second-line antibiotics used but new evidence of their efficacy has led to clinical studies and dose optimization for their inclusion in future MDR-TB treatments (P. [START_REF] Kumar | Repurposing of carbapenems for the treatment of drug-resistant tuberculosis[END_REF].

-Others drugs class: linezolid is an oxazolidinone which binds to the bacterial ribosome (the 50S subunit) and prevents protein synthesis (B. [START_REF] Singh | Linezolid for drug-resistant pulmonary tuberculosis[END_REF]; clofazimine is an antileprosy drug which supposedly acts via the release of reactive oxygen species (ROS) damaging DNA, lipids and proteins in the bacterium [START_REF] Lechartier | Mode of action of clofazimine and combination therapy with benzothiazinones against Mycobacterium tuberculosis[END_REF]; ethionamide and prothionamide are prodrugs activated by EthA which then bind to

InhA and inhibit the cell wall synthesis (F. [START_REF] Wang | Mechanism of thioamide drug action against tuberculosis and leprosy[END_REF].

In 2012, the Food and Drug Administration (FDA) approved bedaquiline (BDQ), a diarylquinoline, as a new anti-TB drug (the first in over 40 years) [START_REF] Cox | FDA Approval of Bedaquiline -The Benefit-Risk Balance for Drug-Resistant Tuberculosis[END_REF]. In 2014, the European Medicines Agency (EMA) also approved BDQ and another antibiotic called delamanid (a nitroimidazo-oxazole derivative) for pulmonary MDR-TB (Zumla et al., 2015a).

BDQ inhibits the mycobacterial F-ATP synthase, a key respiratory chain enzyme [START_REF] Hards | Bactericidal mode of action of bedaquiline[END_REF]; delamanid blocks the mycobacterial cell wall synthesis [START_REF] Xavier | Delamanid: A new armor in combating drug-resistant tuberculosis[END_REF].

As new antibiotics, they are a major part of the treatment of MDR-TB according to WHO 2020 guidelines [START_REF] Mirzayev | World health organization recommendations on the treatment of drug-resistant tuberculosis, 2020 update[END_REF].

The choice of antibiotics used for the MDR-TB treatment depends on many factors: the strain resistance, the previous use of antibiotics, the age of the patient or the side effects profile (World Health Organization, 2020). Each treatment needs to be personalized depending of the case. Success rate of MDR/RR-TB treatments is around 57% (World Health Organization, 2020). Antibiotics used to treat resistant TB are summed up in Table 1.

Improvement of current TB treatments as well as development of new ones remain a major challenge to eradicate TB.

Newly approved drugs and the current pipeline

According to the latest global TB report in 2020, 22 drugs for TB treatment (sensible or resistant) are currently in Phase I, II or III trials. Apart from BDQ and delamanid which are still under review for new regimens/association and new purpose, pretomanid is the latest anti-TB drug approved by FDA in 2019 for adults with pulmonary XDR-TB or non-responsive MDR-TB [START_REF] Deb | Pretomanid: The latest USFDA-approved anti-tuberculosis drug[END_REF]. This antibiotic is a nitroimidazo-oxazine and has the same mechanism of action that delamanid. [START_REF] Silva | Shortened tuberculosis treatment regimens: What is new?[END_REF].

Name

Shorter treatments for drug-resistant TB are also under investigation such as the endTB trial for MDR-TB (with BDQ or delamanid involved) or the Standard Treatment Regimen of Anti-Tuberculosis Drugs for Patients with MDR-TB (STREAM) by which the WHO recommends to shorten the treatment for uncomplicated MDR-TB (without resistance to FQ) from 20 month to 9-month in 2016 (A. [START_REF] Lee | Current and future treatments for tuberculosis[END_REF]. In 2020 and in accordance with WHO guidelines, BDQcontaining regimens are now the most promising regimens for MDR-TB treatments (WHO, 2020b).

In the last few years, there has been an increasing interest to enhance the host immune response as an additional strategy to improve TB treatment. In the current pipeline, many trials are performed to examine the efficacy and the safety of host directed therapies to shorten TB treatment or prevent lung damages (Young et al., 2020). These drugs show interesting promises when adjunct to current TB treatments. This will be discussed further in this thesis.

B. Host-pathogen interaction

I. Cycle of infection

MTB, once inhaled, penetrates deep into the host lungs of the new host and reaches the alveoli. There, MTB is recognized by the host immune system, which triggers an immune response and the recruitment of effector cells. This immune response leads to the formation of a granuloma, a special structure that helps controlling mycobacterial infection (Figure 2).

Mycobacterium tuberculosis

Human TB is mainly caused by bacteria belonging to the MTB complex (MTBC). This group contains many species closely related (> 99% nucleotide sequence identity) including MTB, M. africanum, M. canettii and other animal adapted M. bovis, M. microti or M. caprae [START_REF] Gagneux | Ecology and evolution of Mycobacterium tuberculosis[END_REF]. MTB belongs to the Mycobacteriaceae family which contains the genus Mycobacterium [START_REF] Cook | Physiology of Mycobacteria[END_REF]. In 1998, the genome of MTB H37Rv (laboratory strain) was fully sequenced, allowing a deeper understanding of the biology of this mycobacterium. Its genome size is about 4.4.10 6 base pairs and contains approximately 4,100 genes with a high Guanine-Cytonine (GC) content of 65.6 % (S. T. [START_REF] Cole | Deciphering the biology of mycobacterium tuberculosis from the complete genome sequence[END_REF]. MTB is an obligate human pathogen without animal or environmental reservoirs. It is an aerobe, a facultative intracellular and a slow-growing bacterium (with a generation time of 20 hours in optimal laboratory conditions).

The dominant feature of MTB is its particular cell envelope composed of proteins, abundant lipids and carbohydrates [START_REF] Cook | Physiology of Mycobacteria[END_REF]. The thick cell wall of mycobacteria has low permeability and plays a key role in intrinsic antibiotic resistance [START_REF] Maitra | Cell wall peptidoglycan in Mycobacterium tuberculosis: An Achilles' heel for the TBcausing pathogen[END_REF].

Furthermore, many components of this cell envelope are linked to the virulence of MTB such as proteins inhibiting the antimicrobial functions of macrophages or metal-transporter proteins [START_REF] Maitra | Cell wall peptidoglycan in Mycobacterium tuberculosis: An Achilles' heel for the TBcausing pathogen[END_REF]. This mycobacterial cell envelope is composed of several layers including the cytoplasmic membrane, the cell wall (composed of PG, arabinogalactan and mycolic acids), surface lipids and the capsule:

« élément sous droit, diffusion non autorisée » "copyrighted material, unauthorized distribution" [START_REF] Cambier | Host Evasion and Exploitation Schemes of Mycobacterium tuberculosis[END_REF].

Figure 2. Cell cycle of MTB infection

Cytoplasmic membrane: It contains glycerophospholipids such as phosphatidyl ethanolamine, phosphatidyl inositol and phosphatidylinositol mannosides (PIM). There are also lipomannans (LMs) and lipoarabinomannans (LAMs) which are anchored in the plasma membrane and extend towards the PG. These lipids are imperative for cell wall integrity and play an important role in the interaction with the host immune response [START_REF] Fukuda | Critical Roles for Lipomannan and Lipoarabinomannan in Cell Wall Integrity of Mycobacteria and Pathogenesis of Tuberculosis[END_REF].

Cell wall: It is the middle layer of the envelope and is composed of PG, arabinogalactan and mycolic acids (MA) (Dulberger et al., 2019).

-Peptidoglycan: PG is composed of N-acetylglucosamine (NAG) and N-acetylmuramic disaccharide (NAM) cross-linked by peptides which help maintaining the integrity of the membrane and the cell shape.

-Arabinogalactan: The middle layer of the cell wall core is made of arabinogalactan sugars (arabinose chains linked to galactose residues). Arabinogalactan polymers are connected to PG.

-Mycolic acids: MA are long-chain fatty acids that composed the main part of the mycomembrane. They are free or attached to trehalose sugar forming trehalose monomycolate (TMM) or trehalose dimycolate (TDM) and contribute to the impermeability of the mycobacterial envelope [START_REF] Chiaradia | Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane[END_REF].

Surface lipid phthiocerol dimycocerosate (PDIM): PDIM, located at the surface of MA, are glycolipids crucial for MTB infection and virulence. They are associated with immune escape and attenuation of the host immune response by masking patterns recognized by the immune system [START_REF] Cambier | Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids[END_REF].

Capsule:

The outermost structure is a capsule composed of polysaccharides and proteins. The capsule plays a role in host-pathogen interactions and virulence (Dulberger et al., 2019).

Physiopathology

The infection begins when MTB is inhaled by the host via the respiratory tract and reaches the alveolar space. It is composed of several cell types including epithelial pulmonary alveolar type I cells (which covers more than 95% of the alveolar surface) and type II pneumocytes and immune cell defenses [START_REF] Hussell | Alveolar macrophages: Plasticity in a tissue-specific context[END_REF]. In this alveolar space, MTB encounters the dominant defense cells and the first line of the immune defense: the alveolar macrophages (AM) (S. B. [START_REF] Cohen | Alveolar Macrophages Provide an Early Mycobacterium tuberculosis Niche and Initiate Dissemination[END_REF]. AM are distinct from interstitial macrophages located between airway epithelium and the blood vessels. Indeed, AM reside in a compartment with environmental specificities such as the level of O2, the presence of airway mucus or the permanent contact with environmental antigens [START_REF] Hussell | Alveolar macrophages: Plasticity in a tissue-specific context[END_REF] . The airspace represents a specific environment which needs to be preserved from pathogens and debris but also from structure destruction by the inflammatory response of the immune system. Therefore, AMs have attenuated inflammatory bursts with production of anti-inflammatory mediators such as transforming growth factor-β (TGF-β) and IL-10 to avoid a too strong proinflammatory response in the alveoli [START_REF] Hussell | Alveolar macrophages: Plasticity in a tissue-specific context[END_REF].

Once inhaled and in the alveoli, MTB is phagocytized by AM but also other immune cells such as dendritic cells. More rarely, MTB enters directly into alveolar epithelial cells [START_REF] Scordo | Alveolar Epithelial Cells in Mycobacterium tuberculosis Infection: Active Players or Innocent Bystanders?[END_REF]. Subsequently, dendritic cells transport MTB (or mycobacterial antigens) to pulmonary lymph nodes for the development of the adaptive immune response [START_REF] Pai | Tuberculosis[END_REF]. It was also showed that MTB-infected AMs relocate from the airways to the pulmonary interstitium, allowing the dissemination of MTB to other immune cell types and lymph nodes (S. B. [START_REF] Cohen | Alveolar Macrophages Provide an Early Mycobacterium tuberculosis Niche and Initiate Dissemination[END_REF]. Therefore, recognition and internalization of the pathogen by this line of defense is the first step of the activation of the protective immune response needed for controlling the infection.

Granuloma formation

The migration of MTB-infected AMs initiates the host immune response with the production of different cytokines and chemokines. This response leads to the recruitment of additional cells into the site of infection which form a cell mass: the granuloma [START_REF] Pagán | The Formation and Function of Granulomas[END_REF]. The granuloma architecture is complex, with several layers composed of diverse cell types. At the early stage of its development, the granuloma is highly vascularized, allowing the recruitment of monocytes, macrophages, dendritic cells, neutrophils, lymphocytes CD4 + and CD8 + T cells and B cells [START_REF] Ramakrishnan | Revisiting the role of the granuloma in tuberculosis[END_REF]. At a later stage, the center of the granuloma becomes necrotic area, hypoxic and deprived of nutrients, known as the caseum. This latter is surrounded by different populations of macrophages activated or not, infected or not (such as epithelioid cells and foamy macrophages with lipid droplets), lymphocytes and a peripheral fibrotic layer (synthetized by fibroblasts) (Figure 3) [START_REF] Russell | Foamy macrophages and the progression of the human tuberculosis granuloma[END_REF][START_REF] Pagán | The Formation and Function of Granulomas[END_REF].

The granuloma is an environment of exchange between the pathogen and the host. At first, its structure was thought to be necessary and beneficial for the host to control and clear the MTB infection without excess inflammatory damages [START_REF] Saunders | Restraining mycobacteria: Role of granulomas in mycobacterial infections[END_REF]. However, further studies show that granuloma also contributes to MTB proliferation and dissemination. Indeed, in order to survive inside the granuloma, MTB has developed several defense strategies, mostly demonstrated in studies by using zebrafish or nonhuman primate [START_REF] Cadena | Heterogeneity in tuberculosis[END_REF]. For example, MTB secretes a 6kDa early secreted antigenic target (ESAT-6), which induces host production of matrix metalloproteinase 9 (MMP9) by epithelial cells [START_REF] Volkman | Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium[END_REF]. This MMP9 production results in an enhancement of the recruitment of new macrophages which may in turn be infected by MTB, contributing to the development of the granuloma and the multiplication of the bacterium [START_REF] Volkman | Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium[END_REF]. Furthermore, MTB influences the polarization of macrophages, by increasing the IL-10 production of infected macrophages, driving the differentiation of naïve macrophages into a more permissive antiinflammatory population [START_REF] Mcnab | Type I IFN Induces IL-10 Production in an IL-27-Independent Manner and Blocks Responsiveness to IFN-γ for Production of IL-12 and Bacterial Killing in Mycobacterium tuberculosis -Infected Macrophages[END_REF].

MTB inside the granuloma can enter in a dormant state to avoid recognition and elimination (Peddireddy et al., 2017). More recently, positron emission tomography and computed tomography imaging (PET/CT) was proven to be helpful to study the granuloma dynamics and to distinguish LTBI which are more likely to progress into active TB in macaque [START_REF] Lin | PET CT Identifies Reactivation Risk in Cynomolgus Macaques with Latent M. tuberculosis[END_REF] and in human [START_REF] Esmail | Characterization of progressive HIVassociated tuberculosis using 2-deoxy-2-[18F]fluoro-D-glucose positron emission and computed tomography[END_REF]. Dysregulation of the host immune system may lead to progression of the infection inside the granuloma, collapsing of the caseum and releasing of the pathogen into airways (where MTB can be transmitted by a TB-infected symptomatic individual) or to other tissues (where MTB infects new cell hosts) [START_REF] Ramakrishnan | Revisiting the role of the granuloma in tuberculosis[END_REF].

Another study demonstrates that the fate of the granuloma is also dependent on the inflammatory status inside the granuloma [START_REF] Marakalala | Inflammatory signaling in human tuberculosis granulomas is spatially organized[END_REF]. Indeed, inside the caseum, leukotriene-A4 Hydrolase (LTA4H) synthesizes leukotriene B4 (LTB4), a pro-inflammatory « élément sous droit, diffusion non autorisée » "copyrighted material, unauthorized distribution" Figure 3. Host-MTB interaction influences the granuloma fate [START_REF] Cadena | Heterogeneity in tuberculosis[END_REF]. eicosanoid that is associated with the production of TNF-α [START_REF] Marakalala | Inflammatory signaling in human tuberculosis granulomas is spatially organized[END_REF]. TNF-α is a key cytokine for immune response that triggers granuloma formation. However, an excess of TNF-α is deleterious as it induces mitochondrial ROS (mtROS) production, increasing the microbicidal activity of macrophages, but also inducing programmed necrosis which then increases bacterial proliferation and spreading [START_REF] Roca | TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species[END_REF].

Granuloma therefore represents an extremely dynamic structure where MTB is targeted with a wide range of bactericidal mechanisms. However, over time, MTB has developed several strategies to counteract those bactericidal mechanisms in order to proliferate within the macrophages and further in the granuloma.

II.

Bactericidal mechanisms of macrophages

Macrophages: an overview

There are two major types of macrophages in the lung: the AM and the interstitial macrophage (IM). AM are resident lung macrophages derived from fetal primitive yolk sac [START_REF] Guilliams | Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF[END_REF] whereas IM are thought to be recruited during MTB infection (S. [START_REF] Srivastava | Beyond macrophages: The diversity of mononuclear cells in tuberculosis[END_REF].

They are commonly divided into separate groups M1 and M2 derived from polarization of naïve macrophages [START_REF] Lugo-Villarino | Macrophage polarization: Convergence point targeted by Mycobacterium tuberculosis and HIV[END_REF]. M1 macrophages, classically called activated macrophages or pro-inflammatory macrophages (stimulated by lipopolysaccharides (LPS) or interferon-γ (IFN-γ)), are characterized by a high antigen presentation and a high expression of pro-inflammatory cytokines (IL-12, IL-23 and TFN-α). M2 macrophages, alternatively called activated macrophages or anti-inflammatory macrophages, express different patterns on their surface which trigger a different immune response from M1 (low production of IL-12 and TNF-α and higher production of IL-10). M2 macrophages are divided into subcategories M2a, M2b, M2c, and M2d depending on the origin of their stimulation (IL-4 or IL-13; TLR ligands and IL-1β; glucorticoïdes, IL-10 or TGF-β; IL-10 and vascular endothelial growth factors (VEGF), respectively). They express various surface markers, secrete diverse cytokines and have different biological functions (Y. [START_REF] Wang | M1 and M2 macrophage polarization and potentially therapeutic naturally occurring compounds[END_REF]. In vitro, specific cytokines or stimulating factors are likely to produce one type of macrophages. For example, granulocyte-macrophage colony-stimulating factor (GM-CSF) tends to produce M1 macrophages ("M1-like macrophages") while macrophage colony-stimulating factor (M-CSF) produces M2 macrophages ("M2-like macrophages") [START_REF] Murray | Macrophage Activation and Polarization: Nomenclature and Experimental Guidelines[END_REF]. However, this M1 and M2

polarization must be nuanced because in vivo, macrophages possess a spectrum of different functions regarding their activation and their environmental localization [START_REF] Murray | Macrophage Activation and Polarization: Nomenclature and Experimental Guidelines[END_REF].

Macrophages possess on their surface pattern recognition receptors (PRRs) which recognize conserved microbial motifs, also called pathogen-associated molecular patterns (PAMPs), including MTB's components [START_REF] Hussell | Alveolar macrophages: Plasticity in a tissue-specific context[END_REF]. Among these surfaces: PRRs, TLRs, C-type lectin receptors (CLRs) like mannose receptors (MR), scavenger receptors (SR) and C-type lectin DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) are known to have a role in host-MTB interaction [START_REF] Guirado | Macrophages in tuberculosis: Friend or foe[END_REF]. For example, TLRs (notably TLR2 and TLR4) link the 19kDa lipoprotein LM and other PIMs from MTB [START_REF] Faridgohar | New findings of Toll-like receptors involved in Mycobacterium tuberculosis infection[END_REF]. These recognitions trigger immune signaling cascade within the host, starting from the myeloid differentiation primary response protein 88 (MyD88) and leading to the translocation of the nuclear factor-kappa B (NF-κB), which promotes the expression of pro-inflammatory cytokines and activates genes expression involves in the regulation of the host protection [START_REF] Hossain | Pattern recognition receptors and cytokines in Mycobacterium tuberculosis infection -The double-edged sword?[END_REF]. However, certain PRRs are also used by the pathogen to decrease the host immune response. Indeed, MR interacts with PAMPs mannosylated LAM (ManLAM), LAM and PIMs on MTB surface. This binding triggers an antiinflammatory response of the macrophage by stimulating the release of anti-inflammatory cytokines, inhibiting the production of pro-inflammatory IL-12 and preventing oxidative responses, thereby enhancing MTB survival within the host [START_REF] Killick | Receptor-mediated recognition of mycobacterial pathogens[END_REF]. Also, DC-SIGN has a dual role in MTB infection. Firstly, DC-SIGN was reported to render the cells more prone to be infected by MTB [START_REF] Tailleux | DC-SIGN Induction in Alveolar Macrophages Defines Privileged Target Host Cells for Mycobacteria in Patients with Tuberculosis[END_REF]. However, using DC-SIGN depleted macrophages, it has also been shown that DC-SIGN has an anti-inflammatory role upon MTB infection [START_REF] Lugo-Villarino | The C-type lectin receptor DC-SIGN has an anti-inflammatory role in Human M(IL-4) macrophages in response to Mycobacterium tuberculosis[END_REF].

Recognition is the first interaction between macrophages and MTB. Macrophages are defense immune cells with many bactericidal mechanisms used to kill various pathogens. These mechanisms will be described below. MTB defense mechanisms against them will then be outlined.

Phagosome

Pathogen recognition by macrophage triggers several signaling pathways that result in the uptake of the pathogen. The internalization of the pathogen, also called phagocytosis, forms an intracellular vacuole delimited by a bilayer membrane named the phagosome (Elisabeth [START_REF] Tjelle | Phagosome dynamics and function[END_REF]. This latter then undergoes a maturation process and acquires bactericidal properties and clearance processes [START_REF] Rosales | Phagocytosis: A Fundamental Process in Immunity[END_REF]. The transition between different stages of the phagosome and the regulation of membrane fusion are mostly mediated by the Rab family GTPases ((Elisabeth [START_REF] Tjelle | Phagosome dynamics and function[END_REF][START_REF] Gutierrez | Functional role(s) of phagosomal Rab GTPases[END_REF].

The newly phagosome merges directly with the early endosome mainly via the small GTPase Rab5. Rab5 recruits the early endosome antigen 1 (EEA1) and class III PI-3K human vacuolar protein-sorting 34 (VPS34). VPS34 produces phosphatidylinositol 3-phosphate (PI3P) which retains EEA1 at the cytosolic leaflet of the phagosome and promotes the recruitment of proteins involved in phagosomal maturation, including Rab7 [START_REF] Christoforidis | The rab5 effector EEA1 is a core component of endosome docking[END_REF]. The pH of the early phagosome is estimated between 6.1 -6.5, which too high to kill bacteria [START_REF] Rosales | Phagocytosis: A Fundamental Process in Immunity[END_REF].

As the phagosome matures, Rab5 is replaced by Rab7. This Rab GTPase regulates the fusion of the early phagosome with late endocytic compartments. The transition between the two Rab remains elusive but several studies suggest the implication of the homotypic fusion and protein sorting (HOPS) complex VPS41 subunit or the SAND1/Mon1-Ccz1 complex for the recruitment of Rab7 [START_REF] Barry | Impaired stimulation of p38α-MAPK/Vps41-hops by lps from pathogenic coxiella burnetii prevents trafficking to microbicidal phagolysosomes[END_REF][START_REF] Cabrera | The mon1-ccz1 gef activates the rab7 gtpase ypt7 via a longin-fold-rab interface and association with pi3p-positive membranes[END_REF]. In the meantime, other Rab (Rab 1 and Rab2) regulate phagosome trafficking with the endoplasmic reticulum (ER), post-Golgi and ER-Golgi intermediate compartment [START_REF] Gutierrez | Functional role(s) of phagosomal Rab GTPases[END_REF]. Fusion with late endosomes and elements of the Golgi complex allows the incorporation of lysosomal-associated membrane proteins and luminal proteases (cathepsins, proteases and hydrolases) within the phagosome [START_REF] Kinchen | Phagosome maturation: Going through the acid test[END_REF]. Also, the lumen of the phagosome becomes more acidic (pH 5.5 -6.0) via a gradual accumulation of V-ATPases on the membrane and the translocation of protons (H + ) [START_REF] Kinchen | Phagosome maturation: Going through the acid test[END_REF].

The last stage of the phagosome maturation involves fusion with lysosomes to become the phagolysosome [START_REF] Nguyen | Better Together: Current Insights Into Phagosome-Lysosome Fusion[END_REF]. The phagolysosome has a very acidic lumen (pH around 5.0 -5.5). It contains hydrolytic enzymes including cathepsins, proteases, lysozymes and lipases which have their optimal efficacy at this pH, allowing bacterial clearance [START_REF] Nguyen | Better Together: Current Insights Into Phagosome-Lysosome Fusion[END_REF].

Reactive oxygen species and reactive nitrogen species

ROS are reactive species derived from molecular oxygen. They are implicated in numerous processes in immune cells, such as the regulation of immune pathways (Nathan & Cunningham-Bussel, 2013). ROS also exert an antimicrobial activity against bacteria [START_REF] Sies | Reactive oxygen species (ROS) as pleiotropic physiological signalling agents[END_REF]. They are divided into two subclasses: (i) highly reactive free radicals such as superoxide anion (O2 •-) or radical hydroxyl (HO • ) and (ii) non-radical species including H2O2 (Nathan & Cunningham-Bussel, 2013). ROS are produced in response to various signals,

including some related to the presence of a pathogen (recognition of the PAMPs) or diverse cytokines such as TNF [START_REF] Roca | TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species[END_REF]. There are two major sources of ROS inside macrophages. First of all, NADPH oxidases produce ROS into the phagosomal lumen (precisely the phagocyte NADPH oxidase NOX2 isoform). The enzymatic complex is composed of two membrane-bound subunits, cytosolic components and a small G protein Rac which catalyze the production of superoxide from oxygen and NADPH [START_REF] Babior | NADPH oxidase[END_REF]. Secondly, mitochondria-derived ROS (mtROS) are produced during the generation of ATP by the mitochondrial respiratory electron transport chain (ETC). The most abundant mtROS is O2 •- which will be dismutated by the superoxide dismutases (SOD) into H2O2 [START_REF] Herb | Functions of ros in macrophages and antimicrobial immunity[END_REF]. Other oxidases in subcellular compartments also produce H2O2 such as peroxisome by long chain fatty acid oxidation, endoplasmic reticulum by protein oxidation or the lipoxygenases generating lipid-derived ROS [START_REF] Sies | Reactive oxygen species (ROS) as pleiotropic physiological signalling agents[END_REF].

The generation of ROS within the cell has to be finely regulated. Indeed, a high intracellular level concentration of ROS may be detrimental for the host. ROS toxicity is related to the impairment of structural and functional properties of proteins by binding and damaging the amino-acids like arginine, threonine and lysine [START_REF] Sies | Reactive oxygen species (ROS) as pleiotropic physiological signalling agents[END_REF]. Moreover, they also directly bind and damage DNA or nucleic acids through oxidation of nucleotides pool or trigger lipid peroxidation via oxidation of fatty acid [START_REF] Shastri | Role of oxidative stress in the pathology and management of human tuberculosis[END_REF]. However, a certain level of intracellular ROS is crucial for many cellular processes including cell proliferation, differentiation, apoptosis and defense mechanisms against pathogens. For example, individuals with chronic granulomatous disease, a genetic disease linked to a mutation in the gene encoding for NOX2, have a lack of ROS production and an increased susceptibility to bacterial infection [START_REF] Babior | NADPH oxidase[END_REF]. Moreover, ROS mediate immune pathways, acting as a regulatory agent in the immune system. For example, ROS activate the nucleotide-binding oligomerization domain (NOD)-like receptor containing pyrin domain 3 (NRLP3) inflammasome, responsible for the maturation of pro-inflammatory cytokines such as IL-1β

and IL-18 (see section B.II.c.) [START_REF] Abais | Redox Regulation of NLRP3 Inflammasomes: ROS as Trigger or Effector?[END_REF]. Finally, mtROS impede the pro-inflammatory response of infected macrophages. They induce covalent disulfide linkage of NF-κB essential modulator (NEMO) leading to the activation of the NF-κB pathway and terminally to the secretion of pro-inflammatory cytokines [START_REF] Herb | Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO[END_REF].

Another form of reactive species is used by the macrophage against bacterial pathogens: the nitric oxide (NO) and its reactive nitrogen species (RNS) derivatives. NO is produced by the nitric oxide synthase (NOS). Three isoforms of this enzyme exist including the inducible NOS (iNOS or NOS2) [START_REF] Wink | Nitric oxide and redox mechanisms in the immune response[END_REF]. Expression of iNOS is induced by TLR ligands like LPS and inflammatory cytokines, including IFN-γ. iNOS catalyzes NO production by converting Larginine into L-citrulline, which is subsequently converted into RNS [START_REF] Wink | Nitric oxide and redox mechanisms in the immune response[END_REF]. NO may also be converted into other radicals such as nitrogen dioxide (NO2 . ), nitrous trioxide (N2O3) or very reactive oxidant peroxynitrile (OONO -) [START_REF] Wink | Nitric oxide and redox mechanisms in the immune response[END_REF]. These radicals are known to switch off and on certain cellular pathways, but also to have a direct effect on bacteria. Interestingly, NO plays different roles between human and animal macrophages.

Indeed, NO synthase-deficient mice exhibit increased susceptibility to MTB, while human macrophages generate little NO in response to inflammatory stimuli even though MTBinfected individuals have higher levels of NO in their lungs [START_REF] Macmicking | Identification of nitric oxide synthase as a protective locus against tuberculosis[END_REF]Choi et al., 2012). The effect of NO in human macrophages is still a matter of debate. Findings differ about NO production in human macrophages in vivo and ex vivo [START_REF] Nathan | Role of iNOS in Human Host Defense[END_REF] and nitrites have been suggested to be produced by the bacteria themselves [START_REF] Cunningham-Bussel | Nitrite produced by Mycobacterium tuberculosis in human macrophages in physiologic oxygen impacts bacterial ATP consumption and gene expression[END_REF].

Inflammasome

Inside macrophages, recognition of PAMPs by membrane PRRs (e. i. TLRs) or cytosolic PRRs (e.

i. nucleotide-binding oligomerization domain-like receptors (NOD-like receptors or NLRs)) is the core of the immune responses. Among these responses, activation of NLRs triggers the assembly of protein complexes called inflammasomes [START_REF] Lamkanfi | Mechanisms and functions of inflammasomes[END_REF].

Inflammasomes mainly activate caspase 1 resulting in the production of pro-inflammatory IL-1ß and IL-18 molecules. IL-1β and IL-18 are present in the cell as inactive precursors (pro-IL-1β, not constitutively expressed and pro-IL-18, constitutively expressed) which are then cleaved into mature cytokines (Chong & Sullivan, 2007a). IL-1β binds to its receptor IL-1R which stimulates the release of pro-inflammatory cytokines such as IL-6 and TNF-α [START_REF] Dinarello | A clinical perspective of IL-1β as the gatekeeper of inflammation[END_REF]. Similarly, IL-18 binds to IL-18R. Presence of IL-1R or IL-18R results in the activation of a signaling cascade and transcription factors activation, such as NF-κB, all regulating the inflammation process (P. T. [START_REF] Liu | Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response[END_REF]. Inflammasomes also induce a specific death of infected cells named pyroptosis. Pyroptosis is a cell death which limits the replication of intracellular pathogens by eliminating infected immune cells, exposing released bacteria to other phagocytic cells and releasing inflammatory content such as IL-1β and IL-18 [START_REF] Bergsbaken | Pyroptosis: Host cell death and inflammation[END_REF].

NOD-like receptor family, pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes are two inflammasomes that activate caspase 1 in response to various stimuli, notably upon MTB infection [START_REF] Briken | Mycobacterium Tuberculosis genes involved in regulation of host cell death[END_REF]. NLRP3 inflammasome activation requires a priming signal with the up-regulation of its expression (linked to TLRs or NLRs stimulation of the cell) followed by a stimulus like ionophores (K + , Ca 2+ , Na + and Cl -), mtROS or lysosomal content as a result of membrane damages to be active [START_REF] Kelley | The NLRP3 inflammasome: An overview of mechanisms of activation and regulation[END_REF]. AIM2 is activated following the detection of exogenous double-stranded DNA. This binding leads to a conformational change allowing the recruitment of the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) protein and caspase 1 [START_REF] Saiga | Critical role of AIM2 in Mycobacterium tuberculosis infection[END_REF]. A study shows that Aim2 -/-mice have an increased susceptibility to MTB and decreased levels of IL-1β and IL-18 in the lungs after MTB infection [START_REF] Saiga | Critical role of AIM2 in Mycobacterium tuberculosis infection[END_REF]. MTB activation of AIM2 inflammasome seems to be related to the ESX-1 secretion system, as MTB ESX-1 deficient mutant fails to inhibit the AIM2-inflammasome activation [START_REF] Shah | Cutting Edge: Mycobacterium tuberculosis but Not Nonvirulent Mycobacteria Inhibits IFN-β and AIM2 Inflammasome-Dependent IL-1β Production via Its ESX-1 Secretion System[END_REF]. Other

NODs are implicated in the reconnaissance of pathogen notably NOD2 in the case of MTB, subsequently resulting in NF-κB activation (A. K. [START_REF] Pandey | Nod2, Rip2 and Irf5 play a critical role in the type I interferon response to Mycobacterium tuberculosis[END_REF].

Autophagy

Autophagy is a mechanism of intracellular degradation by which cytoplasmic materials are delivered to the lysosome for degradation. The main form of autophagy is named macroautophagy (refers here as autophagy) (M. Y. [START_REF] Wu | Autophagy and Macrophage Functions: Inflammatory Response and Phagocytosis[END_REF]. Autophagy maintains cell homeostasis by removing defective organelles and protein aggregates, but also by playing a role in cell differentiation and development. Autophagy is also essential for immunity by triggering intracellular pathogens degradation, which is a type of autophagy called xenophagy [START_REF] Mintern | Autophagy and mechanisms of effective immunity[END_REF]; M. Y. [START_REF] Wu | Autophagy and Macrophage Functions: Inflammatory Response and Phagocytosis[END_REF]. Moreover, there is an inter-play between autophagy and the inflammasome. Induction of autophagy is dependent on cytokines levels.

For example, IFN-γ, TNF-α and IL-1 induce autophagy while IL-4, IL-10 and IL-13 are inhibitory [START_REF] Harris | Autophagy and cytokines[END_REF]. Autophagy also self-regulates the production of cytokines including IL-1β and IL-18 via the down-regulation of inflammasomes, thus influencing inflammation inside the cells [START_REF] Saitoh | Regulation of inflammasomes by autophagy[END_REF].

Autophagy is a process involving the formation of a double membrane structure named autophagosome. This formation is a four-step process: (i) autophagosome initiation, (ii) autophagosome elongation, (iii) autophagosome closure and (iv) autophagosome fusion with lysosomes where autophagosomal contents are degraded [START_REF] Mintern | Autophagy and mechanisms of effective immunity[END_REF].

Initiation of the autophagosome formation is highly regulated by signaling pathways involving more than 30 autophagy related proteins (ATG) (M. Y. [START_REF] Wu | Autophagy and Macrophage Functions: Inflammatory Response and Phagocytosis[END_REF]. The first step mostly occurs via activation of the mammalian target of rapamycin (mTOR) that binds to the serine/threonine kinase ULK1 complex. This complex then activates the class III phosphoinositide 3-kinase (PI3K) complex (composed notably of VPS34, Beclin-1 and ATG14L) that generates PI3P, thus marking the autophagosome formation [START_REF] Ktistakis | Digesting the Expanding Mechanisms of Autophagy[END_REF].

Elongation of this structure is directed by covalent linkage between ATG12 with ATG5/ATG16L1 and microtubule-associated light chain 3 (LC3), also known as ATG8. LC3-I, generated by the proteolytic cleavage of pro-LC3 by ATG4, is conjugated to phosphatidylethanolamine, becoming LC3-II, by a series of reactions that involves the ATG5/ATG12/ATG16 complex, ATG7 and ATG3 [START_REF] Mizushima | Autophagy: Renovation of cells and tissues[END_REF]. After closure, autophagosome fuses with lysosome to form an autolysosome where content degradation occurs. This step is mediated by a fusion machinery including the soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) family like syntaxin (Y. Wang et al., 2016).

Xenophagy is a selective autophagy targeting intracellular pathogens [START_REF] Bah | Macrophage autophagy and bacterial infections[END_REF]. In macrophages, xenophagy has been characterized during MTB infection [START_REF] Gutierrez | Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages[END_REF]. MTB has the ability to escape phagosomes and to access cytosol [START_REF] Gutierrez | Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages[END_REF].

Mycobacterial DNA is recognized by the cytosolic sensor named c-GAS (cyclic GMP-AMP synthase). c-GAS triggers the ubiquitination of bacteria by Parkin and Smurf1 [START_REF] Franco | The Ubiquitin Ligase Smurf1 Functions in Selective Autophagy of Mycobacterium tuberculosis and Anti-tuberculous Host Defense[END_REF], whereas galectins fixed on damaged lysosomes or phagosomal membranes trigger TRIM (S. [START_REF] Kumar | Galectins and TRIMs directly interact and orchestrate autophagic response to endomembrane damage[END_REF]. Resulting poly-ubiquitinated proteins bind to autophagy adaptors or receptors such as p62/sequestrosome, neighbor of Bcra1 (NBR1) and nuclear dot protein 52kDa (NDP52). Subsequently, these adaptors recruit LC3 via their LC3-interacting motif and start the autophagy process [START_REF] Bah | Macrophage autophagy and bacterial infections[END_REF]. A study indicated that mice with monocytederived cells lacking Atg5 are more susceptible to MTB and inflammation, showing the role of autophagy in the defense against MTB [START_REF] Castillo | Autophagy protects against active tuberculosis by suppressing bacterial burden and inflammation[END_REF].

More generally, c-GAS is activated to catalyze the production of cyclic GMP-AMP, leading to the activation of the downstream sensor stimulator of IFN genes (STING) [START_REF] Ablasser | CGAS in action: Expanding roles in immunity and inflammation[END_REF]. Upon MTB infection, this process activates the tank-binding kinase 1 (TBK1) and triggers the STING/TBK-1/IRF3 signaling pathway, leading to secretion of type I IFN (with IFNβ production), activation of NF-κB, and production of various Th1 cytokines [START_REF] Abe | Cytosolic-DNA-Mediated, STING-Dependent Proinflammatory Gene Induction Necessitates Canonical NF-B Activation through TBK1[END_REF][START_REF] Watson | The cytosolic sensor cGAS detects Mycobacterium tuberculosis DNA to induce type I interferons and activate autophagy[END_REF]. Interestingly, recent study shows that c-GAS -/-and STING -/-have comparable MTB bacterial burden in the lung and similar inflammation levels compared to WT mice, suggesting other antimycobacterial immune responses in vivo [START_REF] Marinho | The cGAS/STING Pathway Is Important for Dendritic Cell Activation but Is Not Essential to Induce Protective Immunity against Mycobacterium tuberculosis Infection[END_REF].

Apoptosis

Different forms of cell deaths are observed for MTB-infected macrophages. The most wellcharacterized ones are (i) necrosis, commonly described as anarchic cell lysis, and (ii)

apoptosis, a programmed and structured death initiated by the cell [START_REF] Moraco | Cell death and autophagy in tuberculosis[END_REF].

Apoptosis is an important macrophage defense mechanism against pathogens. During apoptosis, plasma membrane remains intact, allowing the containment and the control of the infection. It also provides an important source of bacterial antigens that stimulate other immune cells (J. [START_REF] Lee | Macrophage apoptosis in tuberculosis[END_REF]. MTB-infected macrophages induce apoptosis in different ways:

-Extrinsic pathway activated by the binding of TNF-α to TNF receptor 1 (TNFR1) and Fas ligand (FasL) to Fas. These bindings lead to the formation of a death-inducing signal complex (DISC) by recruitment of the death domain which terminally activates caspases 8 and 10 (Behar et al., 2011).

-Intrinsic pathway is induced by intracellular stresses such as DNA damages, oxidative stress or nutrient deprivation. Those stresses lead to the permeabilization of the outer mitochondrial membrane and the releasing of cytochrome c. This latter binds to the cytosolic apoptotic protease activating factor (APAF1) forming the apoptosome. This complex recruits and activates caspase 9 (J. [START_REF] Lee | Macrophage apoptosis in tuberculosis[END_REF].

The cleavage/activation of these caspases in turn activate the executioner apoptosis caspases and cell reduction and terminal death of the cell [START_REF] Shi | Mechanisms of caspase activation and inhibition during apoptosis[END_REF].

Apoptosis is also induced by arachidonic acid (AA) which is the precursor of eicosanoids lipid mediators such as prostaglandin E2 (PGE2). AA is a part of the cell membrane and is obtained by the action of phospholipases on cell membrane phospholipids. Cyclooxygenases (COX 1

and 2) synthesize prostaglandins H from AA which can be further synthesized in PGE2 [START_REF] Oliveira | Antimicrobial peptides as potential anti-tubercular leads: A concise review[END_REF][START_REF] Rocca | Cyclooxygenases and prostaglandins: Shaping up the immune response[END_REF]. PGE2 acts as an anti-necrosis and pro-apoptotic mediator by initiating the protection of the mitochondria and stimulating the plasma membrane repair from bacteria-induced damage (via induction of synaptotagmin-7 (Syt-7)

which facilitates the fusion of the plasma membrane with lysosomal vesicles) [START_REF] Behar | Apoptosis is an innate defense function of macrophages against Mycobacterium tuberculosis[END_REF]Divangahi et al., 2009a).

Antimicrobial defenses

Antimicrobial peptides (AMPs) are small structures (50-60 amino acid residues) with different properties including antimicrobial and immunomodulatory activities [START_REF] Oliveira | Antimicrobial peptides as potential anti-tubercular leads: A concise review[END_REF].

Several AMPs have been described to play a key role regarding MTB infection such as cathelicidins, defensins, lactoferrins and hepcidins.

Cathelicidins: Cathelicidins are a family of mammalian AMPs with only one representative in human: LL-37/hCAP-18 for human cationic antimicrobial peptide-18 [START_REF] Rivas-Santiago | Expression of cathelicidin LL-37 during Mycobacterium tuberculosis infection in human alveolar macrophages, monocytes, neutrophils, and epithelial cells[END_REF]. This peptide is produced by immune cells including neutrophils and macrophages in response to MTB infection. LL-37 is expressed after stimulation of TLR2 by mycobacterial peptides and upregulation of vitamin D3 receptor [START_REF] Rivas-Santiago | Expression of cathelicidin LL-37 during Mycobacterium tuberculosis infection in human alveolar macrophages, monocytes, neutrophils, and epithelial cells[END_REF]. A study demonstrates that administration of phenylbutyrate and vitamin D3 increases the production of LL-37 resulting in a decrease of the bacterial load [START_REF] Mily | Significant effects of oral phenylbutyrate and Vitamin D3 adjunctive therapy in pulmonary tuberculosis: A randomized controlled trial[END_REF]. Despite its own antibacterial activity, LL-37 also modulates the expression of cytokines in macrophages.

Another study shows that cathelicidin induces the expression of anti-inflammatory IL-10 and TGF-β for balancing inflammatory response without reducing the antimycobacterial activity of the macrophage [START_REF] Torres-Juarez | LL-37 immunomodulatory activity during Mycobacterium tuberculosis infection in macrophages[END_REF].

Defensins: Defensins are small cationic peptides of 29~35 amino acid residues [START_REF] Shin | Antimicrobial Peptides in Innate Immunity against Mycobacteria[END_REF]. They are mainly expressed in epithelial cells and neutrophils but their expression is also detected in monocytes and macrophages [START_REF] Duits | Expression of β-defensin 1 and 2 mRNA by human monocytes, macrophages and dendritic cells[END_REF]Kisich et al., 2001a). Defensins are produced following the recognition of PAMPs by TLRs [START_REF] Shin | Antimicrobial Peptides in Innate Immunity against Mycobacteria[END_REF]. They cause direct bacterial cell membrane disruption but also chemo-attract macrophages to the site of infection (RI et al., 1993).

Lactoferrin: Lactoferrin is an iron binding glycoprotein which belongs to the transferrin family.

It is present in many tissues and is related to iron homeostasis [START_REF] Arranz-Trullén | Host antimicrobial peptides: The promise of new treatment strategies against tuberculosis[END_REF]. A study shows the ability of lactoferrin to decrease the MTB bacterial load in mice [START_REF] Welsh | Influence of oral lactoferrin on Mycobacterium tuberculosis induced immunopathology[END_REF]. It also enhances pro-inflammatory responses in bone marrow-derived monocytes and J774A.1 cells in response to mycobacterial infection, notably by increasing the ratio of IL-12:IL-10 cytokines [START_REF] Hwang | Lactoferrin modulation of IL-12 and IL-10 response from activated murine leukocytes[END_REF].

Hepcidin: Hepcidin is synthesized in hepatocytes following an infectious or inflammatory process [START_REF] Oliveira | Antimicrobial peptides as potential anti-tubercular leads: A concise review[END_REF]. This AMP inhibits iron absorption and macrophage iron release via the degradation of the iron export protein ferroportin 1 (Zumla et al., 2015b). As iron is an essential nutrient for all living organisms, inhibition of iron exportation by macrophages creates a deleterious environment for the bacteria, thus inhibiting MTB growth inside phagosomes (Sow et al., 2007). However, another recent study shows that deficiency of hepcidin in mice does not impede MTB growth in vivo in the TB mouse model, questioning the role of this AMP in MTB infection [START_REF] Harrington-Kandt | Hepcidin deficiency and iron deficiency do not alter tuberculosis susceptibility in a murine M. tb infection model[END_REF].

Nutrients modulation: example of metals

During infection, a dynamic interaction between host and pathogen is established regarding nutrients. In order to proliferate, intracellular pathogenic bacteria need to benefit from host nutrients and metabolites. As a consequence, the host immune system has developed strategies to limit nutrients availability with the aim of starving the pathogen [START_REF] Núñez | Innate Nutritional Immunity[END_REF]. This process of nutrient sequestration is named nutritional immunity [START_REF] Healy | Nutritional immunity: the impact of metals on lung immune cells and the airway microbiome during chronic respiratory disease[END_REF]. Nutritional immunity is most widely used to refer to the sequestration of metallic nutrients such as iron, zinc or copper [START_REF] Healy | Nutritional immunity: the impact of metals on lung immune cells and the airway microbiome during chronic respiratory disease[END_REF]. Metals are essential in various biological processes, for both cells and bacteria [START_REF] Chandrangsu | Metal homeostasis and resistance in bacteria[END_REF]. Hence, nutritional immunity, especially iron and zinc, is a key defense mechanism from immune cells against bacteria.

a. Iron

Tissue resident macrophages such as AMs modulate iron availability into their environment by sequestering and/or releasing iron [START_REF] Winn | Regulation of tissue iron homeostasis: The macrophage "ferrostat[END_REF]. Although essential for many reactions, free iron is potentially toxic by generating reactive hydroxyl radicals. In this context, iron is stored inside macrophages in order to be mobilized during infection [START_REF] Winn | Regulation of tissue iron homeostasis: The macrophage "ferrostat[END_REF].

Uptake of iron by macrophages is mediated through the iron transporters transferrin receptor and divalent metal transporter 1 . Once inside the cell, iron is stored in the iron storage proteins ferritin or hemosiderin [START_REF] Healy | Nutritional immunity: the impact of metals on lung immune cells and the airway microbiome during chronic respiratory disease[END_REF]. The transmembrane protein ferroportin (FPN) exports iron from the cell into the extracellular space [START_REF] Healy | Nutritional immunity: the impact of metals on lung immune cells and the airway microbiome during chronic respiratory disease[END_REF].

Macrophages use several strategies to limit access to iron to the pathogens. In reaction to TLRs stimulation by BCG, macrophages promote intracellular iron sequestration through hepcidin upregulation and FPN downregulation [START_REF] Abreu | Role of the hepcidin-ferroportin axis in pathogenmediated intracellular iron sequestration in human phagocytic cells[END_REF]. FPN is also located in the phagosomal membrane, which can be beneficial for the phagocytized pathogens. As a result, FPN is rapidly removed from early phagosomes and traffics back to the cell membrane, thus depriving the intracellular bacteria from iron [START_REF] Flannagan | Rapid removal of phagosomal ferroportin in macrophages contributes to nutritional immunity[END_REF]. Another study performed on the phagosomes of peritoneal macrophages of C57BL/6 mice infected with M.

avium and MTB, shows that iron concentration inside the phagosome is significantly higher when bacteria are present (Wagner et al., 2005a). In this case, iron is delivered to the phagosome through the transferrin receptor which binds the iron-transferrin complex (Wagner et al., 2005a). However, IFN-γ downregulates this transferrin receptor thus limiting the iron pool inside the phagosome (Wagner et al., 2005a).

During infection, the natural resistance-associated macrophage protein-1 (NRAMP1 or Slc11a1) is recruited to the phagosomal membrane. Several studies demonstrate the role of this protein in host protection mechanisms against mycobacteria [START_REF] Barton | Nramp 1: A link between intracellular iron transport and innate resistance to intracellular pathogens[END_REF]; M. S. [START_REF] Gomes | NRAMP1-or cytokine-induced bacteriostasis of Mycobacterium avium by mouse macrophages is independent of the respiratory burst[END_REF]. For example, NRAMP1 polymorphisms are associated with increased susceptibility to TB in humans (Bellamy et al., 2009). Different modes of action are proposed regarding its activity. First, NRAMP1 acts as a divalent metal efflux pump at the phagosomal membrane, depriving pathogens present in the phagosome from iron [START_REF] Forbes | Divalent-metal transport by NRAMP proteins at the interface of host-pathogen interactions[END_REF]. Another study suggests that NRAMP1 allows the influx of iron inside the phagosome, generating hydroxyl radicals via the Fenton/Haber-Weiss reaction, deleterious for the bacteria [START_REF] Lafuse | Regulation of Nramp1 mRNA stability by oxidants and protein kinase C in RAW264.7 macrophages expressing Nramp1(Gly169)[END_REF]. NRAMP1 also modulates NO production by increasing the expression of iNOS in Raw264.7 cell mice [START_REF] Fritsche | Nramp1 Functionality Increases Inducible Nitric Oxide Synthase Transcription Via Stimulation of IFN Regulatory Factor 1 Expression[END_REF]. However, the role of NRAMP1 in TB infection might be limited as deletion of NRAMP1 in MTB-infected mice does not result in higher bacterial burden [START_REF] North | Consequence of Nramp1 deletion to Mycobacterium tuberculosis infection in mice[END_REF].

Other mechanisms related to iron take place during bacterial infection. Macrophages possess siderophores (low-molecular weight iron chelators) such as lipocalin-2 (LCN-2) that bind to bacterial siderophores, preventing them from iron uptake [START_REF] Flo | Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron[END_REF][START_REF] Dahl | Lipocalin-2 functions as inhibitor of innate resistance to mycobacterium tuberculosis[END_REF].

AMs utilize heme (iron coupled with protoporphyrin IX) to produce ROS and NO, generating a bactericidal effect (Müllebner et al., 2018). Iron is an important nutrient for the bacteria and its control can be used as a major defense mechanism by the cell.

b. Zinc

Besides iron, zinc is another essential metal for cell homeostasis and bacterial defenses. Zinc level inside the cell is mainly regulated by two families of transporters: SLC39A importers (ZIPs family) and SLC30A exporters (ZnTs) [START_REF] Kambe | Current understanding of ZIP and ZnT zinc transporters in human health and diseases[END_REF]. Macrophages have several strategies related to zinc mobilization for bactericidal activity: they can either sequester zinc for zinc starvation or use zinc to intoxicate the pathogen [START_REF] Neyrolles | Zinc and copper toxicity in host defense against pathogens: Mycobacterium tuberculosis as a model example of an emerging paradigm[END_REF]. Zinc starvation by the host is mediated by the S100 superfamily of Ca 2+ -binding proteins, including S100A8/S100A9 heterotetramer named calprotectin (CP) (J. Z. [START_REF] Liu | Zinc sequestration by the neutrophil protein calprotectin enhances salmonella growth in the inflamed gut[END_REF]. Moreover, the pro-inflammatory GM-CSF stimulates zinc import into the cytosol via ZIP2 during fungal infection (Vignesh et al., 2013a). Within the cell, zinc is sequestered with high affinity by metallothioneins (MT), thus limiting the zinc availability to intracellular pathogens (Baltaci et al., 2017). Cytosolic zinc is exported to the Golgi via zinc exporters ZnT4 and Znt7 [START_REF] Vignesh | Zinc Sequestration: Arming Phagocyte Defense against Fungal Attack[END_REF]. Zinc sequestration strategy also increases the phagosomal H + channel function and ROS generation by NOX [START_REF] Gammoh | Zinc in infection and inflammation[END_REF].

Similarly to iron, a high zinc concentration is toxic for living organisms. Therefore, an excess of zinc inside the macrophage is another efficient strategy to fight intracellular pathogens.

Mycobacterial infection causes a burst of free zinc inside the cell [START_REF] Botella | Mycobacterial P 1-Type ATPases mediate resistance to Zinc poisoning in human macrophages[END_REF]. Indeed, following MTB infection, it has been observed that zinc is liberated from MT upon oxidation via NOXs [START_REF] Maret | The function of zinc metallothionein: A link between cellular zinc and redox state[END_REF] but also from intracellular-containing protein called the zincosomes [START_REF] Gammoh | Zinc in infection and inflammation[END_REF]Niederweis et al., 2015). Moreover, during BCG infection, an increase of the extracellular zinc uptake occurs via ZIP8, suggesting an import of zinc for intoxication (Begum et al., 2002). Recently, a study shows that early accumulation of zinc inside Escherichia coli containing phagosomes occurs via the SLC30a1/Znt1 transporter [START_REF] Neyrolles | Antimicrobial zinc toxicity in Mϕs: ZnT1 pays the toll[END_REF][START_REF] Stocks | Frontline Science: LPS-inducible SLC30A1 drives human macrophage-mediated zinc toxicity against intracellular Escherichia coli[END_REF]). Yet, Slc30a1 gene silencing does not impair bacterial clearance (as shown for E.

coli), indicating that this mechanism probably relies on additional pathways.

Nutrients modulation by the host is one of the many strategies used against pathogens and more specifically MTB. However, MTB, as a well-adapted intracellular pathogen, has developed over time efficient mechanisms to counteract these defense mechanisms from the host, in order to survive and to proliferate. The main strategies employed by MTB to fight against the host defenses are detailed below.

III.

Mycobacterium tuberculosis defenses against the host 1. Phagosomal resistance

Once the host invaded, MTB is phagocytized by macrophages and trapped inside a phagosome. The phagosome being a deleterious environment for the bacteria, MTB has developed strategies to avoid degradation such as arrest of the phagosome maturation and inhibition of the phagosomal acidification.

Arrest of phagosomal maturation: MTB-containing phagosome does not possess Rab7 at the surface [START_REF] Chandra | Mycobacterium tuberculosis Inhibits RAB7 Recruitment to Selectively Modulate Autophagy Flux in Macrophages[END_REF]. Absence of this marker is a sign of maturation blockage in the early step phagosome. Hence, MTB prevents Rab7 acquisition through the alteration of the EEA1 recruitment and further activation of the PI3K VPS34 cascade leading to production of PI3P [START_REF] Fratti | Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest[END_REF]. Lack of EEA1 recruitment is related to the Ca 2+ -binding protein calmodulin and the Ca 2+ cytosolic level [START_REF] Vergne | Tuberculosis toxin blocking phagosome maturation inhibits a novel Ca 2+/calmodulin-PI3K hVPS34 cascade[END_REF]. Vergne et al. reported that bacterial surfaces expressing LAM, more specifically ManLAM for MTB, which blocks the activation of the Ca 2+ / calmodulin pathway [START_REF] Vergne | Tuberculosis toxin blocking phagosome maturation inhibits a novel Ca 2+/calmodulin-PI3K hVPS34 cascade[END_REF][START_REF] Vergne | Manipulation of the endocytic pathway and phagocyte functions by Mycobacterium tuberculosis lipoarabinomannan[END_REF]. Moreover, LprG binding to LAM is essential for the increasing surface expression of LAM, therefore giving MTB its virulence ability to arrest phagosomal maturation [START_REF] Shukla | Mycobacterium tuberculosis Lipoprotein LprG Binds Lipoarabinomannan and Determines Its Cell Envelope Localization to Control Phagolysosomal Fusion[END_REF].

Another host protein named coronin 1 (or TACO) has been linked to the Ca 2+ / calcineurin inhibition pathway (Jayachandran et al., 2007). Indeed, the MTB-containing phagosome recruits TACO to block lysosomal delivery of the phagosome (Jayachandran et al., 2007b).

Moreover, MTB has other proteins that inhibit phagosome maturation such as secretory acid phosphatase (SapM). SapM is involved in the maturation arrest of phagosomes through the PI3P pathway [START_REF] Puri | Secreted Acid Phosphatase (SapM) of Mycobacterium tuberculosis Is Indispensable for Arresting Phagosomal Maturation and Growth of the Pathogen in Guinea Pig Tissues[END_REF]. SapM dephosphorylates PI3P thereby inhibiting its activity as a membrane trafficking regulator of lysosomes [START_REF] Fernandez-Soto | Mechanism of catalysis and inhibition of Mycobacterium tuberculosis SapM, implications for the development of novel antivirulence drugs[END_REF][START_REF] Puri | Secreted Acid Phosphatase (SapM) of Mycobacterium tuberculosis Is Indispensable for Arresting Phagosomal Maturation and Growth of the Pathogen in Guinea Pig Tissues[END_REF].

Mycobacterial protein kinase G (PKnG) is a serine/threonine kinase known to block phagosome-lysosome fusion through its activity on host Rab7l1 (it blocks transition of inactive Rab7l1-GDP to active Rab7l1-GTP, enabling its recruitment to phagosome) [START_REF] Pradhan | Mycobacterial PknG Targets the Rab7l1 Signaling Pathway To Inhibit Phagosome-Lysosome Fusion[END_REF]. A recent study also demonstrated that putative lipoprotein LppM is involved in the blocking of the phagosomal maturation [START_REF] Deboosère | LppM impact on the colonization of macrophages by Mycobacterium tuberculosis[END_REF].

Inhibition of the acidification: Along with phagosomal maturation arrest, intracellular MTB has developed different strategies to counteract phagosomal acidification. MTB secretes the protein tyrosine phosphatase (PtpA) which binds to subunit H of the macrophage V-ATPase (D. [START_REF] Wong | Protein tyrosine kinase, PtkA, is required for Mycobacterium tuberculosis growth in macrophages[END_REF]. This binding inhibits V-ATPase trafficking to the mycobacterial phagosome and thus acidification (P. [START_REF] Zhou | Phosphorylation control of protein tyrosine phosphatase A activity in Mycobacterium tuberculosis[END_REF]. Moreover, MTB produces 1tuberculosinyladenosine (1-TbAd) which accumulates in host acidic compartments, neutralizes the pH and swells lysosomes, leading to lysosomal and phagosomal dysfunctions [START_REF] Buter | Mycobacterium tuberculosis releases an antacid that remodels phagosomes[END_REF]. Additionally, interaction of bacterial trehalose-6,6'-dimycolate (TDM) with the monocyte-inducible C-type lectin receptor (Mincle) delays phagosomal maturation and acidification [START_REF] Patin | Trehalose dimycolate interferes with FcγRmediated phagosome maturation through Mincle, SHP-1 and FcγRIIB signalling[END_REF]. Another mechanism of MTB acidic maturation arrest is the depletion of V-ATPase by the cytokine-inducible SH2-containing protein (CISH) (Queval et al., 2017a). MTB-infected macrophage produces GM-CSF, which triggers the expression of CISH via the nuclear STAT5. CISH targets V-ATPase catalytic subunit A (ATP6V1A) for ubiquitination and therefore its degradation by proteasomes (Queval et al., 2017a).

MTB interacts with the host to prevent maturation and acidification of the phagosome.

However, the pathogen itself possesses strategies to resist in acidic environmental conditions.

First of all, the composition of MTB cell wall acts as a natural barrier [START_REF] Vandal | Acid resistance in Mycobacterium tuberculosis[END_REF].

Moreover, certain proteins are overexpressed under low pH conditions such as the functional serine protease Rv3671c, protecting MTB from acidification [START_REF] Biswas | Structural insight into serine protease Rv3671c that protects M. tuberculosis from oxidative and acidic stress[END_REF] or the poreforming outer membrane protein OmpATb, required for MTB survival under low pH conditions [START_REF] Raynaud | The functions of OmpATb, a pore-forming protein of Mycobacterium tuberculosis[END_REF].

Phagosomal escaping

Inhibition of the phagosomal maturation is an important defense used by MTB to survive inside the cell. However, evidence suggests that MTB, contrarily to BCG, is also capable of escaping the phagosome by translocating into the cytosol [START_REF] Vanderven | The Minimal Unit of Infection: Mycobacterium tuberculosis in the Macrophage[END_REF]. Several studies show that ESAT-6 secretion system-1 (ESX-1), a type VII secretion system, is required for the phagosomal rupture process [START_REF] Houben | ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria[END_REF][START_REF] Simeone | Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death[END_REF]. ESX-1 is encoded by a genetic locus region of difference 1 (RD1), which is absent in the vaccine strain BCG in human [START_REF] Pym | Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti[END_REF]. This region comprises genes coding for ESAT-6 and CFP-10 proteins, also known as EsxA and EsxB, respectively [START_REF] Brodin | Functional Analysis of Early Secreted Antigenic Target-6, the Dominant T-cell Antigen of Mycobacterium tuberculosis, Reveals Key Residues Involved in Secretion, Complex Formation, Virulence, and Immunogenicity[END_REF]; K.-W. [START_REF] Wong | The Role of ESX-1 in Mycobacterium tuberculosis Pathogenesis[END_REF].

It has been suggested that once EsxA is released by its putative chaperone EsxB [START_REF] De Jonge | ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity[END_REF], it is inserted into cell host membrane and mediated its membrane lytic activity through pore-forming activity [START_REF] Peng | Characterization of differential pore-forming activities of ESAT-6 proteins from Mycobacterium tuberculosis and Mycobacterium smegmatis[END_REF][START_REF] Smith | Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole[END_REF]. The lytic activity of EsxA was described to depend on the lipid composition of the membrane and its fluidity [START_REF] Ray | Effects of membrane lipid composition on Mycobacterium tuberculosis EsxA membrane insertion: A dual play of fluidity and charge[END_REF]. However, a recent study suggests that EsxA alone does not lyse the host cell membrane and that the phagosomal lysis was due to residual detergent used in the purification protocol [START_REF] Conrad | Mycobacterial ESX-1 secretion system mediates host cell lysis through bacterium contact-dependent gross membrane disruptions[END_REF]. However, another team manages to prove that EsxA still shows membranolytic activity using another purification protocol [START_REF] Augenstreich | Phthiocerol Dimycocerosates From Mycobacterium tuberculosis Increase the Membrane Activity of Bacterial Effectors and Host Receptors[END_REF] Along with ESX-1, mycobacterial lipid phthiocerol dimycocerosate (PDIM) and dimycocerosate (DIM) are also implicated in the phagosomal rupture [START_REF] Barczak | Systematic, multiparametric analysis of Mycobacterium tuberculosis intracellular infection offers insight into coordinated virulence[END_REF][START_REF] Quigley | The cell wall lipid PDIM contributes to phagosomal escape and host cell exit of Mycobacterium tuberculosis[END_REF].

PDIM was previously suggested to prevent phagosomal maturation and acidification [START_REF] Astarie-Dequeker | Phthiocerol dimycocerosates of M. tuberculosis participate in macrophage invasion by inducing changes in the organization of plasma membrane lipids[END_REF]. Recently it has been described that PDIM attenuated mutants have a diminution in virulence and fail to promote host cell death or to induce type I IFN (associated with phagosomal damages) [START_REF] Augenstreich | ESX-1 and phthiocerol dimycocerosates of Mycobacterium tuberculosis act in concert to cause phagosomal rupture and host cell apoptosis[END_REF]. Moreover, MTB escape is increased in presence of PDIM [START_REF] Augenstreich | Phthiocerol Dimycocerosates From Mycobacterium tuberculosis Increase the Membrane Activity of Bacterial Effectors and Host Receptors[END_REF][START_REF] Quigley | The cell wall lipid PDIM contributes to phagosomal escape and host cell exit of Mycobacterium tuberculosis[END_REF]. However, host cell has developed some mechanisms to repair such phagosomal damage. For example, endosomal sorting complex required for transport (ESCRT) machinery is recruited in the MTB-containing phagosomes directly to damaged area for closure of the plasma membrane [START_REF] Jimenez | ESCRT Machinery Is Required for Plasma Membrane Repair[END_REF]. This recruitment occurs in an ESX-1 dependent manner [START_REF] Mittal | Mycobacterium tuberculosis type VII secretion system effectors differentially impact the ESCRT endomembrane damage response[END_REF].

Once in the cytosol, MTB faces host cytosolic defenses such as induction of autophagy.

Damaged phagosomes expose glycans at their surface that are then detected by galectins.

With the rupture of the phagosome, mycobacterial extracellular DNA is found into the host cell cytosol (Manzanillo et al., 2012). This cytosolic mycobacterial DNA is detected by c-Gas of the AIM2 inflammasome but no activation of AIM2 is reported during MTB infection (Ontiveros et al., 2019). AIM2 inhibition is supposedly linked to ESX-1 secretion system because ESX-1 deficient mutant fails to inhibit the AIM2-inflammasome activation [START_REF] Shah | Cutting Edge: Mycobacterium tuberculosis but Not Nonvirulent Mycobacteria Inhibits IFN-β and AIM2 Inflammasome-Dependent IL-1β Production via Its ESX-1 Secretion System[END_REF]. Moreover, they supposed that ESX-1 secrete an unknown effector which inhibits AIM2 activation [START_REF] Shah | Cutting Edge: Mycobacterium tuberculosis but Not Nonvirulent Mycobacteria Inhibits IFN-β and AIM2 Inflammasome-Dependent IL-1β Production via Its ESX-1 Secretion System[END_REF]. This inhibition results in a decrease in IL-1β secretion, which has previously been shown to promote MTB infection in mice [START_REF] Novikov | Mycobacterium tuberculosis Triggers Host Type I IFN Signaling To Regulate IL-1β Production in Human Macrophages[END_REF][START_REF] Novikov | Mycobacterium tuberculosis Triggers Host Type I IFN Signaling To Regulate IL-1β Production in Human Macrophages[END_REF]. The cGas/STING pathway activation has been related to ESX-1. BCG, lacking RD-1 region, does not activate the STING pathway but recombinant BCG expressing ESX-1 T7

secretion system of M. marinum ruptures the phagosome and therefore induces the cGas/STING/TBK1/IRF-3/type I interferon axis [START_REF] Gröschel | Recombinant BCG Expressing ESX-1 of Mycobacterium marinum Combines Low Virulence with Cytosolic Immune Signaling and Improved TB Protection[END_REF].

Isolates from patient with severe TB were shown to produce a lower cytokine response compared to moderate forms of TB [START_REF] Sousa | Mycobacterium tuberculosis associated with severe tuberculosis evades cytosolic surveillance systems and modulates IL-1β production[END_REF]. Interestingly, diminution of IL-1β has been linked to the ability of these strains to escape from cGAS and inflammasomes. Those strains were described as being mutated in the ESX-1 secretion system which consequently restricts phagosome rupture and induce lower release of IL-1β [START_REF] Sousa | Mycobacterium tuberculosis associated with severe tuberculosis evades cytosolic surveillance systems and modulates IL-1β production[END_REF].

Moreover, cytosolic mycobacterial DNA, detected by c-Gas, triggers the autophagy [START_REF] Franco | The Ubiquitin Ligase Smurf1 Functions in Selective Autophagy of Mycobacterium tuberculosis and Anti-tuberculous Host Defense[END_REF][START_REF] Kumar | Galectins and TRIMs directly interact and orchestrate autophagic response to endomembrane damage[END_REF][START_REF] Thurston | Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion[END_REF]. Autophagy being dependent on the fusion with lysosomes like phagosomes, MTB also regulates autophagic flux by, for example, inhibiting the Rab7 recruitment [START_REF] Chandra | Mycobacterium tuberculosis Inhibits RAB7 Recruitment to Selectively Modulate Autophagy Flux in Macrophages[END_REF]. MTB also inhibits autophagy via induction of microRNA-33 (mi-R33) [START_REF] Ouimet | Mycobacterium tuberculosis induces the MIR-33 locus to reprogram autophagy and host lipid metabolism[END_REF]. miRNAs are small non-coding RNAs that bind to the 3ʹ untranslated region (3ʹ UTR) of mRNA, inhibiting their translation or promoting their degradation [START_REF] O'brien | Overview of microRNA biogenesis, mechanisms of actions, and circulation[END_REF]. Presence of mi-RNA33 leads notably to a decrease of key effectors in autophagy pathways such as ATG5, ATG12, LAMP1 and LC3 and enhanced pathogen survival [START_REF] Ouimet | Mycobacterium tuberculosis induces the MIR-33 locus to reprogram autophagy and host lipid metabolism[END_REF].

Reactive oxygen and nitrogen species resistance

MTB possesses several proteins and regulation systems to limit the damages caused by the

ROS production inside the cells including:

KatG: KatG is a catalase-peroxydase produced by MTB in response to oxidative stress for bacterial protection. KatG detoxifies ROS generated by the macrophage by catalyzing the decomposition of H2O2 into O2 and H2O. katG knock-out MTB strain survives less in a mouse infection model, compared to the wild-type, suggesting that KatG is an important virulence factor in a host capable of NOX oxidative burst [START_REF] Ng | Role of KatG catalase-peroxidase in mycobacterial pathogenisis: Countering the phagocyte oxidative burst[END_REF].

Superoxide dismutase (SOD): SOD is a superoxide radical scavenger essential for MTB. It detoxifies superoxide radical O2 .-into O2 and H2O2 and reduces toxic NO through the inhibition of iNOS activity [START_REF] Liao | The role of superoxide dismutase in the survival of Mycobacterium tuberculosis in macrophages[END_REF]. MTB produces two SODs: an iron-dependent enzyme (SodA or FeSOD) and a copper-and zinc-dependent enzyme (SodC or CuZnSOD) [START_REF] Liao | The role of superoxide dismutase in the survival of Mycobacterium tuberculosis in macrophages[END_REF].

SodA is constitutively expressed and is an essential protein of MTB. A study shows that attenuation of sodA expression resulted in attenuated virulence in mice [START_REF] Edwards | Iron-cofactored superoxide dismutase inhibits host responses to Mycobacterium tuberculosis[END_REF].

Moreover, a genome-wide genetic interaction assay has demonstrated that SodA forms a membrane-associated oxidoreductase complex with the integral membrane protein (DoxX) and a predicted thiol-oxidoreductase (SseA). This SodA-DoxX-SseA complex links radical detoxification with cytosolic thiol homeostasis; its defect leads to accumulation of cellular oxidative damage [START_REF] Nambi | The Oxidative Stress Network of Mycobacterium tuberculosis Reveals Coordination between Radical Detoxification Systems[END_REF]. Other studies have investigated the role of sodC for MTB. Deletion of this gene in MTB results in a hypersensitivity to O2 .-and H2O2 in vitro, in mice but not in guinea pigs [START_REF] Dussurget | Role of Mycobacterium tuberculosis copper-zinc superoxide dismutase[END_REF][START_REF] Piddington | Cu,Zn superoxide dismutase of Mycobacterium tuberculosis contributes to survival in activated macrophages that are generating an oxidative burst[END_REF].

Peroxidase: Thiol peroxidase (Tpx) from MTB has a NADPH-linked peroxidase activity and an anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. This enzyme reduces H2O2, t-butyl hydroperoxide, and cumene hydroperoxide [START_REF] Rho | Functional and Structural Characterization of a Thiol Peroxidase from Mycobacterium tuberculosis[END_REF]. A tpx knock-out MTB strain is more sensitive to H2O2 and NO, compared to the wild type strain [START_REF] Hu | Acute and persistent mycobacterium tuberculosis infections depend on the thiol peroxidase TPX[END_REF]. This mutant has a reduced peroxidase activity, its virulence was attenuated and it fails to grow and survive in bone marrow derived macrophages.

Others peroxidases are used by MTB as an antioxidant defense system such as the alkyl hydroperoxide reductase subunit C (AhpC) which is reduced by the alkyl hydroperoxide reductase subunit D (AhpD) or the thioredoxin TrxB and TrxC (C. F. [START_REF] Wong | AhpC of the mycobacterial antioxidant defense system and its interaction with its reducing partner Thioredoxin-C[END_REF]. Strains overexpressing AhpC are more resistant to cumene hydroperoxide than wild type strains [START_REF] Sherman | AhpC, oxidative stress and drug resistance in Mycobacterium tuberculosis[END_REF].

Mycothiol (MSH):

MSH is a low-molecular-mass thiol with an antioxidant activity. It maintains the reducing environment within MTB, protects from oxidants and detoxifies thiol-reactive compounds [START_REF] Buchmeier | A mycothiol synthase mutant of Mycobacterium tuberculosis has an altered thiol-disulfide content and limited tolerance to stress[END_REF]. MSH is required for the peroxiredoxin AhpE peroxidase activity [START_REF] Hugo | Mycothiol/mycoredoxin 1-dependent reduction of the peroxiredoxin AhpE from mycobacterium tuberculosis[END_REF]. Its deletion in M. smegmatis leads to a higher sensitivity to acid, alkylating and oxidative stress, demonstrating its importance for the bacterial growth [START_REF] Rawat | Comparative analysis of mutants in the mycothiol biosynthesis pathway in Mycobacterium smegmatis[END_REF].

Host cell death interference

Several studies investigated the role of apoptosis or necrosis on MTB fate. Classically, avirulent MTB tends to induce apoptosis whereas virulent MTB induces necrosis [START_REF] Behar | Evasion of innate immunity by mycobacterium tuberculosis: Is death an exit strategy?[END_REF][START_REF] Porcelli | Tuberculosis: Unsealing the apoptotic envelope[END_REF]. Virulent MTB can also trigger apoptosis but only in specific conditions, revealing the complexity of cell death induced by MTB [START_REF] Butler | The Balance of Apoptotic and Necrotic Cell Death in Mycobacterium tuberculosis Infected Macrophages Is Not Dependent on Bacterial Virulence[END_REF][START_REF] Danelishvili | Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells[END_REF]. Necrotic macrophages are considered as a replicative niche for MTB. Indeed, releasing of MTB by necrosis allows its survival and its dissemination to other cells while inhibition of necrosis by chemical inhibitors limits MTB multiplication [START_REF] Lerner | Mycobacterium tuberculosis replicates within necrotic human macrophages[END_REF]. Hence, MTB has developed over time many processes to maximize their survival within the macrophage, notably by inducing necrosis and inhibiting apoptosis.

A toxin, named tuberculosis necrotizing toxin (TNT), produced by MTB induces necrosis of infected cells. TNT is secreted by MTB into the cytosol via the ESX-1 secretion-associated proteins EsxE and EsxF [START_REF] Tak | Pore-forming Esx proteins mediate toxin secretion by Mycobacterium tuberculosis[END_REF]. TNT hydrolyzes the cofactor NAD + , depleting the host from this essential cofactor and leading to cell necrosis (J. [START_REF] Sun | The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD[END_REF]. In parallel, MTB produces an immunity factor for TNT (IFT) that inhibits the TNT toxicity within the bacteria (J. [START_REF] Sun | The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD[END_REF].

NuoG is used by MTB to inhibit apoptosis by targeting the ROS production. nuoG encodes a subunit of the type I NADH dehydrogenase that neutralizes ROS produced by NOX2 inside phagosome. Neutralization of ROS inhibits TNF-α secretion and TNF-α induced apoptosis. MTB lacking nuoG does not inhibit apoptosis anymore, which significantly reduced its virulence in mice [START_REF] Velmurugan | Mycobacterium tuberculosis nuoG Is a virulence gene that inhibits apoptosis of infected host cells[END_REF].

Additionally, MTB promotes necrosis by interacting with the lipids of the host cell. PGE2 was previously cited as a pro-apopoptotic eicosanoid regulating the plasma membrane repair and the mitochondrial protection (see section B.II.e.). Virulent MTB suppresses COX2 expression in infected macrophages which leads to PGE2 inhibition (M. [START_REF] Chen | Lipid mediators in innate immunity against tuberculosis: Opposing roles of PGE 2 and LXA 4 in the induction of macrophage death[END_REF]. Instead, the lipoxygenases generate lipoxins (including LXA4) and leukotriene from arachidonic acid.

Production of LXA4 impairs the apoptotic signaling by inhibition of apoptosis mediators (caspases) by the mitochondria and increases the expression of antiapoptotic proteins of the Bcl-2 family [START_REF] Prieto | Lipoxin A4 impairment of apoptotic signaling in macrophages: Implication of the PI3K/Akt and the ERK/Nrf-2 defense pathways[END_REF]. The absence of PGE2 also inhibits the plasma membrane repair by Syt-7, promoting cell necrosis (Divangahi et al., 2009b). Furthermore, virulent MTB disrupts the mitochondrial transmembrane potential, leading to mitochondrial degradation and necrosis (M. [START_REF] Chen | A Mechanism of Virulence: Virulent Mycobacterium tuberculosis Strain H37Rv, but Not Attenuated H37Ra, Causes Significant Mitochondrial Inner Membrane Disruption in Macrophages Leading to Necrosis[END_REF].

Cytokines modulation and macrophages polarization

MTB survival depends on the level of pro-or anti-inflammatory cytokines secreted in the environment. For example, M2 macrophages, which produce more anti-inflammatory cytokines, are more vulnerable to MTB infection than M1 macrophages [START_REF] Khan | Macrophage heterogeneity and plasticity in tuberculosis[END_REF].

Endoplasmic reticulum (ER) stress is increased in M1 polarized macrophages which leads to a more effective clearance of intracellular MTB than M2 macrophages [START_REF] Lim | Roles of endoplasmic reticulum stress-mediated apoptosis in M1-polarized macrophages during mycobacterial infections[END_REF]. In this context, MTB has developed strategies to limit M1 polarization of macrophages and therefore the production of cytokines. Avirulent MTB infection activates M1 macrophages whereas virulent MTB promotes M2 macrophages polarization [START_REF] Lim | Roles of endoplasmic reticulum stress-mediated apoptosis in M1-polarized macrophages during mycobacterial infections[END_REF]. A study also demonstrates that the virulence factor ESAT-6 participates in the transition to the M2 phenotype through the inhibition of TLR2 signaling [START_REF] Lim | Roles of endoplasmic reticulum stress-mediated apoptosis in M1-polarized macrophages during mycobacterial infections[END_REF][START_REF] Pathak | Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages[END_REF]. These observations are corroborated by another study showing that M2 macrophages are predominant in granuloma in vitro [START_REF] Huang | Mycobacterium tuberculosis-induced polarization of human macrophage orchestrates the formation and development of tuberculous granulomas in vitro[END_REF].

IL-10 is one of the main cytokines whose production is beneficial for MTB. Indeed, IL-10 production exacerbates TB disease in mice and promotes MTB growth inside macrophages by blocking phagosomal maturation [START_REF] Beamer | Interleukin-10 Promotes Mycobacterium tuberculosis Disease Progression in CBA/J Mice[END_REF][START_REF] O'leary | IL-10 blocks phagosome maturation in Mycobacterium tuberculosis-infected human macrophages[END_REF]. Transgenic mice overexpressing IL-10 are more susceptible to MTB infection and have a higher M2 macrophages population [START_REF] Schreiber | Autocrine IL-10 Induces Hallmarks of Alternative Activation in Macrophages and Suppresses Antituberculosis Effector Mechanisms without Compromising T Cell Immunity[END_REF]. In a consistent manner, IL-10-deficient mice have reduced bacterial load in their lungs [START_REF] Redford | Enhanced protection to Mycobacterium tuberculosis infection in IL-10-deficient mice is accompanied by early and enhanced Th1 responses in the lung[END_REF]. MTB increases IL-10 production via several pathways including activation of the ERK pathways. MTB activates this pathway via expression of C-C Chemokine Receptor 5 (CCR5) in host macrophages or by stimulating TLR2 to induce extracellular signal-regulated kinases (ERK1/2) phosphorylation which terminally leads to IL-10 production [START_REF] Das | Immune subversion by Mycobacterium tuberculosis through CCR5 mediated signaling: Involvement of IL-10[END_REF][START_REF] Richardson | Toll-like receptor 2-dependent extracellular signal-regulated kinase signaling in Mycobacterium tuberculosis-infected macrophages drives anti-inflammatory responses and inhibits Th1 polarization of responding T cells[END_REF].

Dormancy

Macrophage infection with TB triggers a robust immune response and granuloma formation in lungs. In the granuloma, MTB is exposed to different stresses such as hypoxia, nitric oxide, carbon monoxide and nutrient starvation [START_REF] Saunders | Restraining mycobacteria: Role of granulomas in mycobacterial infections[END_REF]. These conditions limit MTB replication and promote MTB dormancy [START_REF] Lipworth | Defining dormancy in mycobacterial disease[END_REF]. This dormant state allows bacteria to regain metabolic activity as well as the ability to replicate again once the environment is more favorable [START_REF] Lipworth | Defining dormancy in mycobacterial disease[END_REF]. Dormancy of MTB is an immuneevading ability, allowing the pathogen to persist within the host for many years before a putative reactivation.

State of dormancy and reactivation are mainly regulated by the dormancy survival regulator (DosR) which coordinates expression of around 50 genes coding for proteins implicated in hypoxia survival [START_REF] Leistikow | The Mycobacterium tuberculosis DosR regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dormancy[END_REF][START_REF] Sivaramakrishnan | The DosS-DosT/DosR Mycobacterial Sensor System[END_REF]. DosS-DosR form a two-component sensor system, with DosS closely related to the DosT sensor kinase [START_REF] Honaker | Unique roles of DosT and DosS in DosR regulon induction and Mycobacterium tuberculosis dormancy[END_REF]. DosS and DosT under deleterious environment auto-phosphorylate and transfer the phosphate to DosR, resulting in its induction. Induction of DosR regulon enables the expression of genes coding for proteins important for survival during hypoxia [START_REF] Honaker | Unique roles of DosT and DosS in DosR regulon induction and Mycobacterium tuberculosis dormancy[END_REF][START_REF] Sivaramakrishnan | The DosS-DosT/DosR Mycobacterial Sensor System[END_REF]. dosR, dosS and dosT are required for longterm persistence of MTB in the rhesus macaque model [START_REF] Mehra | The DosR Regulon Modulates Adaptive Immunity and Is Essential for Mycobacterium tuberculosis Persistence[END_REF]. The virulence phoP-phoR system also has a direct impact on hypoxia-inducible gene expression of MTB in addition to targeting dosR regulon (P. R. [START_REF] Singh | Metabolic Switching of Mycobacterium tuberculosis during Hypoxia Is Controlled by the Virulence Regulator PhoP[END_REF].

During dormancy, metabolic pathways of MTB are modulated, in reaction to nutrient starvation. Analysis of the transcriptome, proteome and metabolome of MTB during hypoxia emphasizes significant alteration in the lipid catabolism and cholesterol degradation [START_REF] Galagan | The Mycobacterium tuberculosis regulatory network and hypoxia[END_REF]. MTB uses fatty acid as the major source of energy. Host triacylglycerol (TAG) accumulation in the form of intracellular lipid droplets is critical for the survival of MTB in a dormant state [START_REF] Daniel | Mycobacterium tuberculosis Uses Host Triacylglycerol to Accumulate Lipid Droplets and Acquires a Dormancy-Like Phenotype in Lipid-Loaded Macrophages[END_REF]. MTB uses host TAG but also synthesizes his own TAG notably via the triglycerol synthase 1 (Tgs1), whose gene expression is regulated by DosR [START_REF] Daniel | Mycobacterium tuberculosis Uses Host Triacylglycerol to Accumulate Lipid Droplets and Acquires a Dormancy-Like Phenotype in Lipid-Loaded Macrophages[END_REF]. Other lipid changes are observed during hypoxia notably regarding TMM and TDM. Those cord factors, which form the outer lipid barrier of MTB and promote inflammation, are rapidly depleted during hypoxia [START_REF] Eoh | Metabolic anticipation in Mycobacterium tuberculosis[END_REF]. These decreases are reflected in a lower induction of pro-inflammatory TNF-α and IL12p40 cytokines in mouse bone marrow derived macrophages [START_REF] Eoh | Metabolic anticipation in Mycobacterium tuberculosis[END_REF]. This cell envelope remodeling renders MTB less immuno-stimulatory.

Several studies highlight the role of the mycobacterial isocitrate lyase (Icl) during these metabolic changes. Briefly, the degradation of lipids by β-oxidation generates acetyl-coA and the metabolism of cholesterol generates propionyl-coA [START_REF] Mckinney | Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase[END_REF][START_REF] Pandey | Mycobacterial persistence requires the utilization of host cholesterol[END_REF]. Propionyl-coA is toxic at a high concentration and Icl prevents its excessive accumulation (X. [START_REF] Yang | Cholesterol metabolism increases the metabolic pool of propionate in Mycobacterium tuberculosis[END_REF]. Of note it also allows the glyoxylate shunt, a short-cut of the tricarboxylic acid (TCA) cycle, which is essential for MTB dormancy [START_REF] Mckinney | Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase[END_REF].

Regarding its dormant state, the energetic metabolism of hypoxic MTB is also changed towards a decline in ATP production (five to six times lower in hypoxic non-replicating bacteria compared to aerobic one) [START_REF] Rao | The protonmotive force is required for maintaining ATP homeostasis and viability of hypoxic, nonreplicating Mycobacterium tuberculosis[END_REF].

MTB in a dormant state slows down its metabolic activity, its replication and suspension of diverse physiological functions [START_REF] Lipworth | Defining dormancy in mycobacterial disease[END_REF]. As a result, MTB develops a transient resistance or tolerance to antibiotics acting on growing bacteria by targeting cell wall synthesis mechanisms (such as INH), as cell wall synthesis is decreased in dormant MTB [START_REF] Tudó | Examining the basis of isoniazid tolerance in nonreplicating Mycobacterium tuberculosis using transcriptional profiling[END_REF]. A transcriptomic analysis shows that dormant MTB acquires a phenotypic tolerance and that expression of genes coding for proteins involves in INH metabolization is decreased [START_REF] Karakousis | Altered expression of isoniazidregulated genes in drug-treated dormant Mycobacterium tuberculosis[END_REF]. However, mechanisms of INH tolerance remain to be fully elucidated.

Regarding BDQ, the low remaining ATP synthases enzymatic activity of the bacteria allows the antibiotic to be active and kills the pathogen [START_REF] Koul | Diarylquinolines Are Bactericidal for Dormant Mycobacteria as a Result of Disturbed ATP Homeostasis[END_REF]. However, this killing may be delayed due to the utilization of the other metabolic pathways than ATP synthase for energy production [START_REF] Koul | Delayed bactericidal response of Mycobacterium tuberculosis to bedaquiline involves remodelling of bacterial metabolism[END_REF].

Drug tolerance has also been demonstrated on clinical strains, in an in vivo context. A study performed on 245 MTB clinical isolates shows that there is a significant change in first line drug resistance for 38.5% strains under hypoxic conditions (Z. [START_REF] Liu | Impact of hypoxia on drug resistance and growth characteristics of mycobacterium tuberculosis clinical isolates[END_REF]. In a consistent manner, work performed on rabbit caseum indicates that slowly replicating or non-replicating bacteria display an extreme drug tolerance to first-and second-line antibiotics (except RIF which appears to be still active) [START_REF] Sarathy | Extreme drug tolerance of mycobacterium tuberculosis in Caseum[END_REF]. Another important aspect of drug tolerance is related to the accessibility of drugs to the bacteria in granuloma. Indeed, antibiotics differentially diffuse inside the granuloma, rending the combination of antibiotics necessary for a complete sterilization of the caseum [START_REF] Cicchese | Both Pharmacokinetic Variability and Granuloma Heterogeneity Impact the Ability of the First-Line Antibiotics to Sterilize Tuberculosis Granulomas[END_REF].

It has been suggested that reactivation of dormant MTB is dependent of five resuscitationpromoting factors (RpfA-E) [START_REF] Kana | The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro[END_REF]. In a C57Bl/6 mice model, mutants lacking several rpf genes are defective in their ability to disseminate and reactivate even after immunosuppression of the host [START_REF] Biketov | The role of resuscitation promoting factors in pathogenesis and reactivation of Mycobacterium tuberculosis during intra-peritoneal infection in mice[END_REF]. Rpf plays a role in the cell wall hydrolysis of the granulomatous cells, which is an initial step of reactivation [START_REF] Kana | Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling[END_REF].

More recently, it has been shown that Clp is also important for MTB reactivation under DosR control [START_REF] Mcgillivray | The Mycobacterium tuberculosis Clp Gene Regulator Is Required for in Vitro Reactivation from Hypoxia-induced Dormancy[END_REF]. Indeed, a transcriptional analysis comparing gene expression of MTB during hypoxia or undergoing reactivation reveals that Clp (Rv2745c) is induced during those two states [START_REF] Mcgillivray | The Mycobacterium tuberculosis Clp Gene Regulator Is Required for in Vitro Reactivation from Hypoxia-induced Dormancy[END_REF]. Clp is a protease crucial for the degradation of intracellular proteins. However, its role during hypoxic conditions remains to be determined.

Nutrients capture

Lipids

Nutrients inside phagosome are scarce. In this context, MTB has developed several strategies to survive in the phagosome. Inside the macrophage, MTB utilizes the host cholesterol and fatty acids as main sources of carbon (S. T. [START_REF] Cole | Deciphering the biology of mycobacterium tuberculosis from the complete genome sequence[END_REF]. Some studies suggest that MTB deregulates the lipid metabolism of the host cell, leading to the formation of more permissive foamy macrophages [START_REF] Russell | Foamy macrophages and the progression of the human tuberculosis granuloma[END_REF][START_REF] Singh | Mycobacterium tuberculosis-driven targeted recalibration of macrophage lipid homeostasis promotes the foamy phenotype[END_REF]. Recently, it has been demonstrated that differentiation into foamy macrophages is promoted by IL-10 / signal transducer and activator of transcription 3 (STAT3) axis via upregulation of the enzyme acyl

CoA cholesterol acyltransferase (ACAT) [START_REF] Genoula | Formation of foamy macrophages by tuberculous pleural effusions is triggered by the interleukin-10/signal transducer and activator of transcription 3 axis through ACAT upregulation[END_REF]. These foamy macrophages contain many lipid bodies which represent a supply for MTB. Importation of cholesterol and fattu acid through the cell envelope is managed respectively by the Mce4 complex and by

Mce1 and Rv3723/LucA [START_REF] Nazarova | Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis[END_REF][START_REF] Singh | Mycobacterium tuberculosis-driven targeted recalibration of macrophage lipid homeostasis promotes the foamy phenotype[END_REF]. Cholesterol is degraded via β-oxidation. Degradation of the cholesterol rings chains results in the production of acetyl-CoA, propionyl-CoA, succinyl-CoA and pyruvate needed for bacterial metabolism [START_REF] Wilburn | Cholesterol and fatty acids grease the wheels of Mycobacterium tuberculosis pathogenesis[END_REF]. Fatty acids are either directly used into biosynthetic pathways or degraded [START_REF] Wilburn | Cholesterol and fatty acids grease the wheels of Mycobacterium tuberculosis pathogenesis[END_REF]. Furthermore, MTB uses triacylglycerol of the foamy macrophages to enter in a dormancy-like state [START_REF] Daniel | Mycobacterium tuberculosis Uses Host Triacylglycerol to Accumulate Lipid Droplets and Acquires a Dormancy-Like Phenotype in Lipid-Loaded Macrophages[END_REF]. Moreover, degradation of cholesterol has been shown to upregulate the genes involved in the entry of MTB persistent state [START_REF] Pawełczyk | Cholesterol-dependent transcriptome remodeling reveals new insight into the contribution of cholesterol to Mycobacterium tuberculosis pathogenesis[END_REF].

Another important metabolism implicating lipids concerns the eicosanoids. As previously described, they are important signaling molecules notably in the apoptotic/necrotic cell death and modulation of inflammation (see B.I.3 and B.II.6).

Metals

Intracellular MTB faces nutrient starvation, including iron deprivation. To survive, the pathogen acquires iron by stealing it from the host through different mechanisms. First of all, MTB produces siderophores with a high-affinity for iron. There are three types of mycobacterial siderophores: mycobactin, carboxymycobactin and exochelin (K. [START_REF] Patel | Mycobacterial siderophore: A review on chemistry and biology of siderophore and its potential as a target for tuberculosis[END_REF]. Mycobactin is a lipophilic siderophore anchored in the membrane and cell wall of MTB whereas carboxymycobactin has a similar structure but is more hydrophilic (K. [START_REF] Patel | Mycobacterial siderophore: A review on chemistry and biology of siderophore and its potential as a target for tuberculosis[END_REF]. Carboxymycobactin is secreted and released in the extracellular space to chelate and internalize iron. They are also capable of scavenging and removing iron from human transferrin and lactoferrin for the bacteria [START_REF] Luo | Mycobactin-mediated iron acquisition within macrophages[END_REF]. Exochelins have been mostly

described in other mycobacteria such as M. smegmatis and M. avium [START_REF] Horwitz | The Exochelins of Pathogenic Mycobacteria: Unique, Highly Potent, Lipid-and Water-Soluble Hexadentate Iron Chelators with Multiple Potential Therapeutic Uses[END_REF].

Synthesis of mycobacterial siderophores is mediated by cytoplasmic synthases encoded by

mbt-1 and mbt-2 [START_REF] Krithika | A genetic locus required for iron acquisition in Mycobacterium tuberculosis[END_REF]. Regulation of this synthesis is dependent on the irondependent regulator (IdeR). When iron is in a sufficient amount, IdeR represses genes associated with mycobactin synthesis and expresses genes coding for proteins related to ironstorage bacterioferritin, BfrA and BfrB [START_REF] Sritharan | Iron homeostasis in Mycobacterium tuberculosis: Mechanistic insights into siderophore-mediated Iron uptake[END_REF]. Inactivation of ideR is lethal for MTB in macrophages due to its increased sensitivity to oxidative stress (R. [START_REF] Pandey | IdeR is required for iron homeostasis and virulence in Mycobacterium tuberculosis[END_REF].

Export of mycobactin and carboxymycobactin is related to a membrane protein system composed of mycobacterial membrane protein small 4 and 5 (MmpS4-5) and mycobacterial membrane protein large 4 and 5 (MmpL4-5) transporters [START_REF] Wells | Discovery of a Siderophore Export System Essential for Virulence of Mycobacterium tuberculosis[END_REF]. Some studies also suggest the role of ESX-3 type VII secretion system in iron acquisition by MTB [START_REF] Serafini | The ESX-3 Secretion System Is Necessary for Iron and Zinc Homeostasis in Mycobacterium tuberculosis[END_REF][START_REF] Tufariello | Separable roles for Mycobacterium tuberculosis ESX-3 effectors in iron acquisition and virulence[END_REF]. Indeed, ESX-3 mutant shows an overproduction of mycobactins and fails to assimilate iron-linked mycobactin [START_REF] Tufariello | Separable roles for Mycobacterium tuberculosis ESX-3 effectors in iron acquisition and virulence[END_REF]. The precise role of ESX-3 in iron absorption has yet to be fully understood. Another study indicates that microvesicles containing mycobactins are secreted in response to iron restriction; these vesicles are supposed to deliver iron and to promote growth of iron-deficient bacteria (Prados- [START_REF] Prados-Rosales | Role for mycobacterium tuberculosis membrane vesicles in iron acquisition[END_REF]. However, mechanisms of action related to these vesicles are not fully characterized yet. Import of ferric-carboxymycobactins through the inner membrane is mediated by the IrtA/IrtB transporter [START_REF] Rodriguez | Identification of an ABC Transporter Required for Iron Acquisition and Virulence in Mycobacterium tuberculosis[END_REF]. IrtA also possesses a flavin adenine dinucleotide (FAD) domain which catalyzes iron reduction [START_REF] Ryndak | The Mycobacterium tuberculosis High-Affinity Iron Importer, IrtA, Contains an FAD-Binding Domain[END_REF].

However, irtAB mutant still has a residual carboxymycobactin uptake which indicates the presence of another import mechanism not discovered yet [START_REF] Rodriguez | Identification of an ABC Transporter Required for Iron Acquisition and Virulence in Mycobacterium tuberculosis[END_REF].

MTB also captures iron from the host via heme and hemoglobin uptake. Heme, the precursor of hemoglobin, binds to one iron ion. Bacteria are able to scavenge the heme to capture iron [START_REF] Jones | Mycobacterium tuberculosis can utilize heme as an iron source[END_REF]. Two putative systems are implicated in heme uptake: the MmpL3-MmpL11-Rv0203 system [START_REF] Tullius | Discovery and characterization of a unique mycobacterial heme acquisition system[END_REF] and the proline-proline-glutamate 36 (PPE36), PPE62 and the dipeptide transporter Dpp [START_REF] Mitra | PPE surface proteins are required for heme utilization by Mycobacterium tuberculosis[END_REF][START_REF] Mitra | Heme and hemoglobin utilization by Mycobacterium tuberculosis[END_REF]. Once inside the bacteria, the bacterial heme oxygenase MhuD degrades the heme into mycobilin and iron, the latter becoming available for the bacteria [START_REF] Nambu | A new way to degrade heme: The mycobacterium tuberculosis enzyme MhuD catalyzes heme degradation without generating CO[END_REF]. MhuD acts also as a heme storage under iron depleted condition [START_REF] Matthews | MhuD from Mycobacterium tuberculosis: Probing a Dual Role in Heme Storage and Degradation[END_REF].

Besides iron, intracellular MTB also scavenges zinc to survive. Zinc uptake by MTB is mediated by the zinc uptake regulator (Zur or FurB) [START_REF] Maciaģ | Global analysis of the Mycobacterium tuberculosis Zur (FurB) regulon[END_REF]. In a zinc limited environment, zur expression is upregulated. Moreover, in those environmental conditions, MTB also shows an adaptive response with an increased resistance to oxidative stress (with higher KatG expression) [START_REF] Dow | Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis[END_REF]. However, the most described host defense mechanism related to zinc is intoxication rather than starvation [START_REF] Neyrolles | Zinc and copper toxicity in host defense against pathogens: Mycobacterium tuberculosis as a model example of an emerging paradigm[END_REF](Neyrolles et al., , 2015)). To counteract zinc toxicity, MTB extrudes excessive zinc via the P1B-type ATPase efflux pump, CtpC and CtpV [START_REF] Botella | Mycobacterial P 1-Type ATPases mediate resistance to Zinc poisoning in human macrophages[END_REF][START_REF] Ward | CtpV: A putative copper exporter required for full virulence of Mycobacterium tuberculosis[END_REF]. The ctpC mutant is hypersensitive to physiological concentrations of zinc and exhibits impaired growth in human macrophages [START_REF] Botella | Mycobacterial P 1-Type ATPases mediate resistance to Zinc poisoning in human macrophages[END_REF].

During the host-pathogen interaction, maintaining a proper metal homeostasis through acquisition and expulsion is crucial for the pathogen survival. As previously described in this thesis, MTB is a well-adapted pathogen that has developed a number of mechanisms to counteract the host immune defenses. Therefore, if the host cannot manage to kill the pathogen, eradication will rely on the use of antibiotics. However, over time, a rapid increase of MTB resistant strains is observed against the drugs currently used to treat TB. As a result, new strategies have to be developed to fight both susceptible and resistant TB, with the host as the central target.

C. New strategies to fight tuberculosis

I.

Host-directed therapies

Definition

The current anti-TB treatment regimen is challenging for the patient. Indeed, this treatment has to be taken over a long period of time and may cause toxicity, generally leading to a possible non-compliance of the patient. As a consequence, a lack of compliance often promotes the acquisition of antibiotic resistance by MTB and the development of MDR-MTB and XDR-MTB [START_REF] Prasad | Adverse drug reactions in tuberculosis and management[END_REF]. These observations highlight the increased importance for novel or improved strategies to treat TB.

In this context, host-directed therapies (HDT) appear to be promising treatments against TB.

They aim to control an infection by modulating the host immune response [START_REF] Machelart | Host-directed therapies offer novel opportunities for the fight against tuberculosis[END_REF]. Unlike antibiotics, these HDT are less likely to induce bacterial resistance as they solely act on the host cellular pathways, by limiting inflammation or tissue damage for instance [START_REF] Kaufmann | Host-directed therapies for bacterial and viral infections[END_REF]. Moreover, HDT have been shown to be efficient on MDR-/XDR-MTB [START_REF] Zumla | Host-directed therapies for multidrug resistant tuberculosis[END_REF]. Due to the possible combinations and synergistic effects with antibiotics, they can also shorten the TB treatment regimens and thereby increase the patient compliance. HDT encompass many molecules targeting different host cellular pathways that are detailed below (Figure 4).

Granuloma-targeted HDT

As mentioned previously, the granuloma is the site of a balance between the host and the pathogen. While it prevents MTB spreading, granuloma is also described as an environment where bacteria multiply and eventually propagate if not controlled [START_REF] Pagán | The Formation and Function of Granulomas[END_REF]. Granuloma-targeted therapy is a major advance in the fight against TB, designed to prevent tissue damages, to enhance protective immune response and to lead to a better control or a resolution of the infection [START_REF] Hortle | Host-directed therapies targeting the tuberculosis granuloma stroma[END_REF].

« élément sous droit, diffusion non autorisée » "copyrighted material, unauthorized distribution" Figure 4. Host-directed therapies against MTB [START_REF] Kaufmann | Host-directed therapies for bacterial and viral infections[END_REF].

Granuloma has poor vascularization, low availability of nutrients and a hypoxic center [START_REF] Pagán | The Formation and Function of Granulomas[END_REF]. In these environmental conditions, mycobacterial dormancy is promoted, limiting the impact of some antibiotics on bacterial infection [START_REF] Miranda | The tuberculous granuloma: An unsuccessful host defence mechanism providing a safety shelter for the bacteria?[END_REF]. Furthermore, drug penetration is severely impaired in necrotic lesions of granuloma [START_REF] Prideaux | Mass spectrometry imaging of levofloxacin distribution in TB-infected pulmonary lesions by MALDI-MSI and continuous liquid microjunction surface sampling[END_REF][START_REF] Sarathy | Caseum: A niche for mycobacterium tuberculosis drugtolerant persisters[END_REF].

On one hand, granuloma-targeted therapy promotes granuloma's vascularization (with the potential to promote drug diffusion), increases the access of the host immune cells and promotes tissue healing [START_REF] Dartois | The path of anti-tuberculosis drugs: From blood to lesions to mycobacterial cells[END_REF]. On the other hand, promoting angiogenesis without an adequate antibiotic treatment is detrimental as it may facilitate MTB spreading through blood vessels. Interestingly, patients with TB tend to have a higher level of angiogenic factors like VEGF [START_REF] Alatas | Vascular Endothelial Growth Factor Levels in Active Pulmonary Tuberculosis[END_REF][START_REF] Matsuyama | Increased Serum Level of Vascular Endothelial Growth Factor in Pulmonary Tuberculosis[END_REF] as MTB increases the production of VEGF via cis-cyclopropanation of TDM or angiopoietin-2 (another angiogenic factor) [START_REF] Oehlers | Infection-induced vascular permeability aids mycobacterial growth[END_REF][START_REF] Polena | Mycobacterium tuberculosis exploits the formation of new blood vessels for its dissemination[END_REF][START_REF] Walton | Cyclopropane Modification of Trehalose Dimycolate Drives Granuloma Angiogenesis and Mycobacterial Growth through Vegf Signaling[END_REF]). Unlike the previous example, inhibiting angiogenesis is also an HDT strategy to limit MTB growth, by blocking the VEFG production or its VEGFR receptor. Anti-VEGF-like bevacizumab inhibits the growth and the dissemination of MTB in rabbits [START_REF] Datta | Anti-vascular endothelial growth factor treatment normalizes tuberculosis granuloma vasculature and improves small molecule delivery[END_REF] and in mice [START_REF] Polena | Mycobacterium tuberculosis exploits the formation of new blood vessels for its dissemination[END_REF]. Small molecules such as pazopanib, interfering with the kinase activity downstream of VEGFR activation and antibodies blocking VEGFR, reduce bacterial burden in a zebrafish model [START_REF] Oehlers | Infection-induced vascular permeability aids mycobacterial growth[END_REF]. Some studies also report the use of anti-VEFG on ocular TB with good results, showing a potential future for this HDT to treat different types of TB [START_REF] Invernizzi | Optic Nerve Head Tubercular Granuloma Successfully Treated with Anti-VEGF Intravitreal Injections in Addition to Systemic Therapy[END_REF]Jain et al., 2019).

Fibrosis surrounding granuloma is one of the main characteristics of TB disease. Matrix metalloproteinases (MMP) are a key mediator in TB pathology regarding the granuloma degradation and are connected with clinical markers of tissue damages [START_REF] Elkington | MMP-1 drives immunopathology in human tuberculosis and transgenic mice[END_REF].

During the disease, MTB upregulates mmp9 expression which leads to recruitment of uninfected macrophages, thus favoring the bacterial spreading [START_REF] Volkman | Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium[END_REF]. In the MTB-infected human tissue model, global inhibition of MMPs results in smaller and fewer granuloma and reduced bacterial load [START_REF] Parasa | Inhibition of tissue matrix metalloproteinases interferes with Mycobacterium tuberculosis-induced granuloma formation and reduces bacterial load in a human lung tissue model[END_REF]. In addition, a study has demonstrated that MMP inhibition stabilizes the vascular integrity of the granuloma and increases the delivery of anti-TB drugs such as RIF and INH [START_REF] Xu | Matrix metalloproteinase inhibitors enhance the efficacy of frontline drugs against Mycobacterium tuberculosis[END_REF]. A clinical trial has been conducted with doxycycline, a MMP9 inhibitor, to determine the role of MMP inhibition in patients. This trial reveals that doxycycline inhibits matrix destruction and reduces pulmonary cavity volume [START_REF] Miow | Doxycycline host-directed therapy in human pulmonary tuberculosis[END_REF]. While MMP inhibitors are valuable and promising candidates as HDT for TB treatment, deeper investigations are needed to validate the benefits of this HDT. Indeed, MMP are valuable enzymes with many functions in the host.

Therefore inhibiting these proteases could lead to diverse side effects, potentially toxic for human.

Immune cell functions-targeted HDT

Besides granulomas, HDT can directly target the immune cell functions in order to prevent bacterial growth and dissemination, by limiting MTB entry into the host, modulating the ROS production or enhancing autophagy.

Host trafficking

One way to interfere with the MTB infection cycle is to target the entry of mycobacteria inside macrophages. Tyrosine kinases are known to be activated during the internalization of MTB and is therefore a prime target for HDT. The Abelson (Abl) tyrosine kinase is involved in bacterial uptake and modulates the acidification of the phagosome via the V-ATPase expression [START_REF] Bruns | Abelson Tyrosine Kinase Controls Phagosomal Acidification Required for Killing of Mycobacterium tuberculosis in Human Macrophages[END_REF]. Imatinib, an inhibitor of the Abl tyrosine kinase, reduces the number of granulomatous lesions and bacterial load in infected organs [START_REF] Napier | Imatinib-Sensitive tyrosine kinases regulate mycobacterial pathogenesis and represent therapeutic targets against tuberculosis[END_REF]. This inhibitor also impedes the phagosomal acidification and has an antimicrobial activity against BCG [START_REF] Steiger | Imatinib Triggers Phagolysosome Acidification and Antimicrobial Activity against Mycobacterium bovis Bacille Calmette-Guérin in Glucocorticoid-Treated Human Macrophages[END_REF]. Furthermore, a synergistic effect between imatinib and firstline anti-TB drug RIF has been shown in murine macrophages cell lines infected by M. marinum [START_REF] Napier | Imatinib-Sensitive tyrosine kinases regulate mycobacterial pathogenesis and represent therapeutic targets against tuberculosis[END_REF]. The Src tyrosine kinase also regulates phagosomal maturation, acidification and autophagy [START_REF] Karim | Express path analysis identifies a tyrosine kinase Src-centric network regulating divergent host responses to Mycobacterium tuberculosis infection[END_REF]. Its inhibition by the AZD0530 inhibitor decreases the bacterial survival in human cell line THP-1 macrophages and in guinea pigs [START_REF] Chandra | Targeting Drug-Sensitive and -Resistant Strains of Mycobacterium tuberculosis by Inhibition of Src Family Kinases Lowers Disease Burden and Pathology[END_REF]. A larger in silico study with repurposed drugs reveals that dovitinib, AT9283, and ENMD-2076 target receptors of tyrosine kinase. Tyrosine kinase inhibition mediated by these drugs decreases the survival of intracellular MTB [START_REF] Korbee | Combined chemical genetics and data-driven bioinformatics approach identifies receptor tyrosine kinase inhibitors as host-directed antimicrobials[END_REF]. All of these studies identify the tyrosine kinase signaling pathway as a major source of HDT against MTB.

Reactive oxygen species

While ROS production is an important defense mechanism for macrophages against pathogen infection, excess of ROS and low level of antioxidants lead to an uncontrolled oxidative stress and induce necrosis of the cell [START_REF] Morgan | TNFα and reactive oxygen species in necrotic cell death[END_REF]. In this context, using HDT to finely tune the ROS production allows the enhancement of the antimicrobial activity while maintaining the integrity of the cell. N-acetylcysteine (NAC) provides cysteine, necessary for the synthesis of glutathione (GSH), an intracellular antioxidant [START_REF] Ejigu | N-Acetyl Cysteine as an Adjunct in the Treatment of Tuberculosis[END_REF]. GSH creates redox homeostasis that protects cells and tissues from oxidative damage [START_REF] Ejigu | N-Acetyl Cysteine as an Adjunct in the Treatment of Tuberculosis[END_REF]. In addition, S-nitrosoglutathione (GSSNO; reaction of NO with GSH) exerts an anti-mycobacterial effect [START_REF] Venketaraman | Glutathione and nitrosoglutathione in macrophage defense against Mycobacterium tuberculosis[END_REF]. Moreover, NAC itself has an anti-mycobacterial activity [START_REF] Amaral | N-acetyl-cysteine exhibits potent anti-mycobacterial activity in addition to its known anti-oxidative functions[END_REF]. Indeed, a study performed on MTB-infected guinea pigs demonstrates that adjunction of NAC decreases spleen bacterial load as well as the severity of the lungs lesions by limiting oxidative conditions [START_REF] Palanisamy | Evidence for oxidative stress and defective antioxidant response in guinea pigs with tuberculosis[END_REF]. The effect and safety of NAC has been tested in two randomized control trials. The first one shows that patients on intensive anti-TB regimen phase supplemented by NAC, have an improved lung pathology with faster sputum conversion [START_REF] Mahakalkar | Nacetylcysteine as an add-on to Directly Observed Therapy Short-I therapy in fresh pulmonary tuberculosis patients: A randomized, placebo-controlled, double-blinded study[END_REF]. The second one performed on HIVpositive patients concludes that the use of NAC is safe for TB patients with HIV coinfection [START_REF] Safe | Safety and efficacy of Nacetylcysteine in hospitalized patients with HIV-associated tuberculosis: An open-label, randomized, phase II trial (RIPENACTB Study)[END_REF]. However, the result on sputum conversion on this study was not assessed. Further confirmations will be needed to clarify the potential role of NAC as an HDT for TB treatment.

Autophagy

Autophagy is a common mechanism used by the cell to control MTB infection. Enhancement of autophagy is often associated with a better bacterial clearance. Several drugs have been identified to promote autophagic pathways at different levels.

During MTB infection, the PI3P/Akt/mTOR is activated, facilitating the intracellular survival of MTB (P. [START_REF] Singh | Harnessing the mTOR Pathway for Tuberculosis Treatment[END_REF]. PI3P/Akt/mTOR is a signaling pathway that plays a key role in cell homeostasis by regulating apoptosis, cell cycle and autophagy. When activated, mTOR blocks the initiation of the complex that starts the autophagosome formation (P. [START_REF] Singh | Harnessing the mTOR Pathway for Tuberculosis Treatment[END_REF]. Therefore, inhibition of mTOR appears to be a potential target for HDT.

Everolimus, a derivative of rapamycin -a mTOR inhibitor, is efficient against MTB infection inside granulomas and displays additive effects with first line anti-TB drugs [START_REF] Ashley | Antimycobacterial effects of everolimus in a human granuloma model[END_REF]). Bruton's tyrosine kinase (BKT) acts on the PI3P/Akt/mTOR pathway. Ibrutinib, a BKT inhibitor, reduces intracellular MTB in human macrophages and in mice, and restores the autophagy flux for autophagosomes-containing MTB [START_REF] Hu | Ibrutinib suppresses intracellular mycobacterium tuberculosis growth by inducing macrophage autophagy[END_REF].

Vitamins are necessary for regulation of many host cellular functions. Specifically, vitamin D, also called calcitriol, plays a role in controlling TB infection. Vitamin D binds to its receptor VDR which acts as a transcription factor, regulating numerous genes coding for cathelicidin, defensines and MMP enzymes [START_REF] Periyasamy | Vitamin D -A host directed autophagy mediated therapy for tuberculosis[END_REF]. Cathelicidin (LL-37), previously described in this thesis, an antimicrobial peptide produced following MTB infection and TLRs stimulation (P. T. [START_REF] Liu | Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response[END_REF], increases autophagy and the production of protective cytokines [START_REF] Periyasamy | Vitamin D -A host directed autophagy mediated therapy for tuberculosis[END_REF]. Vitamin D adjunctive therapy has been widely tested in trials with no clear conclusion drawn. One trial suggested that vitamin D supplementation promotes radiographic improvement and increases host immune activation but only among patients with a previous vitamin D deficiency [START_REF] Salahuddin | Vitamin D accelerates clinical recovery from tuberculosis: results of the SUCCINCT Study [Supplementary Cholecalciferol in recovery from tuberculosis]. A randomized, placebocontrolled, clinical trial of vitamin D supplementation in patients with pulmonary tuberculosis[END_REF]. A recent meta-analysis on vitamin D supplementation concludes that this vitamin has no beneficial effect on the anti-TB treatment but diminishes the sputum conversion in patients only with a certain polymorphism in VDR (J. [START_REF] Zhang | Effectiveness of vitamin D supplementation on the outcome of pulmonary tuberculosis treatment in adults: A meta-analysis of randomized controlled trials[END_REF]. Observed differences might be linked to the dosage, the type of administration of the vitamin D or the endogenous level of vitamin D among patients between trials. Still, anti-TB treatment associated with vitamin D could be beneficial, but only on a certain population.

Metformin is an anti-hyperglycemic agent with immunomodulatory effects, that promotes autophagy and phagosome-lysosome fusion via the 5'AMP-activated protein-kinase (AMPK) pathway (G. [START_REF] Zhou | Role of AMPactivated protein kinase in mechanism of metformin action[END_REF]. Furthermore, metformin induces mtROS production and promotes phagosome maturation in vitro and in vivo in MTB-infected models [START_REF] Singhal | Metformin as adjunct antituberculosis therapy[END_REF]. Importantly, this drug also enhances anti-TB drug activity of INH and ethionamide in a mice model [START_REF] Singhal | Metformin as adjunct antituberculosis therapy[END_REF]. Regarding clinical data, several studies have been repeatedly demonstrated that metformin is beneficial for diabetic patients by reducing the risk of TB [START_REF] Yu | Impact of metformin on the risk and treatment outcomes of tuberculosis in diabetics: A systematic review[END_REF]M. Zhang & He, 2019). Nowadays, several trials are ongoing to evaluate the use of metformin with other anti-TB treatments among patients with [START_REF] Padmapriyadarsini | Evaluation of metformin in combination with rifampicin containing antituberculosis therapy in patients with new, smear-positive pulmonary tuberculosis (METRIF): Study protocol for a randomised clinical trial[END_REF] or without HIV (Phase II clinical trial). A study on blood and immune cells from healthy individuals shows that metformin decreases the host inflammatory response upon stimulation of MTB [START_REF] Lachmandas | Metformin Alters Human Host Responses to Mycobacterium tuberculosis in Healthy Subjects[END_REF]. However, disparate results have been obtained in nondiabetic versus diabetic mice [START_REF] Sathkumara | Disparate effects of metformin on mycobacterium tuberculosis infection in diabetic and nondiabetic mice[END_REF]. Further non-retrospectives studies in non-diabetic patients are needed to confirm the benefits of metformin in the treatment of TB.

Numerous drugs were shown to induce autophagy and reduce intracellular MTB growth in macrophages such as loperamide, verapamil or valproic acid [START_REF] Abate | New verapamil analogs inhibit intracellular mycobacteria without affecting the functions of Mycobacterium-specific T cells[END_REF][START_REF] Juárez | Loperamide restricts intracellular growth of Mycobacterium tuberculosis in lung macrophages[END_REF]. More recently, BDQ has been found to activate autophagy and improve bacterial clearance [START_REF] Giraud-Gatineau | The antibiotic bedaquiline activates host macrophage innate immune resistance to bacterial infection[END_REF]. Together, these studies suggest that targeting autophagy pathways by HDT could be a valuable strategy to improve TB treatment.

Inflammatory modulators and cytokines modulation by HDT

Corticosteroids

While inflammatory response related to MTB infection is crucial for the granuloma formation, it may also be detrimental for the host, by creating tissue damages if not controlled.

Corticosteroids have anti-inflammatory and immunosuppressive actions and have been extensively employed to treat infections and autoimmune diseases [START_REF] Coutinho | The anti-inflammatory and immunosuppressive effects of glucocorticoids, recent developments and mechanistic insights[END_REF]. The use of corticosteroids as an adjunctive treatment for TB is based on their antiinflammatory capacity, preventing chronic and unproductive inflammation. Mechanisms of action of corticosteroids are based on the regulation of anti-inflammatory genes such as IL-10 via their interaction with the glucocorticoid-responsive element after linkage to glucocorticoid receptors [START_REF] Schutz | Corticosteroids as an adjunct to tuberculosis therapy[END_REF]. Moreover, corticosteroids interact with NF-κB pathways, inhibiting the transcription of inflammatory mediators [START_REF] Scheinman | Characterization of mechanisms involved in transrepression of NF-κB by activated glucocorticoid receptors[END_REF]. They also mediate several signal transduction cascades via their binding to G protein-coupled receptors [START_REF] Schutz | Corticosteroids as an adjunct to tuberculosis therapy[END_REF]. Recently, corticosteroids, notably dexamethasone, have also been shown to inhibit necrotic cell death induced by MTB via the inhibition of the host cell death p38 mitogen-activated protein kinase (MAPK) pathway mediated by mitochondrial membrane integrity [START_REF] Gräb | Corticosteroids inhibit Mycobacterium tuberculosis-induced necrotic host cell death by abrogating mitochondrial membrane permeability transition[END_REF].

Many trials have been conducted to evaluate the role of corticosteroids, like dexamethasone and prednisolone, as adjuvant for TB treatment with very different outcomes [START_REF] Schutz | Corticosteroids as an adjunct to tuberculosis therapy[END_REF]. For instance, a meta-analysis performed on all forms of TB concludes that corticosteroids are effective to reduce mortality [START_REF] Critchley | Corticosteroids for prevention of mortality in people with tuberculosis: A systematic review and meta-analysis[END_REF]. However, another meta-analysis on pulmonary TB only suggests that adjunctive corticosteroids do not improve long-term treatment efficacy but induce more toxic side effects [START_REF] Xie | The efficacy and safety of adjunctive corticosteroids in the treatment of tuberculous pleurisy: A systematic review and meta-analysis[END_REF]. Together, these two analyses point out that localization of TB -pulmonary or extrapulmonary -as well as the patient should be considered before using this adjunct therapy. Finally, it is important to bear in mind that corticosteroids reduce the cytokine responses to TB antigens used in IGRA assays and may interfere with the results [START_REF] Clifford | The impact of anti-tuberculous antibiotics and corticosteroids on cytokine production in QuantiFERON-TB Gold In Tube assays[END_REF]. Dexamethasone is currently the only drug in phase IV trials for the therapy of meningeal TB and further investigations are required to clarify the effect of corticosteroids on TB outcomes.

Phosphodiesterase inhibitors

Phosphodiesterases (PDE) are part of a large family of enzymes with different structures, substrates specificity and tissue distribution [START_REF] Page | Phosphodiesterase inhibitors in the treatment of inflammatory diseases[END_REF]. Host PDE hydrolyze two second messengers: cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). cAMP has an anti-inflammatory and tissue protective effect when highly concentrated in the cell [START_REF] Page | Phosphodiesterase inhibitors in the treatment of inflammatory diseases[END_REF]. In this context, PDE are suitable candidates for HDT, as their inhibition should result in a reduction of inflammation and an improvement of antimycobacterial responses. A study performed on the PDE-3 inhibitor cilostazol and the PDE-5 inhibitor sildenafil shows that these molecules reduce tissue damage and TNF-α, promote bacterial clearance and accelerate lung sterilization in mice [START_REF] Maiga | Successful shortening of tuberculosis treatment using adjuvant hostdirected therapy with FDA-approved phosphodiesterase inhibitors in the mouse model[END_REF]. Other studies on PDE-4 inhibitors such as CC-11050, CC-3052 and roflumilast demonstrate similar properties on rabbits and on mice [START_REF] Koo | Phosphodiesterase 4 inhibition reduces innate immunity and improves isoniazid clearance of Mycobacterium tuberculosis in the lungs of infected mice[END_REF][START_REF] Subbian | Adjunctive Phosphodiesterase-4 Inhibitor Therapy Improves Antibiotic Response to Pulmonary Tuberculosis in a Rabbit Model[END_REF].

More interestingly, these PDE inhibitors associated with INH lead to a higher reduction on the bacterial load and a better lung pathology [START_REF] Koo | Phosphodiesterase 4 inhibition reduces innate immunity and improves isoniazid clearance of Mycobacterium tuberculosis in the lungs of infected mice[END_REF][START_REF] Subbian | Phosphodiesterase-4 inhibition combined with isoniazid treatment of rabbits with pulmonary tuberculosis reduces macrophage activation and lung pathology[END_REF][START_REF] Subbian | Adjunctive Phosphodiesterase-4 Inhibitor Therapy Improves Antibiotic Response to Pulmonary Tuberculosis in a Rabbit Model[END_REF]. A phase II clinical trial on humans is ongoing with CC-11050 to assess its potential role to shorten TB treatment.

Cytokines

Cytokines play a key role in TB outcomes. Depending on their pro-or anti-inflammatory properties, they modulate the host immunity as well as the cell response to the pathogen.

HDT targeting cytokines aim to reduce excessive or inappropriate cytokine responses like with anti-TNF-α in certain cases [START_REF] Mootoo | TNF-α in tuberculosis: A cytokine with a split personality[END_REF]. These HDT also promote Th1 cytokines such as IL-2, IL-12, IFN-γ and GM-CSF, important for the host defense systems against pathogens (Zeng et al., 2017). TNF-α is a cytokine with different effects on the host. An appropriate TNF-α level preserves granuloma integrity whereas high levels worsen the outcome of TB [START_REF] Mootoo | TNF-α in tuberculosis: A cytokine with a split personality[END_REF]. Therefore, TNF-α therapies preferentially focus on reducing the level of this cytokine [START_REF] Morgan | TNFα and reactive oxygen species in necrotic cell death[END_REF]. The use of TNF-α antibodies (adalimumab and infliximab) during the specific paradoxical reactions (exacerbation of the disease and worse inflammation after initiation of anti-TB therapy) has been highly effective for treating this type of patients [START_REF] Blackmore | Therapeutic use of infliximab in tuberculosis to control severe paradoxical reaction of the brain and lymph nodes[END_REF][START_REF] Wallis | Drug tolerance in Mycobacterium tuberculosis[END_REF]. Use of anti-TNF-α as a therapy should therefore be carefully considered and only in certain cases (like paradoxical reactions).

IFN-γ is one of the main cytokines involved in the protective response against MTB and is considered as a good candidate for cytokines therapy [START_REF] Cavalcanti | Role of TNF-Alpha, IFN-Gamma, and IL-10 in the Development of Pulmonary Tuberculosis[END_REF]. IFN-γ activates macrophages, promotes cell proliferation, apoptosis and bacterial killing [START_REF] Cavalcanti | Role of TNF-Alpha, IFN-Gamma, and IL-10 in the Development of Pulmonary Tuberculosis[END_REF]. IFN-γ deficient mice are highly susceptible to MTB infection [START_REF] Flynn | An essential role for interferon γ in resistance to mycobacterium tuberculosis infection[END_REF]. Given the importance of this cytokine, several human trials have been implemented to assess the efficacy of HDT targeting IFN-γ. In one study, patients treated with aerosolized human recombinant IFN-γ1b show an improvement on MTB sputum clearance and a decrease of inflammatory cytokines level at the site of the disease [START_REF] Condos | Recombinant gamma interferon stimulates signal transduction and gene expression in alveolar macrophages in vitro and in tuberculosis patients[END_REF]. Another study has been performed on TB patients with nebulized IFN-γ1b or subcutaneous injections of IFN-γ1b treatment. All patients treated with IFN-γ (nebulized or injected) have better outcomes for TB and increased sputum conversion [START_REF] Dawson | Immunomodulation with recombinant interferon-γ1b in pulmonary tuberculosis[END_REF]. Despite the effectiveness of INF-γ targeting HDT, these latter are expensive and difficult to produce [START_REF] Hawn | Host-Directed Therapeutics for Tuberculosis: Can We Harness the Host?[END_REF].

Several other trials should be performed to confirm the benefit of this cytokine.

Regarding other cytokines, a study has revealed that overexpression of GM-CSF in acute TB contributes to an efficient M1 polarization and effective immune response (Benmerzoug et al., 2018). On one hand, TB patients treated with adjunct subcutaneous GM-CSF show a faster sputum clearance without any adverse effect, displaying its potential role for HDT [START_REF] Pedral-Sampaio | Use of Rhu-GM-CSF in pulmonary tuberculosis patients: results of a randomized clinical trial[END_REF]. On the other hand, a trial using adjunctive recombinant IL-2 has failed to prove a difference in sputum conversion or in bacterial clearance after a couple of months, compared to untreated patients [START_REF] Johnson | Randomized trial of adjunctive interleukin-2 in adults with pulmonary tuberculosis[END_REF].

In conclusion, targeting cytokines as an HDT could have unpredictable outcomes. Use of these therapies should therefore be used with caution to avoid exacerbation or unappropriated host immune response.

Host lipid metabolism

Eicosanoids: Eicosanoids, previously described in this thesis, are important signaling molecules that modulate inflammation and cell death (M. [START_REF] Chen | Lipid mediators in innate immunity against tuberculosis: Opposing roles of PGE 2 and LXA 4 in the induction of macrophage death[END_REF][START_REF] Rocca | Cyclooxygenases and prostaglandins: Shaping up the immune response[END_REF].

The balance between PGE2 and LXA4 / LXB4 is crucial as depending on the predominant eicosanoid, it can lead to apoptosis or necrosis of the cell (M. [START_REF] Chen | Lipid mediators in innate immunity against tuberculosis: Opposing roles of PGE 2 and LXA 4 in the induction of macrophage death[END_REF]. To tilt the balance in favor of PGE2 side, HDT could inhibit the 5-lipoxygenases (5-LOX), responsible of LXA4 synthesis (which could be hydrolyzed in LXB4). Indeed, lipoxins negatively regulate the production of major protective cytokines in TB infection like IL-12 or IFN-γ but also the enzyme NOX2 [START_REF] Bafica | Host control of Mycobacterium tuberculosis is regulated by 5-lipoxygenase-dependent lipoxin production[END_REF]. Inhibition of LXA4 should therefore be beneficial for the host. In this respect, a study has shown that mice treated with zileuton, a 5-LOX inhibitor, associated with PGE2 supplementation, display higher levels of IL-1β production by limiting type I IFN and a decrease of MTB growth [START_REF] Mayer-Barber | Host-directed therapy of tuberculosis based on interleukin-1 and type i interferon crosstalk[END_REF]. Besides being considered as a potential HDT candidate to treat TB, zileuton is also used to treat asthma [START_REF] Thalanayar Muthukrishnan | Zileuton use and phenotypic features in asthma[END_REF].

Despite the apparent benefit of PGE2 in TB, the role of PGE2 remains controversial in MTB infection [START_REF] Moreno | The role of prostaglandin E2 in the immunopathogenesis of experimental pulmonary tuberculosis[END_REF][START_REF] Sorgi | Eicosanoid pathway on host resistance and inflammation during Mycobacterium tuberculosis infection is comprised by LTB4 reduction but not PGE2 increment[END_REF]. Even if PGE2 is important at an early stage of infection, later overexpression of PGE2 downregulates the cell-mediated immunity, especially IFN-γ and TNF-α [START_REF] Moreno | The role of prostaglandin E2 in the immunopathogenesis of experimental pulmonary tuberculosis[END_REF][START_REF] Sorgi | Eicosanoid pathway on host resistance and inflammation during Mycobacterium tuberculosis infection is comprised by LTB4 reduction but not PGE2 increment[END_REF]. Non-steroidal antiinflammatory drugs (NSAID) counteract the overproduction of PGE2. NSAID inhibits COX activity, and therefore the production of prostaglandins and other prostanoids with proinflammatory activity [START_REF] Maitra | Repurposing drugs for treatment of tuberculosis: A role for nonsteroidal anti-inflammatory drugs[END_REF]. Ibuprofen, aspirin, celecoxib and diclofenac significantly increase host cell survival, protect lungs and decrease bacterial loads in mice [START_REF] Kroesen | Non-steroidal anti-inflammatory drugs as host-directed therapy for tuberculosis: A systematic review[END_REF]. Furthermore, combining anti-TB drugs with NSAID leads to a synergic effect on bacterial load [START_REF] Dutta | Activity of diclofenac used alone and in combination with streptomycin against Mycobacterium tuberculosis in mice[END_REF]. Ibuprofen alone is efficient on bacillary loads and on mice survival [START_REF] Vilaplana | Ibuprofen therapy resulted in significantly decreased tissue bacillary loads and increased survival in a new murine experimental model of active tuberculosis[END_REF]. However, a study with patients taking celecoxib and anti-TB drugs together does not highlight a change in bactericidal activity [START_REF] Naftalin | Adjunctive use of celecoxib with antituberculosis drugs: evaluation in a whole-blood bactericidal activity model[END_REF]. Another large population-based study demonstrates that patients using NSAID are associated with increased risk for active TB, emphasizing the divergence between the studies (C. W. [START_REF] Wu | Risk of incident active tuberculosis disease in patients treated with non-steroidal anti-inflammatory drugs: A population-based study[END_REF]. Several trials are currently ongoing to assess the benefit of NSAID for TB treatment and should status for their use or not.

Statins: Statins are lipid-lowering drugs that inhibit the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase [START_REF] Parihar | Statin Therapy Reduces the Mycobacterium tuberculosis Burden in Human Macrophages and in Mice by Enhancing Autophagy and Phagosome Maturation[END_REF]. This enzyme is essential in lipid metabolism and acts on inflammatory pathways [START_REF] Lobato | Statins increase rifampin Mycobactericidal effect[END_REF][START_REF] Skerry | Simvastatin increases the in vivo activity of the first-line tuberculosis regimen[END_REF]. Many studies confirm that statins enhance the in vitro activity of first-line anti-TB drugs like RIF or INH [START_REF] Lobato | Statins increase rifampin Mycobactericidal effect[END_REF][START_REF] Skerry | Simvastatin increases the in vivo activity of the first-line tuberculosis regimen[END_REF]. Statins contribute to macrophage-bactericidal activity against MTB by increasing phagosomal maturation and by promoting host-induced autophagy in murine macrophages [START_REF] Parihar | Statin Therapy Reduces the Mycobacterium tuberculosis Burden in Human Macrophages and in Mice by Enhancing Autophagy and Phagosome Maturation[END_REF]. These effects are supposedly linked to the reduction of total cholesterol in host macrophages, thus limiting the favorable lipid environment for MTB [START_REF] Parihar | Statin Therapy Reduces the Mycobacterium tuberculosis Burden in Human Macrophages and in Mice by Enhancing Autophagy and Phagosome Maturation[END_REF]. A study reports that hospitalized patients treated with anti-TB drugs and statins at the same time display a reduction of mortality after admission [START_REF] Jeeraaumponwat | Statin with standard antituberculosis regimen and mortality in hospitalized pulmonary tuberculosis[END_REF]. A national representative cohort has confirmed that these drugs decrease the risk of active TB, suggesting a real benefit of these lipid-lowering drugs [START_REF] Lai | Statin treatment is associated with a decreased risk of active tuberculosis: an analysis of a nationally representative cohort[END_REF]. However, a recent population-based cohort study affirms that statins do not improve anti-TB treatment (Y.-T. [START_REF] Chen | Use of lipid-lowering agents is not associated with improved outcomes for tuberculosis patients on standard-course therapy: A population-based cohort study[END_REF]. These inconsistencies may be attributed to the differences in the cohort. Thus, further trials are needed to conclude on statins effects.

HDT can be a useful tool in the fight against TB, including the MDR-TB and XDR-TB. Used in combination with anti-TB drugs, they could shorten the TB treatment, reduce side and toxic effects as well as improve patient compliance. While the impact of HDT on TB treatment could be major, further studies and trials are needed to fully include HDT as part of the TB regimen.

Recently, many studies have focused on the ability of pathogens to modify host epigenetics.

Thus, it would be interesting to investigate whether targeting the host epigenome as a novel HDT may give the host an advantage on the fight against MTB.

II.

Targeting epigenetics

Definition

The concept of epigenetics was first introduced in 1942 by Conrad Waddington [START_REF] Deichmann | Epigenetics: The origins and evolution of a fashionable topic[END_REF]. He defined epigenetics as "the complex of developmental processes between the genotype and phenotype". Over the years, this concept has evolved and now refers to the research on chromatin modifications such as DNA or histone modifications and RNA related changes, without a change in the DNA base sequence [START_REF] Deichmann | Epigenetics: The origins and evolution of a fashionable topic[END_REF]. Recently, it has been reported that pathogens target and modulate the host epigenome (epigenetic status of the host cell) to their advantage to enhance their intracellular survival [START_REF] Fatima | Epigenetic code during mycobacterial infections: therapeutic implications for tuberculosis[END_REF]. Three main categories of epigenetic changes are modulated by pathogens: DNA methylation, histone modifications and RNA modifications (M. Singh et al., 2018) (Figure 5).

DNA methylation: DNA methylation is a chemical process in which a methyl group (CH3) is added to a cytosine residue [START_REF] Adhikari | DNA methyltransferases and epigenetic regulation in bacteriaa[END_REF]. This methylation tag is mostly common on the CpG sequence in the genome and is regulated by various DNA methyltransferase (DNMTs) enzymes DNMT1, DNMT2, DNMT3a, and DNMT3b [START_REF] Adhikari | DNA methyltransferases and epigenetic regulation in bacteriaa[END_REF][START_REF] Bird | DNA methylation patterns and epigenetic memory[END_REF]. Among them, isoform DNMT1 is the most abundant and methylates already hemimethylated CpG dinucleotides whereas DNMT3a/b performs "de novo" methylation or hemi-« élément sous droit, diffusion non autorisée » "copyrighted material, unauthorized distribution" [START_REF] Gartstein | Prenatal influences on temperament development: The role of environmental epigenetics[END_REF].

methylation [START_REF] Bird | DNA methylation patterns and epigenetic memory[END_REF]. DNA methylation leads to gene silencing by preventing the recruitment of transcriptional factors or by recruiting some methyl-binding proteins that bind transcriptional corepressor complexes [START_REF] Miller | The role of DNA methylation and histone modifications in transcriptional regulation in humans[END_REF]. DNA methylation is stable and usually influences the development, the differentiation and the gene expression of the host cell [START_REF] Miller | The role of DNA methylation and histone modifications in transcriptional regulation in humans[END_REF].

Histone modifications: Chromatin is formed at its basic unit by the association of proteins called histones with eukaryotic DNA. This unit called the nucleosome is composed of approximately 147 base pairs of DNA attached to an octamer of four core histones proteins (H2A, H2B, H3, and H4) (Tollefsbol, 2011). The linker histone H1 for its part, links nucleosomes with each other and around the DNA. The structure of the chromatin is dynamic and is divided into euchromatin or heterochromatin, that are transcriptionally active or inactive states, respectively [START_REF] Tjelle | Phagosome dynamics and function[END_REF]. In the euchromatin region, DNA is accessible and contains genes that can be transcribed; DNA in heterochromatin is highly condensed and inaccessible.

Histone modifications change chromatin status and therefore modulate various processes such as gene expression or DNA repair and DNA replication [START_REF] Miller | The role of DNA methylation and histone modifications in transcriptional regulation in humans[END_REF]. Histones are positively charged and interact with the negatively charged DNA. Modification on histone neutralizes these positive charges, allowing the exposition of DNA to transcription factors [START_REF] Miller | The role of DNA methylation and histone modifications in transcriptional regulation in humans[END_REF]. Histone modifications are post-translational modifications and include methylation, acetylation, phosphorylation, ubiquitination and sumoylation (Bowman & Poirier, 2014). They are performed by different enzymes like histone methyltransferases / demethylase, histone acetyltransferases / deacetylases, kinases or phosphatases (Bowman & Poirier, 2014). Histone acetylation leads to transcriptional activation whereas deacetylation leads to the opposite. Depending on the amino acid that is methylated and the degree of methylation, histone methylation activates or silences gene expression. In conclusion, epigenetic modifications on histone are wide, diverse and have an important impact on gene expression.

RNA modifications: Noncoding RNA are small RNA (21-24 nucleotides) that regulate gene expression at the post-transcriptional level (Treiber et al., 2018). These noncoding RNA, also called microRNA (miRNA), have an important role in cell proliferation, differentiation or apoptosis (Treiber et al., 2018). They are highly conserved endogenous gene silencers which target, along with the RNA-induced silencing complex (RISC), the protein-coding mRNA [START_REF] Tjelle | Phagosome dynamics and function[END_REF]. This binding leads to either mRNA degradation or the inhibition of its translation. Thus, miRNA are major regulators of many cellular pathways, including those related to immunity. Recent studies have focused also on long non-coding RNA (lncRNA) and their role in host response to mycobacterial infection. They are transcripts with more than 200 nucleotides which do not code for proteins but act as chromatin remodelers and regulators of innate immunity [START_REF] Hadjicharalambous | Long Non-Coding RNAs and the Innate Immune Response[END_REF].

Epigenetics during tuberculosis infection

MTB, like other bacterial pathogens, modulates the host epigenome to its advantage, promoting its adaptation to the host environment, latency or growth inside the host cells.

Alteration of the host DNA methylation profile as well as histone modifications or RNA interferences by MTB and their impact on the mycobacterial virulence will be described in this thesis.

DNA methylation

A few years ago, it has been demonstrated that the host DNA methylation profile is modified by MTB during infection in macrophages and in DCs. DNA methylation is an important epigenetic modification whose roles include regulation of chromosome stability, DNA mismatch repair and gene transcription [START_REF] Marinus | Roles of DNA adenine methylation in host-pathogen interactions: Mismatch repair, transcriptional regulation, and more[END_REF]. These methylations are involved in the regulation of transcriptional responses to the infection [START_REF] Pacis | Bacterial infection remodels the DNA methylation landscape of human dendritic cells[END_REF][START_REF] Zheng | Unraveling methylation changes of host macrophages in Mycobacterium tuberculosis infection[END_REF]. MTB possesses several methyltransferases including Rv2966c which localizes in the host cell nucleus and modifies host DNA methylation on non-CpG regions [START_REF] Sharma | The interaction of mycobacterial protein Rv2966c with host chromatin is mediated through non-CpG methylation and histone H3/H4 binding[END_REF]. By comparing genome-wide DNA methylation profiles between THP-1 macrophages infected or not with MTB, a study demonstrates an enrichment of DNA hypermethylation hotspots (associated with gene repression) in MTB-infected macrophages for specific gene families including HLA complex, cytokines and inflammatory associated genes [START_REF] Sharma | Genome-wide non-CpG methylation of the host genome during M. tuberculosis infection[END_REF]. Moreover, a study performed on TB patients shows a higher methylation level on TLR2 promoter's regions and lower TLR2 expression on monocytes, compared to healthy people [START_REF] Kisich | Antimycobacterial agent based on mRNA encoding human β-defensin 2 enables primary macrophages to restrict growth of Mycobacterium tuberculosis[END_REF]. In this study, a TLR2 hypermethylation is associated with active pulmonary TB. Interestingly, TLR2 gene expression is reverted after a six-month anti-TB treatment suggesting this hypermethylation is somehow important for the infection. Similarly, hypermethylation in the CpG island of the VDR gene contributes to the risk of TB for African and European patients [START_REF] Jiang | The methylation state of VDR gene in pulmonary tuberculosis patients[END_REF]. Another study describes that patients with TB have DNA hypermethylation on several genes related to IL-2, NF-κB, and IFN-γ signaling pathways [START_REF] Dinardo | DNA hypermethylation during tuberculosis dampens host immune responsiveness[END_REF]. These methylations are associated with a decreased production of major protective cytokines including TNF-α, IL-12 or IFN-γ (DiNardo et al., 2020). In addition, macrophages isolated from TB patients present a DNA hypermethylation in the inflammatory protective IL-17 gene promoter region [START_REF] Zheng | Unraveling methylation changes of host macrophages in Mycobacterium tuberculosis infection[END_REF]. On the other side, the host modulates its own DNA methylation profile to fight against bacterial infection. For instance, a demethylation of the promoter region of NLRP3 during infection activates the NLRP3 inflammasome and increases the levels of pro-inflammatory cytokines IL-1β and IL-18 to control the infection (M. [START_REF] Wei | NLRP3 Activation Was Regulated by DNA Methylation Modification during Mycobacterium tuberculosis Infection[END_REF].

In conclusion, maintaining host DNA methylation and demethylation profile is crucial in order to have proper immune responses and to fight infections. Modifying this methylation/demethylation patterns could modulate TB outcomes in favor of the host or MTB.

Histone modifications

Histone modifications, by chromatin remodeling, are responsible for the accessibility of DNA to transcription factors, thereby modulating gene expression. These modifications are targeted by pathogens to modulate the host immune machinery for their own survival [START_REF] Fatima | Epigenetic code during mycobacterial infections: therapeutic implications for tuberculosis[END_REF]. For instance, MTB secretes ESAT-6 within the host, which then inhibits histone methylation at the lysine 4 of histone 3 (H3K4) site induced by IFN-γ. This hypomethylation results in the suppression of the Class II transactivator (CIITA) protein expression and thus diminution of antigen presentation within macrophages (P. [START_REF] Kumar | ESAT6 differentially inhibits IFN-γ-inducible class II transactivator isoforms in both a TLR2-dependent andindependent manner[END_REF]. Apart from ESAT-6, the enhanced intracellular survival (Eis) acetyltransferase of MTB induces a high level of IL-10 expression via acetylation of histone H3 [START_REF] Duan | Mycobacterium tuberculosis EIS gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3[END_REF]. This mechanism was presented by the authors as part of the MTB escape mechanism from autophagy [START_REF] Duan | Mycobacterium tuberculosis EIS gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3[END_REF]. The methyltransferase secreted Rv1998 is implicated in host histone modulation by MTB [START_REF] Yaseen | Mycobacteria modulate host epigenetic machinery by Rv1988 methylation of a non-tail arginine of histone H3[END_REF]. Rv1988 di-methylates the arginine at the position 42 of the H3 (H3R42me2), which in turn represses the expression of genes involved in ROS production NOX1, NOX4, NOS2 and the tumor necrosis factor receptor-associated factor (TRAF3) controlling type I IFN production [START_REF] Häcker | Expanding TRAF function: TRAF3 as a tri-faced immune regulator[END_REF][START_REF] Yaseen | Mycobacteria modulate host epigenetic machinery by Rv1988 methylation of a non-tail arginine of histone H3[END_REF]. Deletion of Rv1988 gene in MTB results in reduced survival in the host, highlighting the major role of this methylase during the infection process [START_REF] Yaseen | Mycobacteria modulate host epigenetic machinery by Rv1988 methylation of a non-tail arginine of histone H3[END_REF].

In addition, MTB equally regulates host histone deacetylase (HDAC) to reduce cytokines expression. Indeed, a study shows that MTB infection induces HDAC1 expression with a concomitant decrease of H3 acetylation level observed [START_REF] Chandran | Mycobacterium tuberculosis infection induces HDAC1-mediated suppression of IL-12B gene expression in macrophages[END_REF]. HDAC1 is subsequently recruited to the IL-12B promoter and represses the expression of this cytokine [START_REF] Chandran | Mycobacterium tuberculosis infection induces HDAC1-mediated suppression of IL-12B gene expression in macrophages[END_REF]. IL-12B plays a role in the initiation of the Th1 immune response of the host [START_REF] Cooper | Interleukin 12 (IL-12) is crucial to the development of protective immunity in mice intravenously infected with mycobacterium tuberculosis[END_REF]. In a consistent way, knocking-down HDAC1 gene in macrophages reduces MTB survival and promotes colocalization of MTB and LC3 in autophagosomes [START_REF] Madhavan | Transcription Repressor Protein ZBTB25 Associates with HDAC1-Sin3a Complex in Mycobacterium tuberculosis-Infected Macrophages, and Its Inhibition Clears Pathogen by Autophagy[END_REF]. Furthermore, a study demonstrates that MMP-1, responsible for matrix degradation upon MTB infection, is regulated by histone acetylation and deacetylation.

In MTB-infected macrophages, MMP-1 is upregulated and chemical inhibition of histone acetyltransferases suppressed the MTB-driven MMP-1 (and MMP-3) induction [START_REF] Moores | Epigenetic regulation of matrix metalloproteinase-1 and -3 expression in Mycobacterium tuberculosis infection[END_REF]. This study demonstrates that MTB has the ability to induce changes in host HDAC expression for its own benefice.

Therefore, the previous studies demonstrate that epigenetic mechanisms regulating histone modifications contribute broadly to the MTB arsenal against the host immune response. miRNA miRNA are important regulators of gene expression including cytokines related genes. They act as a key player in host-MTB interaction during infection. It has been shown that many miRNAs are manipulated by MTB to alter cytokines expression. Some miRNA implicated in TB infection are listed in Table 3.

miRNA-125a

UV radiation resistanceassociated gene Inhibition of autophagy (J. K. [START_REF] Kim | MicroRNA-125a Inhibits Autophagy Activation and Antimicrobial Responses during Mycobacterial Infection[END_REF] miRNA-125b TNF-α Inhibition of TNF mRNA and reduction of host inflammatory response [START_REF] Rajaram | Mycobacterium tuberculosis lipomannan blocks TNF biosynthesis by regulating macrophage MAPK-activated protein kinase 2 (MK2) and microRNA miR-125b[END_REF] miRNA-140 TNF receptor -associated factor 6 (TRAF6)

Reduction of pro-inflammatory cytokines levels of IL-6, TNF-α, IL-1β (X. [START_REF] Li | MiR-140 modulates the inflammatory responses of Mycobacterium tuberculosis-infected macrophages by targeting TRAF6[END_REF] miRNA-144 DNA damage-regulated autophagy modulator 2 Inhibition of autophagosome formation (J. K. [START_REF] Kim | MIR144* inhibits antimicrobial responses against Mycobacterium tuberculosis in human monocytes and macrophages by targeting the autophagy protein DRAM2[END_REF] miRNA-146a miRNA are important actors during MTB infection and may represent potential therapeutic targets to fight TB. Furthermore, circulating miRNA in serum are undergoing evaluation to be prognostic biomarkers for TB infection [START_REF] Chakrabarty | Host and MTB genome encoded miRNA markers for diagnosis of tuberculosis[END_REF]. Altogether, miRNA studies represent a various and promising field regarding MTB infection.

Modulation of the host epigenome is a mechanism used by MTB to promote its chances of survival within the host cell. Therefore, inhibiting these mechanisms could be valuable for HDT to fight the mycobacterial infection. For example, several HDACs inhibitors are in clinical trials in the treatment of breast cancer [START_REF] Hontecillas-Prieto | Synergistic Enhancement of Cancer Therapy Using HDAC Inhibitors: Opportunity for Clinical Trials[END_REF]. These molecules being already tested in humans, their benefits could be extended to patients with TB. Recently, a study performed in vitro in human macrophages and in vivo in zebrafish reveals that inhibition of HDACs by trichostatin A enhances the antimicrobial response of M1 and M2 macrophages [START_REF] Moreira | Functional Inhibition of Host Histone Deacetylases (HDACs) Enhances in vitro and in vivo Anti-mycobacterial Activity in Human Macrophages and in Zebrafish[END_REF]. Furthermore, a selective inhibition of class IIa HDACs by TMP195 and TMP26 decreases bacterial growth in M2 macrophages, showing the potential of molecules targeting HDAC in MTB HDT [START_REF] Moreira | Functional Inhibition of Host Histone Deacetylases (HDACs) Enhances in vitro and in vivo Anti-mycobacterial Activity in Human Macrophages and in Zebrafish[END_REF]. Recently, a growing number of studies focuses on specific HDAC upon MTB infection: the sirtuins family.

Example of sirtuins

The sirtuins family is composed of seven proteins called sirtuin 1 to sirtuin 7 (SIRT1-7), with different modes of action and targets [START_REF] Houtkooper | Sirtuins as regulators of metabolism and healthspan[END_REF]. They are divided into four main classes: SIRT1-3 belong to class I, SIRT4 to class II, SIRT5 to class III and SIRT6-7 to class IV [START_REF] Michan | Sirtuins in mammals: Insights into their biological function[END_REF]. The sirtuins have different cellular localizations: SIRT1 is localized in the nucleus and cytosol, SIRT2 in the cytosol, SIRT3-5 in mitochondria, SIRT6 in the nucleus and SIRT7 in the nucleolus [START_REF] Michan | Sirtuins in mammals: Insights into their biological function[END_REF]. SIRT1, 2 and 3 are nicotinamide adenine dinucleotide (NAD + )-dependent class III histone deacetylase. They target histone and nonhistone proteins [START_REF] Houtkooper | Sirtuins as regulators of metabolism and healthspan[END_REF]. The most described SIRT involved upon MTB infection are the SIRT1, 2, 3 and will be described further in this thesis.

SIRT1: this sirtuin is involved in different cellular processes such as host metabolism and stress responses [START_REF] Dang | The controversial world of sirtuins[END_REF]. SIRT1 targets transcription factors including p53, NF-κB, FOXO1 but also histones, thereby modulating gene expression [START_REF] Houtkooper | Sirtuins as regulators of metabolism and healthspan[END_REF]. For example, deacetylation of p53 impairs its activity as an inducer of apoptosis and autophagy [START_REF] Kitada | Chapter 3 -Role of Sirt1 as a Regulator of Autophagy[END_REF]. The importance of SIRT1 in gene regulation has been investigated upon bacterial infection. First of all, disruption of SIRT1 pathway leads to impairment of autophagy in cells infected by Salmonella Typhimurium [START_REF] Ganesan | Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy[END_REF]. In a consistent way, it has been observed that MTB infection down-regulates SIRT1 expression and thus impairs the SIRT1mediated autophagy [START_REF] Cheng | Host sirtuin 1 regulates mycobacterial immunopathogenesis and represents a therapeutic target against tuberculosis[END_REF] . Macrophages treated with resveratrol, an activator of SIRT1, better control intracellular bacterial load through an increase in the autophagy pathway. Moreover, Sirt1 +/-mice exhibit higher inflammation with enhancement of proinflammatory cytokines and are more susceptible to MTB infection (H. [START_REF] Yang | Role of Sirt1 in innate immune mechanisms against Mycobacterium tuberculosis via the inhibition of TAK1 activation[END_REF]. SIRT1

activation allows the regulation of this overt inflammation through the inhibition of transforming growth factor-β-activated kinase 1 (TAK1) pathway (which leads to the subsequent NF-κB activation) (H. [START_REF] Yang | Role of Sirt1 in innate immune mechanisms against Mycobacterium tuberculosis via the inhibition of TAK1 activation[END_REF]. SIRT1 activation also normalizes the MTBinduced inflammation via RelA/p65 deacetylation [START_REF] Cheng | Host sirtuin 1 regulates mycobacterial immunopathogenesis and represents a therapeutic target against tuberculosis[END_REF]. Moreover, a study has recently demonstrated that SIRT1 prevents the activation of the B-cell lymphoma 2-associated X protein (Bax) pro-apoptotic factor, which is normally increased upon MTB infection, and thus limits excessive apoptosis (H. [START_REF] Yang | Sirt1 activation negatively regulates overt apoptosis in Mtb-infected macrophage through Bax[END_REF]. Another team suggests that SIRT1 directly promotes glycogen synthase kinase 3 beta (GSK3β) phosphorylation by its deacetylation, which also prevents MTB-induced apoptosis in macrophages (H. [START_REF] Yang | Sirtuin inhibits M. tuberculosis -induced apoptosis in macrophage through glycogen synthase kinase-3β[END_REF]. Together, these studies demonstrate that SIRT1 has an important role in the MTB pathogenesis and could be a potential target for HDT.

SIRT2: SIRT2 targets proteins (histone or not) according to their subcellular localization and influences different biological processes. For example, this sirtuin deacetylates tubulin and hence regulates cytoskeleton dynamics, modulates metabolic pathways or regulates skeletal muscle differentiation [START_REF] Houtkooper | Sirtuins as regulators of metabolism and healthspan[END_REF]. SIRT2 has been implicated in various processes such as inhibition of autophagy or regulation of NF-κB signaling via p65 deacetylation (P. [START_REF] Gomes | Emerging Role of Sirtuin 2 in the Regulation of Mammalian Metabolism[END_REF]. SIRT2 is highly expressed in myeloid cells including macrophages and its role in bacterial infection has been investigated. Listeria monocytogenes induces deacetylation of H3K18 by regulating SIRT2 function and localization [START_REF] Eskandarian | A role for SIRT2-dependent histone H3K18 deacetylation in bacterial infection[END_REF][START_REF] Pereira | Infection Reveals a Modification of SIRT2 Critical for Chromatin Association[END_REF]. Inactivation of SIRT2 results in a decrease of bacterial infection in vivo and in vitro, showing that activity of SIRT2 is necessary for bacterial infection [START_REF] Eskandarian | A role for SIRT2-dependent histone H3K18 deacetylation in bacterial infection[END_REF]. Moreover, enhanced bacterial phagocytosis upon staphylococcal infection is observed in mice deficient for SIRT2 [START_REF] Ciarlo | Sirtuin 2 Deficiency Increases Bacterial Phagocytosis by Macrophages and Protects from Chronic Staphylococcal Infection[END_REF].

Similarly, SIRT2 -/-mice show a better S. Typhimurium clearance after infection, compared to wild-type mice [START_REF] Gogoi | Salmonella escapes adaptive immune response via SIRT2 mediated modulation of innate immune response in dendritic cells[END_REF]. A study suggests that MTB induces SIRT2 translocation into the nucleus and deacetylation of H3K18 [START_REF] Bhaskar | Host sirtuin 2 as an immunotherapeutic target against tuberculosis[END_REF]. Moreover, inhibition of SIRT2 leads to a decrease of intracellular MTB growth and significantly reduces MTB pathogenicity in mice [START_REF] Bhaskar | Host sirtuin 2 as an immunotherapeutic target against tuberculosis[END_REF]. However, an opposite result has been published by another team, where SIRT2 deletion in the myeloid lineage increases MTB load in the lungs and liver in mice [START_REF] Cardoso | Myeloid sirtuin 2 expression does not impact long-term Mycobacterium tuberculosis control[END_REF].

SIRT2 also modulates the expression of inflammatory genes via the deacetylation and inhibition of NF-κB (described in a sepsis mice model) (X. Wang et al., 2016). Hsp90 is also deacetylated by SIRT2, resulting in a disassociation of Hsp90 with the glucocorticoid receptor (GR). This disassociation allows the translocation of GR into the nucleus and the repression of pro-inflammatory cytokines expression by its binding to NF-κB (K. Sun et al., 2020).

SIRT2 is an important sirtuin which is involved in the process of many bacterial infections.

Thus, its regulation by HDT could better control bacterial infection outcomes, including MTB infection.

SIRT3: SIRT3 is the major mitochondrial sirtuin deacetylase. It regulates various processes like amino acid metabolism, fatty acid oxidation, energy balance, ETC activity and cellular redox homeostasis [START_REF] Houtkooper | Sirtuins as regulators of metabolism and healthspan[END_REF]. In MTB infection, SIRT3 deficiency is associated with an enhanced inflammatory response and mitochondrial dysfunctions (T. S. [START_REF] Kim | SIRT3 promotes antimycobacterial defenses by coordinating mitochondrial and autophagic functions[END_REF].

sirt3 -/-mice present an increased susceptibility to MTB infection and an exaggerated lungs inflammatory response with damaged mitochondria in macrophages (T. S. [START_REF] Kim | SIRT3 promotes antimycobacterial defenses by coordinating mitochondrial and autophagic functions[END_REF].

MTB down-regulates SIRT3 in a TLR2-dependent manner [START_REF] Smulan | Sirtuin 3 downregulation in mycobacterium tuberculosis-infected macrophages reprograms mitochondrial metabolism and promotes cell death[END_REF]. This decrease results in the destabilization of the TCA cycle and ETC, which then increases mtROS and proinflammatory cytokines and may lead to cell death [START_REF] Smulan | Sirtuin 3 downregulation in mycobacterium tuberculosis-infected macrophages reprograms mitochondrial metabolism and promotes cell death[END_REF]. Activation of SIRT3 also enhances bacterial autophagy during MTB infection (T. S. [START_REF] Kim | SIRT3 promotes antimycobacterial defenses by coordinating mitochondrial and autophagic functions[END_REF]. Finally, a recent study shows that the single-nucleotide polymorphisms rs3782118 on sirt3 was associated with an increased risk of TB, showing the implication of SIRT3 in TB pathogenesis (T. [START_REF] Wu | The dominant model analysis of Sirt3 genetic variants is associated with susceptibility to tuberculosis in a Chinese Han population[END_REF]. Altogether, SIRT3 seems to be a modulator of the mycobacterial infection outcomes. Activation of SIRT3 leads to better bacterial control and can therefore be considered as a new target for HDT. Of note, it seems that SIRT3 deficiency does not affect immune cell development or defenses against other pathogens like Escherichia coli, Klebsiella pneumoniae, or L. monogenes (Ciarlo, Heinonen, Lugrin, et al., 2017).

Sirtuins are key factors in many cellular processes but also during MTB infection. Small molecules targeting sirtuins could therefore prevent intracellular mycobacterial growth. In this context, sirtuin modulators could be valuable news drugs for the treatment of MTB infection.

III. News drugs or regimen for tuberculosis treatment

In fifty years, only three news drugs have been approved for treating TB [START_REF] Cox | FDA Approval of Bedaquiline -The Benefit-Risk Balance for Drug-Resistant Tuberculosis[END_REF][START_REF] Ryan | Delamanid: First global approval[END_REF][START_REF] Keam | Pretomanid: First Approval[END_REF]. Emergence of antibiotic resistance to first-and second-line anti-TB drugs urge the development or the discovery of new treatments. In this optic, two main techniques can be used to address this problem: the repurposing of already approved drugs (which could shorten the approval of TB treatment), or the discovery of new drugs such as HDT or new antibiotics. In addition, given the difficulty of treating TB and MDR-TB, studying the combination of old and new drugs may be another promising approach.

Drug screening

Bacterial genome sequencing has enhanced the understanding of bacterial evolution and physiology. Indeed, it has allowed the identification of new targets essential for pathogen survival inside the host and has paved the way for the discovery of novel antibacterial drugs.

Subsequently to these findings, large high-throughput screening (HTS) assays were designed to assess the "druggable potential" of these targets (Payne et al., 2006).

A screening for drug discovery is the search of interaction between a chemical (natural or synthetic) and a defined biological target [START_REF] Entzeroth | Overview of high-throughput screening[END_REF]. For example, classical HTS assay evaluates the drug binding to a receptor or monitors enzyme activities by measuring fluorescence intensity modifications or by using reporter gene expression assays [START_REF] Entzeroth | Overview of high-throughput screening[END_REF]. Introduction of automated devices over the years has enabled the automatization of screening and a high throughput testing of compounds, compared to manual methods. The tested compounds are often grouped in compound libraries, some of which contain several hundred thousand compounds [START_REF] Entzeroth | Overview of high-throughput screening[END_REF]. This method also allows the multiplication of screening conditions with different parameters (media, nutrients, pH, infection time) and the use of different MTB strains with a significant time saving [START_REF] Ollinger | A highthroughput whole cell screen to identify inhibitors of Mycobacterium tuberculosis[END_REF].

In parallel, automatization of cellular imaging techniques has enabled the development of high-content screening (HCS). This approach combines automated images coupled with data analysis in a high-throughput format (Wagner et al., 2005b). HCS, for instance, allows the visualization of intracellular bacterial growth, the exclusion of cytotoxic compounds and the quantification of the effect of compounds via computer analysis of the data [START_REF] Queval | A microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening[END_REF][START_REF] White | Mycobacterium tuberculosis highthroughput screening[END_REF]. The purpose of HTS/HCS screening is to find active compounds/molecules, also called "hits", for which the activity/potential has to be confirmed with ulterior assays to be considered as a "lead". This hit-to-lead phase is based on the potential of the molecule in terms of toxicology, pharmacology and structure-activity relationships (SAR) [START_REF] Temml | Structure-based molecular modeling in SAR analysis and lead optimization[END_REF]. The selected lead(s) will then be optimized, possibly with the synthesis of more potent or less toxic analogues. Target identification or validation could be done in parallel with this optimization. The efficacy of the molecule can be evaluated in an in vivo model for further development in the TB drug pipeline.

HCS can also be associated with RNA interference (RNAi) genetic screening. In this technique, short interfering RNA (siRNA) are employed to silence specific genes during the experiment [START_REF] Krausz | High-content siRNA screening for target identification and validation[END_REF]. It can be used to assess the importance of certain genes during the infection and increases the understanding of factors involved in host-pathogen interaction [START_REF] Queval | Mycobacterium tuberculosis Controls Phagosomal Acidification by Targeting CISH-Mediated Signaling[END_REF]Wagner et al., 2005b).

High-throughput approaches on MTB have been conducted in different conditions such as nutrient starvation, stress, infection or in live cells and have provided amount of information (intracellular growth, phagosomal modification, oxidative stress production, lipid accumulation) [START_REF] Christophe | High-content imaging of Mycobacterium tuberculosis-infected macrophages: An in vitro model for tuberculosis drug discovery[END_REF][START_REF] Deboosere | High-Content Analysis Monitoring Intracellular Trafficking and Replication of Mycobacterium tuberculosis Inside Host Cells[END_REF]. They have revealed many natural or synthetic compounds that enhance host defenses against MTB [START_REF] Ollinger | A highthroughput whole cell screen to identify inhibitors of Mycobacterium tuberculosis[END_REF]. Due to their results, these high-throughput techniques can serve as a starting point for the discovery of new molecules against MTB. However, the major limitations of development of new drugs are the duration of the optimization phase and the resources devoted to the project.

Drug repurposing

The discovery of new drugs takes time considering they have to go through different clinical phases before they are approved by the competent authority. Considering this, alternative approaches such as drug repurposing is a valuable strategy for implementing a new TB regimen.

Drug repurposing concerns drugs that have been already approved for a pathology and revealed to be beneficial for another disease [START_REF] Maitra | Repurposing-a ray of hope in tackling extensively drug resistance in tuberculosis[END_REF]. Those drugs have the advantage of having already known safety and toxicity profiles due to their previous approval.

In this context, drug repurposing has a faster evaluation of the drugs for treating other diseases (an average of 14 years clinical trial for a new drug versus 5 years for a repurposed drug), but also a reduced development time and a lower cost (Chong & Sullivan, 2007b).

Before a drug is repurposed to treat MTB, various evaluations of the drug have to be carried out on MTB in vitro and in vivo (An et al., 2020a). Antibiotics but also antifungal or antiviral drugs are now under evaluation for TB treatment repurposing (An et al., 2020a). It is the case of mefloquine, normally used as an antimalarial drug, that has been shown to be an alternative to treat MDR-TB clinical strains in vitro and in vivo in mice, making it a potential candidate for drug repurposing (An et al., 2020a).

Facing the current low number of new drugs approved, tools for rapid identification of a potential drug to be repurposed have emerged. One of these tools is the in silico screening of already approved drugs. Computational analysis provides information on putative interaction(s) with new or old cellular targets, essential for MTB survival [START_REF] Passi | RepTB: a gene ontology based drug repurposing approach for tuberculosis[END_REF]. In silico screening is a structure-based phenotypic screening that assesses the potential binding between a known drug with a defined target (Battah et al., 2019). This approach is highly valuable to find potential new candidates. Moreover, it is also an interesting method to predict the target of the molecules and possibly their mechanisms of action [START_REF] Passi | RepTB: a gene ontology based drug repurposing approach for tuberculosis[END_REF].

However, a major problem with drug repurposing is that even if these drugs target MTB components, the bacteria can still develop resistance.

To tackle this issue, drugs that directly target the host is an alternative solution. Indeed, drug repurposing can be closely linked to HDT. It relies on the fact that a drug developed originally for a disease may exhibit secondary beneficial biological effects on the host [START_REF] Maitra | Repurposing-a ray of hope in tackling extensively drug resistance in tuberculosis[END_REF]. Utilization of those drugs on the host could result in a decrease of bacterial load or a better control of the infection. Several previously cited drugs are under evaluation for TB drug repurposing including metformin, NSAID drugs or statins (see section C.I.). Repurposed drugs

for HDT could be a potential way to include old drugs in a new TB regimen.

Despite the benefits of drug repurposing, they still require evaluations and clinical trials before being approved for the treatment of TB. Then, several investigations are performed to enable the expansion of TB treatment with approved drugs in combination.

Drugs combinations

New techniques, such as high-throughput approaches, combined with known chemical libraries, have allowed the testing of drug combinations for the approval of new anti-TB treatment regimens [START_REF] Ramón-García | Synergistic drug combinations for tuberculosis therapy identified by a novel high-throughput screen[END_REF]. Using a combination of already described drugs together may have the potential to better control bacterial infection. Firstly, drugs can target different pathways like the regimen used to treat TB with RIF, INH, EMB and PZA [START_REF] Fischbach | Combination therapies for combating antimicrobial resistance[END_REF]. Secondly, drugs may have different targets within the same pathway, like preventing pathways preventing cell wall synthesis [START_REF] Worthington | Combination approaches to combat multidrugresistant bacteria[END_REF]. Thirdly, drugs may have the same targets but act with different mechanisms (for example association of streptogramins and virginiamycin which alone are bacteriostatic but together have bactericidal activity -not used in TB treatment) [START_REF] Mast | Streptogramins -Two are better than one![END_REF]. Results of compound combinations are usually either additive, synergistic or antagonist. Additive effect is the combination of effects of each drug individually; synergy occurs when the effect produced is greater than the additive effects of the molecules and antagonism is the opposite of synergy [START_REF] Bulusu | Modelling of compound combination effects and applications to efficacy and toxicity: State-of-the-art, challenges and perspectives[END_REF] .

Combination of drugs is especially needed against MDR-TB because of their preexisted antibiotic resistance(s) and the risk of developing/acquiring new ones [START_REF] Brooks | Therapeutic strategies to combat antibiotic resistance[END_REF]. Interestingly, some drug combinations restore the efficacy of certain antibiotics in drug-resistant strains. A study has shown that addition of spectinomycin with first-line anti-TB drug INH and RIF restores their activities in vitro against resistant MTB [START_REF] Omollo | Developing synergistic drug combinations to restore antibiotic sensitivity in drug-resistant mycobacterium tuberculosis[END_REF].

Recently, drug combinations with adjuvants such as small molecules, have been investigated

for the design of new drug regimens against sensible and resistant MTB. For example, boosters were designed and tested for their ability to potentiate the activity of ethionamide (ETH) [START_REF] Willand | Synthetic EthR inhibitors boost antituberculous activity of ethionamide[END_REF]Flipo et al., 2012;[START_REF] Blondiaux | Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420[END_REF]. Furthermore, drug combinations allow an increased efficacy of drugs without the need of a high dose (and the significant toxicity usually associated with a high drug concentration) [START_REF] Brooks | Therapeutic strategies to combat antibiotic resistance[END_REF].

Finally, drug combinations may use ineffective antibiotics against the pathogen, like β-lactam for MTB. Those ineffective antibiotics may present a certain antimicrobial activity once combined with another drug. For example, β-lactam are rapidly hydrolyzed by MTB but meropenem, belonging to carbapenem family, combined with clavulanate, a β-lactamase inhibitor, shows potent activity against laboratory MTB strains [START_REF] Hugonnet | Meropenem-clavulanate is effective against extensively drug-resistant Mycobacterium tuberculosis[END_REF].

It is worth mentioning that there is a risk that drug combinations promote the evolution of drug resistance by causing higher sensitivity to other antibiotics or by blocking their activities [START_REF] Brooks | Therapeutic strategies to combat antibiotic resistance[END_REF]. Depending on the drug combinations as well as the drug concentrations and duration of the treatment, this strategy may also be toxic for the host cell.

Therefore, toxicity assays should be performed to find the best drug combinations and concentrations [START_REF] Yilancioglu | Design of high-order antibiotic combinations against M. tuberculosis by ranking and exclusion[END_REF] .

Several trials are ongoing to find new treatment regimens with old and new drugs (An et al., 2020a;[START_REF] Brooks | Therapeutic strategies to combat antibiotic resistance[END_REF]. Treatment of TB is complex and becomes even more difficult to treat with the apparition of MDR/XDR-TB strains [START_REF] Seung | Multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis[END_REF]. Other strategies can be used to improve the efficacy of current TB treatment, notably with the use of nanoparticles.

Nanoparticles

Nanoparticles (NPs) are small particles made of lipids, liposomes, polymers or polysaccharides for example [START_REF] Costa-Gouveia | How can nanoparticles contribute to antituberculosis therapy?[END_REF]. There was an increasing interest for the use of NPs in anti-TB therapy due to their ability to either have a direct antimycobacterial activity or to act as a vehicle for enhancing the delivery of already known anti-TB drugs [START_REF] Costa-Gouveia | How can nanoparticles contribute to antituberculosis therapy?[END_REF]. For instance, booster molecules, which potentiate ETH, have limited solubility in aqueous media. Moreover, ETH tends to rapidly crystallize, limiting the effect of this combination. To solve this issue, the poly-β-cyclodextrins (pβCD) NPs was used to co-encapsulate these two compounds. Administration of this suspension shows no toxicity for the mice and is efficient to decrease pulmonary mycobacterial load, compared to untreated mice [START_REF] Costa-Gouveia | Combination therapy for tuberculosis treatment: Pulmonary administration of ethionamide and booster coloaded nanoparticles[END_REF]. Another study has assessed the intrinsic antibacterial properties of the unloaded pβCD. These NPs impair MTB-macrophages invasion by disrupting cell surface lipid rafts and contribute to the depletion of alveolar macrophages, diminishing the pool of potential reservoir for MTB [START_REF] Pastor | A novel codrug made of the combination of ethionamide and its potentiating booster: Synthesis, self-assembly into nanoparticles and antimycobacterial evaluation[END_REF]. More recently, ETH and its booster BDM43266 were synthetized as codrug (association of both molecules via a glutaric linker) and designed to self-assemble into NPs [START_REF] Pastor | A novel codrug made of the combination of ethionamide and its potentiating booster: Synthesis, self-assembly into nanoparticles and antimycobacterial evaluation[END_REF]. The resulted NPs can then be intranasally administered. Finally, NPs encapsulation of booster or anti-TB drug is thought to be very interesting for the treatment of drug-resistant TB. Indeed, the higher delivery of the drug or the ability to combine it with a booster may lead to a decrease of the dose needed for treatment, diminishing the risk of toxicity and side-effects [START_REF] Pelgrift | Nanotechnology as a therapeutic tool to combat microbial resistance[END_REF].

Taken together, these findings highlight the potential of using NPs as a strategy to improve current TB treatment and to fight more efficiently MDR-TB. In addition to NPs, discovery of news drugs, including HDT, drug repurposing and drug combinations represent valuable strategies to overcome MDR-TB and XDR-TB and to finally eradicate TB.

TB is one of the top ten causes of human death worldwide and remains a major public health threat. An effective vaccine against TB is still not available. Nowadays, people developing TB can be cured with a 6-month anti-TB treatment regimen, involving 4 antibiotics. This antibiotic treatment has been introduced 40 years ago, and multidrug resistant strains of MTB are continually emerging. Since then, and despite important investments by scientists and funding agencies, only a few new anti-TB drugs have been approved and released recently. Although several antibiotics are currently under clinical trials, it is most likely that MTB will develop resistance against these new molecules. In this context, innovative strategies are urgently needed to eradicate TB.

In addition to the development of "classical" anti-TB drugs targeting mycobacterial key factors necessary for MTB survival, host-directed therapies (HDT) are considered as a promising strategy to control MTB infection. HDT are drugs that target the host instead of the bacteria by either (i) modulating host anti-mycobacterial pathways that are blocked or altered by MTB to promote its survival, or (ii) potentiating antimicrobial host immune defense mechanisms.

Because HDT do not target mycobacteria directly, the development of resistance to HDTs by MTB is expected to be more difficult than for antibiotics [START_REF] Kaufmann | Host-directed therapies for bacterial and viral infections[END_REF]. Furthermore, HDTs are predicted to be effective on drug-sensitive and drug-resistant TB strains. Taken in combination with antibiotics, HDT may also reduce the duration of current anti-TB treatments [START_REF] Kilinç | Host-directed therapy to combat mycobacterial infections*[END_REF]. Indeed, host factors may influence the pharmacokinetics and pharmacodynamics of antimicrobials, impacting drug efficacy and/or toxicity. Therefore, combining antibiotics with host-targeted molecules to improve their efficacy, holds great promise for improving the treatment against TB. Taken together, these evidences suggest that HDT could be one of the future strategies used to eradicate TB.

MTB modulates host signaling and effector pathways to its own advantage [START_REF] Koul | Interplay between mycobacteria and host signalling pathways[END_REF].

In particular, MTB modifies the host epigenome to evade the immune response (M. Singh et al., 2018). Thus, by reprogramming the host immune system epigenome using HDT, the host cell may be able to better control intracellular MTB growth. This hypothesis is the starting point of this thesis, where I screened new compounds targeting the host epigenome that could render human macrophages more resistant to MT. Macrophages are important innate immune cells that detect and eliminate invading microbes including MTB and instruct the immune system for appropriate adaptive responses. Macrophages are the main cell target of MTB which has developed numerous strategies to survive and replicate inside them.

The main objectives of my thesis were the following:

-Identification of a molecule that directly enhances the capacity of macrophages to control MTB infection.

-In vitro characterization of the host target(s) of the molecule and its mechanism of action.

-Determination of hit optimization by SAR studies.

-Investigating the impact of the molecules or its analogues on the efficacy of TB drugs.

-Evaluation of the efficacy of the molecules in vivo, in a mouse model of MTB infection.

Previous studies have identified novel HDTs in TB through intracellular high-throughput screening methods [START_REF] Jayaswal | Identification of host-dependent survival factors for intracellular Mycobacterium tuberculosis through an siRNA screen[END_REF][START_REF] Korbee | Combined chemical genetics and data-driven bioinformatics approach identifies receptor tyrosine kinase inhibitors as host-directed antimicrobials[END_REF][START_REF] Stanley | Identification of Host-Targeted Small Molecules That Restrict Intracellular Mycobacterium tuberculosis Growth[END_REF], and led to the identification of host factors impacting MTB intracellular replication. Here we used primary cells, namely human monocyte-derived macrophages. HCS coupled to image-based analysis represent a rapid and reliable way to test molecules in an intracellular model of infection. Based on this assumption, the beginning of my thesis was devoted to acquiring knowledge and skills to use this technique. I was hosted in Priscille Brodin's team at the Pasteur Institute in Lille to be trained in the use of HCS. She has developed a phenotypic assay relying on the quantification of the growth of GFP-expressing MTB within cells using automated confocal microscopy. Back in my unit, I adapted this technique using human macrophages infected with MTB. After some adjustments, I was able to screen a library of non-commercialized molecules provided by a team of chemists from the University of Sapienza in Rome (Dante Rotili and Antonello Mai). The number of molecules in this library was relatively modest compared to others (<300 compounds), but they were developed to inhibit or activate enzymes regulating the host epigenome. Another advantage, these molecules have never been tested on MTB.

From this screening, I was able to identify a promising hit on which I based the following results of my thesis.

GlutaMAX; Gibco) and 10% FBS. The cells were incubated at 37°C, 5% CO2, and the culture medium was changed every two days.

Bacterial strains

Listeria monocytogenes strain wildtype EGD, number BUG600 was provided by Melanie

Hamon and grown in brain-heart infusion medium (BHI; Difco Laboratories) at 37°C. The strain E. coli MG1655 was cultivated in Luria Bertani (LB) medium at 37°C. Laboratory mycobacterial strains used in this study include MTB strain H37Rv (ATCC 27294) transformed with an Ms6based integrative plasmid pNIP48 harboring GFP (MTB H37Rv-GFP) provided by Priscille

Brodin [START_REF] Christophe | High-content imaging of Mycobacterium tuberculosis-infected macrophages: An in vitro model for tuberculosis drug discovery[END_REF], M. bovis BCG Pasteur 1173P2, M. abscessus (ATCC 19977) and BDQ-resistant MTB strain H37Rv (Giraud-Gatineau et al., 2020). Clinical resistant strains were provided by Nathalie Grall, Anne-Laure Roux and Jean-Louis Herrmann from the "Assistance Publique Hôpitaux de Paris". Mycobacteria were grown at 37°C, 5% CO2 in Middlebrook 7H9 medium (Becton-Dickinson) supplemented with 10% albumin dextrose catalase (ADC, Difco Laboratories), and 0.05% Tween 80. Hygromycin B was added to the culture medium of strains containing a plasmid encoding GFP.

Infections

L. monocytogenes was grown to exponential phase at an optical density 600 nm (OD600 nm) of 1. Bacteria were washed twice in PBS and added to cells at a multiplicity of infection (MOI) of 50:1. After 1 hour, cells were washed with medium, and 10 µg/mL of gentamycin was added to kill extracellular bacteria. Mycobacterial cultures were washed and aggregates were removed by filtration through a 10 μm filter. Number of bacteria was estimated by measuring OD600nm. Cells were infected at a MOI of 0.5:1, 2:1 or 5:1 for MTB, BCG and M.abscessus respectively for two hours in complete medium. The cells were washed and incubated at 37°C

for one hour in complete medium containing amikacin (50 μg/mL) to remove extracellular bacteria. Infected cells were then incubated in fresh complete medium and were ready to use.

System (Perkin Elmer Technology), with a 20x air objective NA 0,4. Images were analyzed using the software Columbus Conductor™ Database (Perkin Elmer Technologies).

RNA isolation, library preparation and sequencing

Total RNA from macrophages was extracted using QIAzol lysis reagent (Qiagen) and purified by RNeasy columns (Qiagen). RNA integrity of the samples was assessed with an Agilent 2100 bioanalyzer (Agilent Technologies). Only RNA with integrity number >9 were selected for further experiments. cDNA libraries were prepared with the Illumina TruSeq Stranded mRNA and were sequenced on llumina NovaSeq 6000 system at the CHU Sainte-Justine Integrated Centre for Pediatric Clinical Genomics (Montreal, Canada).

Gene ontology (GO) enrichment analyses were performed using the Cytoscape app ClueGO (version 3.8.2) [START_REF] Bindea | ClueGO: A Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks[END_REF]. The following parameters were used: only pathways with pV ≤0.01, Minimum GO level = 3, Maximum GO level = 8, Min GO family >1, minimum number of genes associated to GO term = 3, and minimum percentage of genes associated to GO term = 4. Enrichment p-values were calculated using a hypergeometric test (p-value<0.05, Bonferroni corrected).

Quantitative reverse transcription PCR (RT-qPCR)

mRNA was reverse transcribed into cDNA SuperScript III Reverse Transcriptase (Thermo Fisher). Each standard dilution and each sample were tested in duplicate/triplicate and negative controls were included in each experiment. Amplification of cDNA was performed using Power SYBR Green PCR Master Mix (Thermo Fisher) in a StepOnePlus Real-Time PCR System Thermal Cycling block (Applied Biosystems). To perform RT-qPCR expression analyses, 2-fold dilutions of genomic DNA were used as PCR templates for construction of standard curve. Based on the CT values obtained, a linear regression line was plotted and the resulting equation is used to calculate the efficiency of amplification. Results were normalized using reference gene. The expression level of each gene was reported as ratio of expression (fold induction) according to the 2 -ΔCt method [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF].

RNA silencing of SIRT2

siRNA silencing was performed using a previously described protocol [START_REF] Troegeler | An efficient siRNA-mediated gene silencing in primary human monocytes, dendritic cells and macrophages[END_REF].

Briefly, macrophages were transfected using the lipid-based HiPerfect system (Qiagen), siRNA targeting SIRT2 (ON-TARGETplus SMARTpool at 50 nM) and a scramble siRNA (ON-TARGETplus Non-targeting Pool) (Dharmacon). After 6 hours, complete medium supplemented with M-CSF (final concentration of 10ng/mL) was added to the cells, before the cells were allowed to recuperate for an additional 2 days. The inactivation of SIRT2 was confirmed by RT-qPCR 48 hours after the transfection.

Western blot analysis

Cells were lysed with RIPA buffer (Thermo Fisher) supplemented with protease inhibitor cocktails (Complete Protease Inhibitor Cocktail; Sigma) and phosphatase inhibitors cocktails (Thermo Scientific). Protein concentration was determined using the BCA protein assay kit (Thermo Fisher) according to the manufacturer instructions. 20 µg of total proteins were loaded and separated by SDS-PAGE on a NuPage 4-12% Bis-Tris polyacrylamide gel (Thermo Fisher). Proteins were transferred into a nitrocellulose membrane using the iBlot dry blotting system (Invitrogen). The membranes were blocked with TBS-0.1% Tween20, 5% non-fat dry milk for 30 min at RT and incubated overnight with primary antibodies against α-β-Tubulin, anti-acetyl-histone H3K18 (Cell Signaling) and anti-histone H3 antibody (Abcam). Membranes were washed with TBS and incubated with the appropriate secondary antibody coupled to horseradish peroxidase-conjugated antibody (GE Healthcare) at RT for 1 hour. Membranes were washed and exposed to SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher). Detection and quantification of band intensities were performed using Azure Imager C400 (Azure Biosystems) and ImageJ software.

Indirect immunofluorescence

Macrophages (4 × 10 5 cells/mL) were grown on 12 mm circular coverslips in 24-well tissue culture plates for 24 hours in cell culture medium. Cells were infected and treated during the desired amount of time. Cells were fixed with 4% PAF for 1 hour at RT, and were then incubated for 30 min in 1% BSA (Sigma-Aldrich) and 0.075% saponin (Sigma-Aldrich) in PBS, to block nonspecific binding and to permeabilize the cells, respectively. Cells were incubated with anti-LC3 (MBL) during 2 hours at RT. Cells were washed and incubated with Alexa Fluor 555 secondary antibody (Thermo Fisher) for 2 hours. Nuclei were stained with DAPI (1 µg/mL) during 10 min. After labeling, coverslips were set in Fluoromount G medium (SouthernBiotech) on microscope slides. LC3B puncta were analyzed using Leica TCS SP8 confocal microscopy and quantified using ImageJ. Dot plots represent the mean values of at least 100 cells. Error bars depict the standard deviation.

Synergy degree of drugs combination

Bliss independence model was used to calculate the excess over Bliss model for growth inhibition (Q. Liu et al., 2018). The Bliss model has been suggested as the best model of choice for drugs with supposed different modes of action and targets [START_REF] Baeder | Antimicrobial combinations: Bliss independence and loewe additivity derived from mechanistic multi-hit models[END_REF]. The

In vivo experiments

Compounds were solubilized daily in 0.5% DMSO + 4.5% Cremophor EL® + 90%NaCl. 8 weeksold female C57BL/6J mice (Charles River Laboratories) were treated by intraperitoneal injections during 2 weeks, 6 days per weeks. Asparatate aminotransferase (AST) and alanine aminotransferase (ALT) were quantified using Reflover®Plus. Mice were infected via aerosol generated from a suspension containing 5 × 10 6 CFUs/ml of H37Rv to obtain an expected inhaled dose of 50-100 bacilli/lungs. After 7 days, mice were treated with compounds by intraperitoneal injections and/or with antibiotic by gavage during 2 weeks, 6 days per weeks.

Colony forming units (CFUs) in lungs and spleen of infected animals were determined after 21 days post-infection.

Identification of a compound by HCS that inhibits intracellular MTB growth.

In order to identify new compound(s) that inhibit(s) the intracellular MTB growth, we performed HCS, a technique that has already been proven successful for the discovery of compounds with a potent intracellular anti-mycobacterial efficacy [START_REF] Christophe | High-content imaging of Mycobacterium tuberculosis-infected macrophages: An in vitro model for tuberculosis drug discovery[END_REF][START_REF] Ollinger | A highthroughput whole cell screen to identify inhibitors of Mycobacterium tuberculosis[END_REF][START_REF] Shapira | High-Content Screening of Eukaryotic Kinase Inhibitors Identify CHK2 Inhibitor Activity Against Mycobacterium tuberculosis[END_REF]. Because MTB manipulates epigenetic host-signaling pathways to subvert host immunity (M. Singh et al., 2018), we hypothesized that reprogramming the host immune system by a compound targeting the host epigenome may lead to a better control of the bacterial infection (J. [START_REF] Cole | The therapeutic potential of epigenetic manipulation during infectious diseases[END_REF]. To tackle this hypothesis, we screened a library of 157 compounds with putative epigenetic targets to identify a hit.

Human monocytes-derived macrophages were first infected by MTB expressing GFP, before being seeded in a 384-well plate; each well contained a different compound at a final concentration of 10µM. Using automated confocal microscopy, cell toxicity was determined by comparing the number of nuclei stained by Hoechst 33342 in treated wells to the control well with DMSO. Compounds associated with a cell viability superior to 75% were considered as non-toxic (Figure 6A). Following this viability analysis, 106 compounds were selected for further testing.

Inhibition of intracellular MTB growth in presence of the remaining compounds was assessed by determining the spot area within the cells. The spot area is representative of the GFP area per cell, which is correlated to the amount of intracellular MTB expressing GFP. By comparing the spot area intensity of cells in presence of different compounds to the DMSO condition, we selected the most promising hit, MC3465, for its ability to reduce the bacterial infection by 60% compared to the control (Figures 6A-B). To validate these initial screening results, MC3465 efficacy was then confirmed by enumerating the number of bacteria inside macrophages (measured by colony forming units (CFUs) assay). A decrease of 25% and 55% of CFUs is obtained when MTB-infected cells are cultivated in presence of MC3465 for 1 or 4 days, respectively (Figure 6C). Moreover, MC3465 seems to have a bacteriostatic effect as the number of CFUs remains constant over time once MTB is treated with this compound, compared to a bactericidal INH treatment, where the number of CFU rapidly drops after 24 hours (Figure S1). To extrapolate the potential applicability of this compound to a larger scale, we then determined if MC3465 also exhibits an intracellular bacterial growth inhibitory activity against other bacteria. Our results show that MC3465 does not impede the intracellular growth of L. monocytogenes, S. Typhimurium and E. coli (Figures 6D andS2).

However, the intracellular growth of BCG and M. abscessus, a slow and a fast-growing mycobacterium respectively, is affected in presence of MC3465 (Figures 6D andS2).

To verify the host-directed effect of this molecule, we then monitored MTB growth in liquid medium, with the same concentration used in the cell-based infection model (10 µM) but also at a higher concentration (50µM). In any case, MC3465 does not affect MTB growth (Figure 6E). This observation suggests that MC3465 does not directly act on bacteria, but on the host cell. Finally, to confirm MC3465 targets only intracellular MTB, we performed a transwell experiment, where MTB was cultivated in presence of macrophages, without any direct contact with the host cells (Figure 6F). Our data shows that bacterial growth is not affected by MC3465 when bacteria are cultivated in the transwell condition, compared to the control condition, where bacteria are able to infect the host cell (Figure 6F). This observation suggests that MC3465 is not metabolized by macrophages in an active form that could target extracellular bacteria.

SIRT2 is not the target of MC3465.

MC3465 belongs to an uncommercialized library of compounds designed to target the host epigenome. This molecule has been described to inhibit specifically sirtuin 2 (SIRT2) (Gu et al., 2009a). In a recent study, it has been shown that inhibition of SIRT2 by AGK2 leads to a decrease of intracellular MTB survival [START_REF] Bhaskar | Host sirtuin 2 as an immunotherapeutic target against tuberculosis[END_REF]. To determine if MC3465 acts in a similar way as SIRT2 inhibitors, we incubated MTB-infected macrophages with MC3465, but also with two well-known SIRT2 inhibitors: AGK2 and SirReal2. Our results show that only MC3465 has a stronger effect on the intracellular MTB growth inside human macrophages, while no effect on bacterial growth is observed in the tested conditions for both AGK2 and SirReal2 (Figure 7A). This experiment suggests that MC3465 has probably a different mechanism of action than AGK2 and SirReal2. To keep investigating if SIRT2 is the putative target of MC3465, we then silenced the SIRT2 gene by using a siRNA and managed to reduce its expression by 70% in human macrophages (Figure 7B). As a control, the relative expression of SIRT2 when cells are incubated with a scramble RNA (scRNA) is similar to the control condition (Figure 7B). We then infected the silenced SIRT2 macrophages with MTB, with or without MC3465, and counted the number of intracellular bacteria after 4 days of infection. Even when 70% of SIRT2 gene is silenced, MC3465 has a similar effect on intracellular MTB, compared to when cells express SIRT2

(control and scRNA conditions) (Figure 7C). Moreover, MC3465 is still active on intracellular MTB in mouse embryonic fibroblasts (MEFs) SIRT2 -/-cell line, to a similar level as the wild-type cells (Figure 7D).

Finally, we tested the impact of MC3465 on macrophages infected with L. monocytogenes.

Indeed, it has been shown that this bacterium uses the host SIRT2 at its advantage during infection, by deacetylating the lysine 18 on histone H3 [START_REF] Eskandarian | A role for SIRT2-dependent histone H3K18 deacetylation in bacterial infection[END_REF]. However, upon L. monocytogenes infection, MC3465 does not inhibit the deacetylation of H3K18 compared to AGK2 or nicotinamide, another sirtuin inhibitor (Figures 7E-F).

Taken altogether, these results demonstrate that SIRT2 is not the target of MC3465.

Transcriptome analysis of uninfected or MTB-infected macrophages reveals host pathways modulated by MC3465.

As SIRT2 is not the target of MC3465, we decided to use an unbiased approach to investigate which host pathways are differentially regulated when macrophages are cultivated in presence or in absence of MC3465. Concretely, macrophages from four individual donors were infected by MTB or left uninfected before being treated with either DMSO (control condition) or MC3465 at a final concentration of 10 µM. We then characterized the genomewide gene expression profiles by RNAseq after 4 hours or 24 hours of treatment. The analysis revealed that the expression of 1,383 genes is affected by MC3465 (p-value < 0.05). We focused the analysis on the most differentially expressed genes (with log FC ≥ 0.5 and ≤ -0.5

for upregulated and downregulated genes, respectively). 53 genes were upregulated and 128 genes downregulated at 4 hours post-infection (Figure 8A). Moreover, 3,702 genes are differentially expressed at 24 hours post-infection, including 224 upregulated genes and 652 genes downregulated (Figure 8A). Interestingly, MC3465 also modulates the gene expression of uninfected cells with a total of 1,346 genes are differentially expressed with 103 genes upregulated and 97 genes downregulated at 4 hours post-treatment and 3,364 genes differentially expressed with 147 genes upregulated and 240 genes downregulated at 24 hours post-treatment (Figure S3). We classified those differentially expressed genes by performing gene-set enrichment analysis using ClueGO cluster analysis [START_REF] Bindea | ClueGO: A Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks[END_REF].

The gene set downregulated by MC3465 in MTB-infected cells is mainly composed of genes associated with cellular zinc ion homeostasis and cell chemotaxis at 4 hours (Figure 8B). At 24 hours, most of the downregulated genes for MTB-infected cells treated with MC3465 belongs to the innate immune response and cytokine response (Figure 8C). At 4 hours, the expression of genes belonging to the metallothionein (MT) family is downregulated in presence of MC3465 (for both uninfected and MTB-infected cells), compared to DMSO, as shown on the heatmap (Figure 8D). At 24 hours, genes related to the type I IFN pathways are also downregulated when the molecule is added (for both uninfected and infected cells), compared to the control condition (Figure 8D).

MC3465 effect on intracellular MTB growth is not related to autophagy.

To further investigate the activity of MC3465 on the inhibition of the intracellular MTB growth, we explored if the compound has an effect on the host defense mechanisms, and more specifically on autophagy. Indeed, autophagy is a well-established key factor for host defense against MTB [START_REF] Gutierrez | Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages[END_REF]. Furthermore, several drugs are known to activate autophagy upon MTB infection such as PZA, INH and more recently BDQ [START_REF] Giraud-Gatineau | The antibiotic bedaquiline activates host macrophage innate immune resistance to bacterial infection[END_REF][START_REF] Kim | Host cell autophagy activated by antibiotics is required for their effective antimycobacterial drug action[END_REF]. To test if MC3465 somehow triggers the autophagy in MTBinfected macrophages, we performed some confocal microscopy to evaluate the presence of LC3B puncta, a cellular marker for the formation of autophagosomes and autolysosomes.

Presence of LC3B puncta was detected using secondary antibody Alexa Fluor 555 linked to anti-LC3 antibody. Our result shows a slight decrease of LC3B puncta per cell at 24 hours and 48 hours post-MC3465 treatment (only significative for 24 hours) in MTB-infected macrophages, suggesting that MC3465 effect is not related to autophagy (Figures 9A-B). Structure-activity relation (SAR) study is a key aspect of drug design and optimization [START_REF] Temml | Structure-based molecular modeling in SAR analysis and lead optimization[END_REF]. In our study, we screened 29 analogues of MC3465 with the aim of finding a compound with a higher activity than MC3465. Using automated confocal microscopy, we showed that MC3466 inhibits 70% of intracellular MTB growth, compared to the control condition (Figure 10A). Effect of MC3466 was confirmed by a viability assay, with a decrease of one log of CFU, compared to the control, when MTB-infected macrophages are treated with MC3466 for 4 days (Figure 10B). Similarly to MC3465, the analogue does not have an effect on MTB growth in a liquid culture medium at 10 µM nor at a higher concentration (50 µM) (Figure 10C).

We next assessed the IC50 of MC3466. IC50 represents the concentration required to inhibit 50% of the bacterial colonization [START_REF] Christophe | High content screening identifies decaprenyl-phosphoribose 2ʹ epimerase as a target for intracellular antimycobacterial inhibitors[END_REF]. We used two different techniques to estimate the IC50: (i) an image-based analysis with the dose-response curve (DRC) (Figure 10D) and (ii) a CFU-based assay (Figure 10E). Both techniques gave us similar IC50 for MC3465 and MC3466, with an estimated IC50 for MC3465 between 4.5-5.9 µM and 2.5-3.2 µM for MC3466 (Figure 10F).

Moreover, the SAR experiments allowed us to define that the 5-(3-bromopropyl)-3-(4-Rphenyl)-1,2,4-oxadiazole (R being bromine for 3465 and methoxyle for MC3466) is the active site of the molecule (marked in red) (Figure 10F).

As HDT drugs act on the host rather than on bacterial targets, our compounds are expected to inhibit the growth of different MTB strains in a similar manner. To test this assumption, we determined the effect of MC3465 and MC3466 on human macrophages infected with hypervirulent clinical MTB strains belonging to the Beijing family, namely GC1237, CDC1551 and Myc5750 [START_REF] Millán-Lou | Rapid test for identification of a highly transmissible Mycobacterium tuberculosis beijing strain of sub-Saharan origin[END_REF][START_REF] Polena | Mycobacterium tuberculosis exploits the formation of new blood vessels for its dissemination[END_REF][START_REF] Valway | An Outbreak Involving Extensive Transmission of a Virulent Strain of Mycobacterium tuberculosis[END_REF]. Our data show that both compounds are effective in inhibiting the intracellular growth of each MTB strains (Figure 10G).

Both MC3465 and MC3466 protect MTB-infected cells from cell death.

MTB is known to induce macrophages death in order to proliferate [START_REF] Moraco | Cell death and autophagy in tuberculosis[END_REF].

To evaluate the role of MC3465 and MC3466 on the cell death of MTB-infected macrophages, we treated MTB-infected cells with both molecules and we stained the cells with LIVE/DEAD 11). In addition, less GFP positive MTB-infected cells are PB450 positive when treated with the compounds.

Taken together, these results show that both MC3465 and MC3466 have a protective effect on the cells.

MC3466 potentiates the activity of antibiotics on susceptible and mono-resistant strains.

HDT are not stand-alone drugs and are mostly considered as adjunctive drugs to standard antibiotic treatments for TB [START_REF] Kaufmann | Host-directed therapies for bacterial and viral infections[END_REF][START_REF] Machelart | Host-directed therapies offer novel opportunities for the fight against tuberculosis[END_REF]. We therefore investigated whether there is a synergistic or additive effect of MC3466 and antibiotics on a drug susceptible MTB. To do so, we combined MC3466 at different concentrations with RIF, BDQ, MOX or INH and we analyzed the bacterial growth by image-based analysis (Figures 12A andS4). The synergy or antagonist effect of the drug combination was determined using the excess over Bliss score and Bliss independence model (Q. Liu et al., 2018). These effects were represented by a heatmap ranging from antagonism (blue) to synergy (red). For the RIF condition, we observe a strong synergy at low concentrations (below 0.031 µg/mL) with MC3466 at concentrations superior to 2.5 µM (Figure 12A). A synergy is also observed with MC3466 (≥ 2.5µM) in presence of MOX and INH but at lower concentrations (≤ 0.016 µg/mL for MOX and ≤ 0.002 µg/mL for INH) (Figure S4). For BDQ, lower concentrations of MC3466

show a higher synergy with BDQ (Figure 12A). These results may be explained by the fact that at a high concentration, each drug is very effective on its own and the combination with MC3466 does not increase the antibiotic effect than when the antibiotic is used alone.

After having shown that there is a synergy between MC3466 and anti-TB drugs on drug susceptible MTB, we then investigated the impact of these drug combinations on monoresistant clinical strains. Macrophages were infected with RIF MTB resistant strains, before evaluating the effect of a MC3466-RIF treatment on the bacterial infection. Our data shows that the highest synergy occurs when MC3466 is used at a concentration of 2.5 µM (Figure 12B). The effect of MC3466 alone was then evaluated on this clinical RIF resistant strain by confocal microscopy and image analysis. We observed that the compound has a direct dosedependent efficacy on this resistant strain (Figure 12B). We confirmed the image-based synergy result by performing a CFU assay on MTB-infected macrophages. The number of CFU remains constant when MTB-infected macrophages are cultivated in presence of suboptimal concentrations for a RIF resistant strain (from 0.1 to 20 µg/mL). However, the number of CFUs decreases when cells are cultivated in presence of both MC3466 (10 µM) and RIF, with a higher impact on the intracellular RIF resistant MTB survival when the RIF concentration is increased (Figure 12C). In parallel, we have also tested the effect of MC3466 on a BDQ resistant MTB strain [START_REF] Giraud-Gatineau | The antibiotic bedaquiline activates host macrophage innate immune resistance to bacterial infection[END_REF]. Synergy effect on this strain is also observed with MC3466 (at a concentration superior to 5 µM) and BDQ (at a concentration superior to 0.31µg/mL) (Figure 12D). However, an antagonist effect of the combination is observed at lower concentrations (Figure 12D). We tested the effect of MC3466 at a concentration of 10µM in combination with different concentrations of BDQ by a CFU assay (Figure 12E).

MC3466 was also proved to impede the intracellular BDQ-resistant strain growth, in a dose dependent manner (Figure 12E).

All these results show thatMC3466 can potentiate the effect of anti-TB drugs when it is used at a concentration ≥ 5µM. MC3466 inhibit MTB growth in mice and potentiate RIF effect in vivo.

After having shown that both MC3465 and MC3466 inhibit the intracellular MTB growth, without any toxicity for the cell, we then performed an in vivo experiment on C57BL/6J mice.

We first assessed if the compounds are toxic or not for the mice. To do so, we intraperitoneally injected MC3465 and MC3466 at different concentrations, six days a week for two weeks in uninfected mice. No loss of weight was observed for the mice treated with the molecules, as well as for the control group that was treated with the vector alone (Figure 13A). After the two weeks treatment, total blood of each mouse was collected to quantify the level of alanine aminotransferase (ALT) and the aspartate aminotransferase (AST), which could reveal a hepatic dysfunction. No difference between the groups treated with the compounds and the control group is observed for the level of ALT (Figure 13B) and AST (Figure 13C), suggesting there is no toxicity associated to both MC3465 and MC3466 treatments.

From these results, we then decided to test the compounds on MTB-infected mice in an acute model of infection. One week after the mycobacterial infection, mice were treated for two weeks, six days a week. Lungs and spleens were harvested, and the number of bacteria were estimated by CFUs. No significative effect on the bacterial load in the lung was observed for the MC3465 treatment (Figure 13D). However, the number of bacteria in the lung after the MC3466 treatment drops by 5 times, compared to the control condition (Figure 13D). No significant difference between the three conditions was observed in the spleen of the mice (Figure 13E).

As we have previously shown that MC3466 potentiates the effect of RIF on intracellular MTB in vitro, we then investigated whether we could observe a similar effect in vivo. To do so, mice were treated for two weeks with RIF, with or without MC3465 and MC3466. MC3465 does not potentiate the effect of RIF, contrarily to MC3466 in the lung (Figure 13F). However, no effect is observed on the number of bacteria in the spleen (Figure 13G).

Taken together, our results demonstrate that both MC3465 and MC3466 are non-toxic to mice over a 15-day period. Furthermore, MC3466 exerts an effect on intracellular MTB growth in vivo and potentiates the effect of RIF on the bacterial load within the lungs. The aim of this thesis was to discover new compounds that enhance TB treatment by targeting the host epigenome. To do so, we have performed a high-content screening to evaluate the impact of a non-commercialized library of molecules on the intracellular growth of MTB.

HTS has been extensively used to identify potential targets for antimycobacterial inhibitors [START_REF] Christophe | High content screening identifies decaprenyl-phosphoribose 2ʹ epimerase as a target for intracellular antimycobacterial inhibitors[END_REF] or drug candidates that inhibit MTB growth [START_REF] Ollinger | A highthroughput whole cell screen to identify inhibitors of Mycobacterium tuberculosis[END_REF][START_REF] Shapira | High-Content Screening of Eukaryotic Kinase Inhibitors Identify CHK2 Inhibitor Activity Against Mycobacterium tuberculosis[END_REF]. Phenotypic-based approaches were first used to screen a large number of compounds for their ability to kill MTB in culture medium [START_REF] Entzeroth | Overview of high-throughput screening[END_REF]. However, this approach has rapidly showed some limitations as drugs inhibiting MTB growth in culture medium may be ineffective on intracellular MTB. For instance, those drugs may be inactivated by some host cell enzymes or they may not reach their bacterial target in those conditions. In this context, intracellular MTB drug screening models were developed to address these limitations, notably with the widespread use of image-based HCS. Intracellular screens are used to select antimicrobial drugs that are effective on intracellular bacteria, with no toxicity for the cells. Furthermore, MTB being known to interfere with many host cell processes, the use of HCS on intracellular MTB has revealed number of novel targets for the development of anti-TB drugs related to host-pathogen interaction [START_REF] Sorrentino | Development of an intracellular screen for new compounds able to inhibit Mycobacterium tuberculosis growth in human macrophages[END_REF].

In this thesis, we performed an HCS with human-monocyte derived macrophages. This model is closer to the human host, more physiological than immortalized tumor cells and allows to evaluate the efficacy of molecules on the host with inter-individual variability. Because MTB subverts host functions through epigenetic modifications (M. Singh et al., 2018), we performed HCS with a library of molecules that presumably target the host epigenome to promote the discovery of a host-directed compound. Our research hypothesis was that by reprogramming the host epigenetic system, the immune cells may better control a mycobacterial infection. This hypothesis is supported by several studies showing that epigenetic modifications of histones modulate the anti-mycobacterial activity of the cells [START_REF] Cheng | Host sirtuin 1 regulates mycobacterial immunopathogenesis and represents a therapeutic target against tuberculosis[END_REF][START_REF] Moreira | Functional Inhibition of Host Histone Deacetylases (HDACs) Enhances in vitro and in vivo Anti-mycobacterial Activity in Human Macrophages and in Zebrafish[END_REF]. Through our screening, we discovered a new molecule, MC3465, which prevents MTB growth inside macrophages, without any effect on MTB growth in culture medium. This compound is active against the laboratory strain H37Rv as well as against clinical isolates belonging to the Beijing lineage and mono-resistant strains.

To determine the effect of MC3465 on MTB intracellular growth, we have performed a kinetic experiment on MTB-infected macrophages. Our results have shown that MC3465 seems to be bacteriostatic rather than bactericidal. Interestingly, the Live/Dead flow cytometry experiment revealed that this molecule decreases infected and uninfected bystander cells death, subsequently preventing the spread of the infection. The efficacy of this molecule on MTB-infected macrophages prompted us to further investigate its mechanism of action.

MC3465 was described to be a potent SIRT2 inhibitor (Moniot et al., 2017b). However, we have shown that MC3465 mechanism of action is independent of the host SIRT2. SIRT2

inhibition by AGK2, a commercialized inhibitor of SIRT2, has been described to reduce the intracellular survival of MTB [START_REF] Bhaskar | Host sirtuin 2 as an immunotherapeutic target against tuberculosis[END_REF]. However, in our experiment with human macrophages AGK2 did not show any effect on inhibiting intracellular MTB growth. This difference between Bhaskar et al. study and ours could be explained by the use of different cell types. Unlike us, they also pre-treated their cells with AGK2 2 hours before infection.

Furthermore, upon L. monocytogenes infection, we did not see any inhibition of the deacetylation of the H3K18 following the addition of MC3465, nor a decrease of the bacterial load, compared to an AGK2 treatment [START_REF] Eskandarian | A role for SIRT2-dependent histone H3K18 deacetylation in bacterial infection[END_REF][START_REF] Pereira | Infection Reveals a Modification of SIRT2 Critical for Chromatin Association[END_REF]. Moniot [START_REF] Greenwood | Subcellular antibiotic visualization reveals a dynamic drug reservoir in infected macrophages[END_REF] and then in vivo [START_REF] Fearns | Correlative light electron ion microscopy reveals in vivo localisation of bedaquiline in Mycobacterium tuberculosis-infected lungs[END_REF]. They also have shown that the lipid droplet represents a reservoir of BDQ.

Subsequently, MTB interacts with these droplets, resulting in an increase of the antibacterial efficacy of the antibiotic. MC3465 also possesses a bromine atom and a similar approach could be used to determine where the molecule accumulates within the cells. In a synergic context, it may also be interesting to investigate a co-localization of MC3465 with anti-TB drugs, like it was recently assessed with PZA and BDQ [START_REF] Santucci | Intracellular localisation of Mycobacterium tuberculosis affects efficacy of the antibiotic pyrazinamide[END_REF].

Based on our data, the host SIRT2 inhibition is not the mechanism of action of MC3465. The non-validation of the target made us question whether the molecule was indeed an HDT. To prove that MC3465 only targets intracellular bacteria, we have performed a transwell experiment, showing that extracellular MTB are not affected by the compound nor by a potential metabolite secreted by the cells following the treatment. However, at this stage, we cannot rule out that MC3465 is not metabolized. A quantification of the compound within the cells by liquid chromatography with tandem mass spectrometry might allow us to determine if macrophages metabolized the compound [START_REF] Xiao | Metabolite identification and quantitation in LC-MS/MS-based metabolomics[END_REF]. Meanwhile, the transwell experiment allow us to conclude that MTB has to be localized inside the cell for MC3465 to have its effect. From this, we then have investigated the efficacy of MC3465 on other bacteria to see if the molecule also prevents the growth of other intracellular pathogens with different lifestyles. Our compound has shown no activity on S. Typhimurium, L. monocytogenes and E.coli. However, MC3465 limits the intracellular growth of BCG and M. abscessus, a rapidly growing mycobacterium which is also an opportunistic pathogen [START_REF] Johansen | Non-tuberculous mycobacteria and the rise of Mycobacterium abscessus[END_REF].

These results suggest that MC3465 efficacy is related to mycobacteria.

Mycobacteria have a gene coding for a Sir2 homolog, named Rv1151c. This latter acts as a NAD + -dependent protein deacetylase (Gu et al., 2009b). Rv1151c does not seem to be important for MTB infection and is a non-essential gene for growth in vitro [START_REF] Dejesus | Comprehensive essentiality analysis of the Mycobacterium tuberculosis genome via saturating transposon mutagenesis[END_REF]. However, it has been shown that nicotinamide, a SIRT inhibitor, also inhibits the bacterial Sir2 (Gu et al., 2009b). From those observations, we have hypothesized that MC3465 may target Rv1151c. We have generated a strain lacking Rv1151c and we are planning to perform growth and virulence assays on this mutant compared to the WT strain, in presence of MC3465. These experiments will help us determine whether the activity of the compound is dependent of the bacterial Sir2. More generally, it might be interesting to perform a transcriptomic analysis of intracellular MTB, with or without MC3465. This could help us to determine whether MTB differently expresses certain genes in response to the presence of the molecule [START_REF] Benjak | Whole-transcriptome sequencing for highresolution transcriptomic analysis in Mycobacterium tuberculosis[END_REF]. For example, because MC3465 has a bacteriostatic effect on the bacteria, we could hypothesize that the molecule, somehow, triggers a dormancy state of MTB. Assessing the general gene expression in MTB could broad us valuable knowledge on the state and response of intracellular MTB to MC3465.

Furthermore, macrophages have other sirtuins that may be MC3465 targets. Interestingly, SIRT1 and SIRT3 have been shown to be regulated during MTB infection. More specifically, activation of host SIRT1 reduces intracellular growth of MTB, which is similar to what we have observed with MC3465 [START_REF] Cheng | Host sirtuin 1 regulates mycobacterial immunopathogenesis and represents a therapeutic target against tuberculosis[END_REF]. SIRT1 reduces MTB intracellular growth by inducing autophagy and phagosome-lysosome fusion. Moreover, several drugs also regulate the host immune defense and activate autophagy such as PZA, INH and more recently BDQ (Giraud-Gatineau et al., 2020; J. J. [START_REF] Kim | Host cell autophagy activated by antibiotics is required for their effective antimycobacterial drug action[END_REF]. In this context, we have assessed whether the addition of MC3465 leads to an increase in autophagy activity. Our experiments did not prove that this pathway was involved in the inhibition of MTB growth, which limits the possible role of the host SIRT1 in our case. One way to assess whether MC3465 has a role on SIRT3 is to determine the ROS level inside the cell. Indeed, ROS are one of the main defense mechanisms of the cell against pathogens and SIRT3 is an important regulator of cellular redox homeostasis during MTB infection [START_REF] Herb | Functions of ros in macrophages and antimicrobial immunity[END_REF][START_REF] Smulan | Sirtuin 3 downregulation in mycobacterium tuberculosis-infected macrophages reprograms mitochondrial metabolism and promotes cell death[END_REF]. Thus, quantification of the total ROS level with CellROX™ or mtROS with MitoSOX™ dye by flow cytometry [START_REF] Giraud-Gatineau | The antibiotic bedaquiline activates host macrophage innate immune resistance to bacterial infection[END_REF] is also relevant to link or not ROS production to the mechanism of action of MC3465. Moreover, as MC3465 is supposed to target a histone deacetylase, it would also be interesting to analyze histone modifications by chromatin immunoprecipitation followed by sequencing (ChIP-seq) to highlight a possible histonerelated modification [START_REF] O'geen | Using ChIP-seq technology to generate highresolution profiles of histone modifications[END_REF].

At this stage, we cannot conclude whether the molecule has a direct effect on the host or on compounds and targets (S. [START_REF] Wang | Advanced activity-based protein profiling application strategies for drug development[END_REF]. ABPP is more focused on analyzing enzymatic functions while CCCP identifies interacting components via drug affinity chromatography and mass spectrometry techniques (S. [START_REF] Wang | Advanced activity-based protein profiling application strategies for drug development[END_REF]. In principle, a small group is added to the molecule of interest which will then allow the isolation of the molecule with its target, also called "target fishing". Click-chemistry regroups the reactions by which groups are added to the molecules [START_REF] Hein | Click chemistry, a powerful tool for pharmaceutical sciences[END_REF]. Target fishing is a valuable technique that then allows the design of drug inhibitors [START_REF] Sharpless | In situ click chemistry: A powerful means for lead discovery[END_REF]. Reporter groups are commonly biotin, fluorescent groups, alkynes or azide. For example, biotin is used for biotin-streptavidin pulldown assays (capture of the tagged protein with its target) followed by identification of the target by mass spectrometry (S. [START_REF] Wang | Advanced activity-based protein profiling application strategies for drug development[END_REF]. Identification of the target with fluorescent reporters is performed via imaging-based detection (S. [START_REF] Wang | Advanced activity-based protein profiling application strategies for drug development[END_REF].

However, these techniques have some limitations. First of all, efficacy of the modified molecules has to be assessed before being used, to verify that the molecules are still active.

In addition, non-specific binding may lead to the "fishing" of many off-targets, preventing the identification of the true target(s). Discriminating the false positive targets from the real target(s) may be performed by comparing the target pool between the molecule of interest versus an inactive similar analogue [START_REF] Wright | Chemical proteomics approaches for identifying the cellular targets of natural products[END_REF]. Another issue associated with these techniques is that the probe-labeled molecule may be metabolized by the cells, therefore leading to the loss of the binding and the efficacy. Despite these limitations, this technique has been shown to be a valuable tool to determine possible targets of molecules.

Moreover, potential identified targets have then to be confirmed by other techniques, such as use of recombinant proteins to verify the interaction of the compound with the target ex vivo; to a co-localization or a co-immunoprecipitation by microscopy or western blot analysis, respectively (if an antibody of the target is available); to silence the gene expression by siRNA and determine if the compound is still active. Identification of a drug target remains challenging. For example, there is no strong consensus for the target(s) of PZA which has been discovered more than 65 years ago [START_REF] Gopal | Pharmacological and Molecular Mechanisms Behind the Sterilizing Activity of Pyrazinamide[END_REF].

Beside finding the target of MC3465, it is also important to investigate its mechanism of action. In order to unravel the mode of action of this molecule, we used an undirect approach to map the host pathways differentially expressed after the addition of MC3465. We performed RNAseq on uninfected and MTB-infected cells at different time points, with or without the molecule. We found that several pathways are downregulated in presence of MC3465 in uninfected and infected cells, including zinc related pathways and cytokines regulation with notably modulation of INF signaling pathways. Metals are essential for all living organisms and thereby represent highly important elements in host-pathogen interaction [START_REF] Hood | Nutritional immunity: Transition metals at the pathogenhost interface[END_REF]. The host cell has deployed many mechanisms to limit the availability of essential nutrients to starve the pathogen or, on the contrary, to intoxicate it with an excess of metals (Neyrolles et al., 2015b). Upon TB infection, mechanisms controlling the intracellular zinc concentration are highly regulated by the host [START_REF] Lonergan | Nutrient Zinc at the Host-Pathogen Interface[END_REF].

In the host, zinc is bound to metallothioneins (MT). During the infection, zinc is released from the host and accumulates in the MTB-containing vacuole [START_REF] Botella | Mycobacterial P 1-Type ATPases mediate resistance to Zinc poisoning in human macrophages[END_REF]. Results of our

RNAseq have showed a downregulation of several MT at 4h in MTB-infected and uninfected cells. Based on these results, two hypotheses for the mechanism of action of the molecule involving zinc are considered. First, MC3465 may generate a high burst of zinc which is then mobilized in the phagosome to intoxicate the bacteria. If zinc is redirected to the vacuole, the level of zinc in the cytosol should subsequently be low, which must lead to down-regulation of MT. Secondly, MC3465 could act as a zinc binder, and the MT are less required by the host.

The zinc binding by MC3465 may lead to zinc starvation for the bacteria, therefore affecting bacterial growth inside the cell. These opposite hypotheses could be tested by different methods. For example, Botella et al. used the FluoZin™-3 to demonstrate a higher zinc concentration inside the phagocytic vacuole using confocal microscopy [START_REF] Botella | Mycobacterial P 1-Type ATPases mediate resistance to Zinc poisoning in human macrophages[END_REF].

A similar technique could be used on MTB-infected macrophages, with or without MC3465.

Furthermore, MTB under zinc deprivation or intoxication has distinct changes in its transcriptome [START_REF] Dow | Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis[END_REF]. We could therefore assess the expression of different mycobacterial genes by qRT-PCR to determine whether MTB is intoxicated (e.g. with the expression of the metal efflux pump linked to the cpcC gene [START_REF] Botella | Mycobacterial P 1-Type ATPases mediate resistance to Zinc poisoning in human macrophages[END_REF]) or under starvation (with Zur-regulated genes [START_REF] Dow | Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis[END_REF]).

The host transcriptomic analysis has also pointed out a differential regulation of several pathways that are implicated in the modulation of cytokines at 24 hours in uninfected and infected cells. Particularly, our data have shown that genes involved in type I IFN pathway are downregulated. Several studies have demonstrated that Type I IFN overexpression is deleterious for the host during MTB infection [START_REF] Dorhoi | Type I IFN signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics[END_REF][START_REF] Mcnab | Type I IFN Induces IL-10 Production in an IL-27-Independent Manner and Blocks Responsiveness to IFN-γ for Production of IL-12 and Bacterial Killing in Mycobacterium tuberculosis -Infected Macrophages[END_REF][START_REF] Moreira-Teixeira | Type I interferons in tuberculosis: Foe and occasionally friend[END_REF]. In patients with active TB, blood transcriptional gene signature with overexpression of type I IFN-related genes have been correlated with disease severity and is downregulated following successful treatment [START_REF] Moreira-Teixeira | Type I interferons in tuberculosis: Foe and occasionally friend[END_REF]. MTB induces IFN-related gene expression to promote its survival [START_REF] Novikov | Mycobacterium tuberculosis Triggers Host Type I IFN Signaling To Regulate IL-1β Production in Human Macrophages[END_REF][START_REF] Stanley | The Type I IFN Response to Infection with Mycobacterium tuberculosis Requires ESX-1-Mediated Secretion and Contributes to Pathogenesis[END_REF]. Indeed, type I IFN cytokines are linked to the inhibition of TNF-α, IL-12, and IL-1β while it promotes the production of IL-10 (McNab et al., 2014;[START_REF] Novikov | Mycobacterium tuberculosis Triggers Host Type I IFN Signaling To Regulate IL-1β Production in Human Macrophages[END_REF]. However, type I IFN may be occasionally beneficial for the host in absence of IFN-γ [START_REF] Moreira-Teixeira | Type I IFN Inhibits Alternative Macrophage Activation during Mycobacterium tuberculosis Infection and Leads to Enhanced Protection in the Absence of IFN-γ Signaling[END_REF]. Some host-directed therapies (using zileuton) manipulate host eicosanoid by increasing PGE2 levels which negatively regulates type I IFN in vivo [START_REF] Mayer-Barber | Host-directed therapy of tuberculosis based on interleukin-1 and type i interferon crosstalk[END_REF].

Thus, it might be interesting to dose type I IFN cytokines level, PEG2 and the related cytokines IL-ß, to determine if macrophages treated with MC3465 better control the infection through cytokine or eicosanoid modulations.

While the mechanism of action of MC3465 remains elusive and needs further investigation, we continued to focus on the efficacy of the molecule and its analogue MC3466. In addition to their own growth inhibition activity on MTB, our results have underlined important observations regarding the combination of the analogue MC3466 with antibiotics used to treat TB. Suboptimal concentrations of RIF, INH, MOX or BDQ in combination with MC3466 had more efficacy on preventing MTB intracellular growth than used alone. These effects were also demonstrated in vivo for RIF. In the fight against TB, there is growing interest in the development of effective antibacterial drug combinations for better therapeutic results [START_REF] Ramón-García | Synergistic drug combinations for tuberculosis therapy identified by a novel high-throughput screen[END_REF][START_REF] Yilancioglu | Design of high-order antibiotic combinations against M. tuberculosis by ranking and exclusion[END_REF]. Experimental drug regimens have been tested in vitro and optimized for their use in vivo (B.-Y. [START_REF] Lee | Drug regimens identified and optimized by output-driven platform markedly reduce tuberculosis treatment time[END_REF]. Many in vivo studies have led to trials to determine the efficacy of different combinations of molecules to shorter current treatments (World Health Organization, 2020). Recently, drug combinations using an already approved drug with an adjuvant such as small molecules, have been integrated for the design of new drug regimens. These molecules are also called boosters, activators or enhancers depending on the study [START_REF] Guieu | Desirable drug-drug interactions or when a matter of concern becomes a renewed therapeutic strategy[END_REF]. For example, several 1,2,4-oxadiazoles were tested for their potency to boost the antibacterial activity of ETH (Flipo, Desroses, et al., 2012;[START_REF] Flipo | Ethionamide boosters: Synthesis, biological activity, and structure-activity relationships of a series of 1,2,4-oxadiazole EthR inhibitors[END_REF]Flipo, Willand, et al., 2012). Efficacy of small molecules is based on the enhancement of the pharmacokinetic profile of the approved drug, the increasing of its activity or the reversion of the resistance of a strain [START_REF] Guieu | Desirable drug-drug interactions or when a matter of concern becomes a renewed therapeutic strategy[END_REF][START_REF] Wambaugh | Highthroughput identification and rational design of synergistic small-molecule pairs for combating and bypassing antibiotic resistance[END_REF]. Reversion of antibiotic resistance represents a breakthrough for the fight against MDR-TB. As an example, the spiroisoxazoline small molecules aborting resistance (SMARt)-420 was described to boost the effect of ETH but also to revert the ETH-acquired resistance via its interaction with the transcriptional regulator EthR2 (SMARt-420 inhibits the EthR2 repression of EthA2 and lead to the activation of ETH) [START_REF] Blondiaux | Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420[END_REF]. More interestingly, in our study, the anti-TB drug used with our small molecule MC3466 also shows a better effect on mono-resistant strains.

Our results support the fact that MC3466 may be a potential molecule for adjunctive therapy for TB treatment. Pre-clinical studies, related to the pharmacodynamic and pharmacokinetic of the molecule, are very important for the development of a potential drug. In this context, it might be relevant to test the genotoxicity of MC3466, the concentration of the molecule found in the serum and the safer dose for long-term administration, the half-life of the molecule and its clearance, the possible sign of toxicity with a higher dose on the liver or the induction of cytochrome P450 (CYP450) especially if this drug is used in combination with other anti-TB drug including RIF (which is a known inducer of CYP450 [START_REF] Kanebratt | Cytochrome P450 induction by rifampicin in healthy subjects: Determination using the Karolinska cocktail and the endogenous CYP3a4 marker 4β-hydroxycholesterol[END_REF]).

In this thesis, we have tested the impact of MC3465 and MC3466 on MTB in an acute model of infection in vivo. Therefore, testing these compounds in a chronic infection model should determine the possible interaction with the development of the adaptive immune response.

Several in vivo tests have to be performed before properly considering this molecule as an adjuvant for anti-TB treatment.

HCS has allowed the discovery of promising new drugs that inhibit the intracellular growth of MTB in vitro, on susceptible and resistant strains of MTB, but also in vivo. So far, we have not discovered the target of MC3465 or its mechanism of action, but we have carried out many experiments that have given some interesting leads. An important result of our study is the efficacy of MC3466 with known anti-TB drugs on both sensible and resistant strains. Recently, the combination of drugs and the use of adjunctive therapy have been extensively studied for the development of new treatment regimens against MTB and MDR-TB [START_REF] Blondiaux | Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420[END_REF][START_REF] Guieu | Desirable drug-drug interactions or when a matter of concern becomes a renewed therapeutic strategy[END_REF]. However, many drugs have different effects on the host. Indeed, some drug effects may be deleterious and cause toxic side effects. However, some drugs may also have beneficial effects and be candidates for drug repurposing. Drug repurposing represents one of the main elements of HDT (An et al., 2020b). HDT are not stand-alone drugs.

They act through various pathways inside the cell in combination with already known and characterized antibiotics. The use of HDT leads to very different outcomes, especially among individuals. Indeed, as described in the introduction, results from human trials which include HDT are often contradictory. These inconsistencies among patients represent the major limitation for the use of HDT and these different responses underlying the importance of drug combination studies. However, HDT still represent a very interesting alternative for overcoming antimicrobial resistance. Use of HDT for TB treatment is clearly at its early stages and requires deeper studies and analysis to become a main part of TB treatment. In the meantime, development of new regimens for TB treatment with adjunctive, drug repurposing or new molecules represents one of the best ways to eradicate TB.

Title: Enhancing tuberculosis treatment with host-targeting strategies.

Abstract: Tuberculosis (TB) is one of the top ten causes of human death worldwide and remains a major public health threat. This disease can be cured with a 6-month treatment regimen, combining up to four antibiotics. Alarmingly, multidrug resistant (MDR) strains of Mycobacterium tuberculosis (MTB), resistant to the first-line anti-TB drugs, are constantly emerging and spreading. Treatment options to cure MDR-TB are limited, expensive and associated with a lower success rate. In addition, they are based on the use of more toxic second-and third-line drugs for a longer duration. Despite considerable research efforts, the vaccine is not sufficient to control the epidemic. Recently, host-directed approaches have emerged as an innovative and promising strategy to eradicate TB. These therapies, unlike antibiotics which specifically target bacteria, aim to improve the host defenses. Their combination with existing or future antibiotics can be a major improvement for eradicating TB. MTB manipulates some host signaling pathways to subvert innate and adaptive immunity.

For instance, MTB modulates the host gene expression by targeting the host epigenome to allow immune evasion. My thesis project aims to determine whether pharmacological interference with these MTB-induced driven changes of the epigenome decreases bacterial survival within the host. Using an automated confocal microscopy, we have developed a high throughput screen to evaluate the impact of compounds that target the host epigenome. With this technique, we have identified a new molecule that prevents GFP-expressing MTB replication within macrophages. This molecule is a putative inhibitor of Sirtuin 2 (SIRT2). SIRT2 is an NAD+-dependent deacetylase that regulates many cellular processes and plays a role in controlling bacterial infections such as listeriosis. We have demonstrated that this compound is not toxic to human cells. Moreover, it does not affect bacterial growth in liquid medium, suggesting a host-dependent mechanism of action. This molecule potentiates the activity of several anti-TB antibiotics, on both drug-susceptible and resistant MTB strains, in human macrophages and in mice. These promising results pave the way for future research, evaluating the potential of molecules targeting host epigenome as a new weapon in the arsenal against TB.

Key words: Tuberculosis, Mycobacterium tuberculosis, macrophages, high throughput screening, host-directed therapy, epigenetics, sirtuin, antibiotic, resistance Titre : Améliorer le traitement de la tuberculose grâce à des stratégies dirigées vers l'hôte.

Résumé : La tuberculose (TB) est l'une des dix premières causes de mortalité dans le monde et représente une menace majeure pour la santé publique. Cette maladie peut être guérie grâce à un traitement de 6 mois associant jusqu'à quatre antibiotiques. Cependant, il est alarmant de constater que des souches multirésistantes (MDR) de Mycobacterium tuberculosis (MTB) ne cessent d'apparaître et de se propager. Leur résistance aux antibiotiques de première ligne rende les traitements beaucoup plus difficiles. Les options thérapeutiques pour guérir la TB MDR sont limitées, coûteuses et associées à un taux de réussite plus faible. De plus, elles sont basées sur l'utilisation de médicaments de deuxième et troisième intention plus toxiques et sur une durée plus longue. Malgré des efforts de recherche considérables, le vaccin quant à lui, ne suffit pas à enrayer cette épidémie. Récemment, les approches dirigées vers l'hôte sont apparues comme une stratégie innovante et prometteuse pour éradiquer la tuberculose. Ces thérapies, contrairement aux antibiotiques qui ciblent spécifiquement les bactéries, visent à améliorer les défenses immunitaires de l'hôte. Leur combinaison avec les antibiotiques existants ou futurs peut se révéler d'une utilité majeure pour améliorer le traitement de la TB. MTB a su développer différentes stratégies pour contourner l'immunité innée et adaptative de l'hôte qu'elle colonise. Pour exemple, MTB module l'expression des gènes de l'hôte en ciblant l'épigénome ce qui lui permet de se développer dans les cellules immunitaires. Mon projet de thèse vise à déterminer si l'interférence pharmacologique de ces modifications de l'épigénome induites par MTB diminue la survie bactérienne intracellulaire chez l'hôte. Dans cette optique, nous avons mis au point une technique de criblage de composés épigénétiques qui modifient l'expression des gènes de la cellule. Nous avons testé l'efficacité des composés sur des macrophages infectés par une bactérie MTB exprimant la GFP grâce à l'utilisation d'un microscope confocal automatisé à haut débit. Ce criblage nous a permis de découvrir une molécule qui réduit considérablement l'infection bactérienne dans ces cellules en empêchant MTB de se multiplier. Cette molécule est un inhibiteur putatif de la Sirtuine 2 (SIRT2). La SIRT2 est une désacétylase dépendante du NAD+ qui régule de nombreux processus cellulaires et joue un rôle dans le contrôle des infections bactériennes telle que la listériose. Nous avons démontré que ce composé n'était pas toxique pour les cellules humaines. De plus, il n'affecte pas la croissance bactérienne en milieu liquide, suggérant un mécanisme d'action dépendant de l'hôte. Aussi, cette molécule potentialise l'activité de plusieurs antibiotiques antituberculeux sur des souches de MTB sensibles et résistantes aux médicaments, aussi bien in vitro dans des macrophages humains qu'in vivo chez la souris. Ces travaux de thèse pourraient permettre la création d'un nouveau traitement contre la tuberculose.

Mots clés : Tuberculose, Mycobacterium tuberculosis, macrophages, criblage à haut débit, thérapie dirigée vers l'hôte, épigénétique, sirtuine, antibiotique, résistance

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 . 76 Figure 6 .Figure 7 .Figure 8 .Figure 9 .Figure 10 .Figure 11 .Figure 12 .Figure

 12345766789101112 Figure 1. Global representation of countries with at least 100,000 incident cases of TB reported in 2019.. ................................................................................................................ 10 Figure 2. Cell cycle of MTB infection. ................................................................................... 32 Figure 3. Host-MTB interaction influences the granuloma fate. .......................................... 36 Figure 4. Host-directed therapies against MTB. ................................................................... 65 Figure 5. Schematic representation of the three different epigenetic mechanisms: DNA methylation, histone modification and non-coding RNAs. .................................................. 76 Figure 6. Identification of new compounds inhibiting the intracellular growth of MTB. ... 104 Figure 7. MC3465 constrained the intracellular growth of MTB independently of SIRT2. . 106 Figure 8. Differentially expressed genes upon MC3465 treatment.................................... 108 Figure 9. MC3465 did not induce autophagy in MTB-infected macrophages. ................... 110 Figure 10. Structure-Activity Relationship (SAR) analysis of MC3465 and identification of MC3466 as a more efficient analogue. .............................................................................. 112 Figure 11. MC3465 and MC3466 protected MTB-infected cells from death. ..................... 114 Figure 12. MC3466 potentialized the efficacy of anti-TB drugs on sensible-and resistant-MTB strains. ....................................................................................................................... 116 Figure 13. MC3466 increased the efficiency of rifampicin in the mouse model of TB. ...... 118 Figure S1. MC3465 blocked the intracellular growth of MTB.……………………………………………121 Figure S2. Effect of MC3465 on other bacteria.…………………………………………………………………121 Figure S3. Differentially expressed genes upon MC3465 treatment.…………………………………122 Figure S4. MC3466 increased the efficiency of anti-TB drugs. ………………………………….………122

  is based on the antigenic detection of LAM, an immunogenic glycolipid component of the mycobacterial cell wall excreted in the urine. According to the WHO, this technic is implemented in 13 countries for the diagnosis of TB(World Health Organization, 2020) In 2010, the WHO endorsed a novel molecular technique for TB detection: the Xpert® MTB/RIF assay (World Health Organization, 2020a).This assay is based on an automatized nucleic acid amplification for rapid TB diagnosis and simultaneous identification of rifampicin (RIF -an antibiotic routinely used in the TB treatment) resistance. It can be used on bronchoalveolar lavage, sputum or induced sputum, gastric or nasopharyngeal aspirates. Since then, nextgeneration versions of this system have been developed like the Xpert® MTB/RIF Ultra with a higher sensitivity. Other tests, like the Xpert® XDR cartridge or the MDR/MTB ELITe MGB ® Kit

  ): INH is a prodrug that first needs to be activated by KatG, a mycobacterial enzyme, to be active. Once activated, INH produces intracellular INHderived damaging species including superoxide, hydrogen peroxide and nitric oxide (NO), deleterious for MTB. It also affects several key cellular pathways such as the bacterial cell wall synthesis by inhibiting InhA (enoyl acyl carrier protein reductase),

  TB preventive treatments recommended by the WHO are: six to nine months of daily INH; four months of daily RIF; three months of weekly rifapentine plus INH; three months of daily INH plus RIF or a one-month regimen of daily rifapentine plus INH.

3 and 7 .

 7 They then initiate the degradation phase of apoptosis with proteolytic cleavage of different proteins such as structural components of the cell like actin or DNA-dependent protein. Their action leads to DNA fragmentation, mitochondrial remodeling, ROS production

Figure 5 .

 5 Figure 5. Schematic representation of the three different epigenetic mechanisms: DNA methylation, histone modification and non-coding RNAs[START_REF] Gartstein | Prenatal influences on temperament development: The role of environmental epigenetics[END_REF].

  formula ŷab=ya+yb-yayb, was used to determine the Bliss predicted inhibition rate ŷab, where ya and yb are the observed inhibition rates with drug A alone at dose a and drug B alone at dose b. Excess over Bliss score (I), I=yab-ŷab, was used to determine the difference between the observed response (yab) and the Bliss predicted response (ŷab) at the same combination dose. If I = 0, the two drugs act independently. I < 0 indicates antagonistic drug interactions, while I > 0 indicates synergy.

Figure 6 .

 6 Figure 6. Identification of new compounds inhibiting the intracellular growth of MTB. (A) Human macrophages were infected with GFP-MTB at a MOI 0.5:1 and were incubated with the epigenetic library at 10 µM. Four days post-infection, images were acquired by automated confocal microscopy followed by image analysis. Only compounds which were not toxic to the cell (cell viability>75%, upper panel) were kept for further analysis. The spots area per cell (lower panel) was expressed as the percentage of GFP area in compound-treated cells compared to cells incubated with DMSO. (B) Representative confocal images of macrophages infected with MTB (green) and treated with MC3465. Hoechst and HCS CellMask (blue) were used to visualize respectively nuclei and cytoplasm. Scale bar: 10 μm (C) MTB-infected cells were treated with different concentrations of MC3465. After one or four days, cells were lysed and bacteria were enumerated by CFU. (D) Macrophages were infected with Listeria monocytogenes, Salmonella Typhimurium or Bacille Calmette Guerin (BCG). Intracellular bacteria were enumerated at the indicated time post-infection. (E) Growth of MTB in culture liquid medium in the presence of MC3465 at different concentrations. (F) On the left panel, illustration of the Transwell® assay. Briefly, MTB were plated in the upper chamber of the Transwell plate with or without macrophages in the lower chamber. The co-culture system was then treated with MC3465 or vehicle (DMSO) for four days and the number of bacteria was enumerated. MTB-infected macrophages were seeded as control. One representative experiment (of three) is shown. Error bars represent the mean ± SD. One-way ANOVA test was used. * p < 0.05, ** p < 0.01, *** p < 0.001, ****p<0.0001.

Figure 7 .

 7 Figure 7. MC3465 constrained the intracellular growth of MTB independently of SIRT2. (A) MTB-infected macrophages were treated with different concentrations of MC3465 and of SIRT2 inhibitors (namely AGK2 and SirReal2). After four days, cells were lysed and bacteria were enumerated by CFU. (B) siRNA-mediated Silencing of SIRT2. The relative gene expression of SIRT2 was measured by RT-qPCR and normalized to the GADPH gene. (C) SIRT2-silenced macrophages were infected with MTB and treated with MC3465. After 4 days, the number of intracellular bacteria was counted. (D) WT MEFs or Sirt2 -/-MEFs were infected with MTB. After 48 hr, the number of intracellular bacteria was enumerated. (E) Acetylation levels in Hela cells infected with L. monocytogenes and treated with MC3465 or with sirtuin inhibitors for 24 hr, as detected by immunoblotting. Uninf: uninfected, NAM: nicotinamide. (F) Quantification of acetylated H3K18 immunoblots in L. monocytogenes-infected Hela cells treated with MC3465 or with sirtuin inhibitors. Error bars represent the mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 8 .

 8 Figure 8. Differentially expressed genes upon MC3465 treatment. Uninfected and MTB-infected macrophages derived from four individual donors were treated with MC3465 (10µM) for 4 hr and 24 hr. Differentially expressed genes were identified by mRNAseq. (A) Volcano plot showing differentially expressed genes due to MC3465 treatment (p-value <0.05, fold change <-0.5 and >0.5). (B-C) Gene ontology enrichment analysis using the Cytoscape app ClueGO, of genes whose expression is downregulated by MC3465 treatment at 4 hr (B) and at 24 hr (C) (p-value ≤ 0.05; LogFC ≤ -0.5). (D) Heatmap showing differential expression of genes differentially expressed by MC3465 in naive and MTB-infected cells. Genes related to cellular zinc ion homeostasis and to type I interferon signaling pathway were respectively represented at 4 hr and 24 hr. Genes which were not differentially expressed were represented by a grey square.

Figure 9 .

 9 Figure 9. MC3465 did not induce autophagy in MTB-infected macrophages. (A) Detection by indirect immunofluorescence of LC3 (red) in MTB (green) infected macrophages, treated with MC3465 for 24 hr and 48 hr (scale bar: 10 μm). DAPI (blue) was used to visualize nuclei. (B) Determination of the number of LC3-positive puncta per cell (unpaired t-test). (C) MTB-infected macrophages were left untreated or incubated with MC3465 plus bafilomycin (BAF), 3-methyladenine (3-MA) or chloroquine (CQ). After four days, the number of intracellular bacteria was enumerated. Error bars represent the mean ± SD. One-way ANOVA test was used. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 10 .

 10 Figure 10. Structure-Activity Relationship (SAR) analysis of MC3465 and identification of MC3466 as a more efficient analogue.(A) Macrophages were infected with GFP-MTB were incubated with 29 analogues at 10 µM. Four days post-infection, images were acquired by automated confocal microscopy followed by image analysis. The spots area per cell was expressed as the percentage of GFP area in compound-treated cells compared to cells incubated with DMSO. (B) MTB-infected macrophages were treated with MC3465 or MC3465 (10µM). After four days, the number of intracellular bacteria was enumerated. One representative experiment (of at least three) is shown. (C) MTB growth in culture liquid medium in the presence of the analogue MC3466 at different concentrations, determined by OD600. (D) The dose-response curves (DRC) for MC3465 and MC3466 were calculated by automated confocal microscopy followed by image analysis. Tfhe ratio of GFP area in compound-treated macrophages compared to cells incubated with DMSO, was normalized with the negative control DMSO (0% inhibition) and the positive control RIF (100% inhibition). (E) MTB-infected cells were treated with different concentration of MC3465 or MC3465. After four days, bacteria were enumerated by CFU. (F) The IC50 of each compound was determined by automated confocal microscopy (as described in (D)) and by counting the CFU. Representation of the molecules. (G) Macrophages were infected with a panel of clinical isolates of MTB (GC1237, CDC1551 and Myc5750) and were treated with MC3465 and MC3466. The number of intracellular bacteria was determined after four days of treatment. Error bars represent the mean ± SD. *p<0.05, **p<0.01, ***p<0.001.

Figure 11 .

 11 Figure 11. MC3465 and MC3466 protected MTB-infected cells from death. Macrophages were infected with GFP-MTB at a MOI 5:1 and were treated with DMSO, MC3465 and MC3466. After four days, cells were labeled with Dead/Live fixable violet dead cell dye. The fluorescence intensity was quantified by flow cytometry.

Figure 12 .

 12 Figure 12. MC3466 potentialized the efficacy of anti-TB drugs on sensible-and resistant-MTB strains. (A) Heatmaps representing variation in drug combination, using the Bliss independence model, ranging from antagonism (blue) to synergy (red). Cells were infected with GFP-MTB and were treated with a range of concentration of MC3466 and RIF or BDQ. Four days postinfection, images were acquired by automated confocal microscopy followed by image analysis as described in Figure 6. (B-C) Cells were infected with a rifampicin-resistant MTB strain and treated with MC3466 during four days. (C) Heatmaps of the combination of RIF and MC3466, using the Bliss independence model (D) Macrophages were infected with a rifampicin-resistant MTB strain. RIF was added at different concentrations with or without MC3466 at 10µM. The number of intracellular bacteria was enumerated 4 days post-infection. (D-E) Cells were infected with a bedaquiline-resistant MTB and treated with MC3466 during four days. The effects of MC3466 were analyzed as in (B-C). Error bars represent the mean ± SD.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 13 .

 13 Figure 13. MC3466 increased the efficiency of rifampicin in the mouse model of TB. (A) Eight-week-old female C57BL/6J mice received different concentration of MC3465 and MC3466 by intraperitoneal injection six days per week during two weeks. Weight was control before every injection. (B-C) Estimation of the enzymatic activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) using total blood of mice treated during two weeks. (D-E) Mice were infected via the aerosol route with <50 CFU of MTB H37Rv. After a week of infection, mice were treated six times per week with MC3465 or MC3466. Bacterial loads in the lungs (D) and spleen (E) were measured after two weeks of treatment. Five mice per group were included. (F-G) Seven days after infection, mice were treated during two weeks with MC3466 in combination with RIF. Lungs and spleens harvested were and the number of bacteria were determined by CFUs. Error bars represent the mean ± SD.*p<0.05, **p<0.01, ***p<0.001

Figure S4 .

 S4 Figure S4. MC3466 increased the efficiency of anti-TB drugs. Heatmaps of the combination of MC3466 and MOX or INH, using the Bliss independence model, ranging from antagonism (blue) to synergy (red).

Fold

  et al. tested the inhibitory effect of the compound by the determining the level of cytosolic acetyl-α-tubulin in U937 cell (pro-monocytic, human histiocytic lymphoma cell line)(Moniot et al., 2017b). It might be possible that once inside the cells, MC3465 has a more selective and potent target than SIRT2 or that it is metabolized. Difference between inhibition of the deacetylation of α-tubulin or H3 may be due to the fact that the molecule may allegedly inhibit SIRT2 inside the cytosol but does not have any effect on SIRT2 in the nucleus. Determining the localization of the molecule within the cell may therefore provide insights regarding its mechanism of action. Using correlative electron and ion microscopy,Greenwood et al. has determined the localization of the BDQ (which contains a bromine atom), by measuring the intensity of 79 Br ion signal in vitro

a

  bacterial component used by mycobacteria to grow intracellularly. Identification of the targets could provide us valuable insights for the characterization of this molecule. By performing SAR studies, where we have tested several analogues of MC3465, we have identified the active group of MC3465. With this knowledge, we can possibly modify the molecule or its analogues to identify its target. Activity-based protein profiling (ABPP) and compound-centric chemical proteomics (CCCP) are chemical proteomic approaches which use small-molecule chemical probes for the evaluation of interaction mechanisms between

Drugs Targets Modes of action A

		Levofloxacin or moxifloxacin	DNA gyrase and topoisomerase IV	Inhibition of DNA replication, transcription and repair
		Bedaquiline	F-ATP synthase	ATP synthase inhibition
		Linezolid	23S ribosomal RNA of the 50S subunit	Inhibition of bacterial protein synthesis
			Generation of
		Clofazimine	intracellular reactive	DNA damages
	B	Cycloserine or terizidone	oxygen species L-alanine racemase and D-alanine: D-alanine ligase	Inhibition of cell-wall synthesis
		Ethambutol	Arabinosyl transferases	Inhibition of cell-wall formation
			Formation of radical
		Delamanid	intermediates which inhibit mycolic acid	Inhibition of cell-wall synthesis
			synthesis
				Inhibition of protein
	C	Pyrazinamide	S1/RpsA, GpsI, PanD	translation process, nucleic acid metabolism, coenzyme A synthesis
		Imipenem-cilastatin or meropenem	L,D-transpeptidase	Inhibition of cell-wall synthesis
		Amikacin	30S ribosomal subunits	Inhibition of protein synthesis
		Ethionamide or prothionamide	InhA	Inhibition of synthesis of mycoloic acids
		p-aminosalicyclic acid Pteridine synthetase	Inhibition of folic acid synthesis

Table 1 . Antibiotics used for MDR-TB treatment. MDR

 1

	-TB regimens are usually composed of

Clinical trial phase Mode of action

	BTZ-043	Ib/IIa	Inhibition of the DprE1 enzyme
	Delpazolid	II	Inhibition of bacterial protein synthesis
	GSK-3036656	IIa	Inhibition of protein synthesis
	Macozinone	I/II	Inhibition of DprE1 enzyme
	OPC-167832	I/II	Inhibition of DprE1 enzyme
	Telacebec (Q203)	I/IIa	Inhibition of cytochrome bc1 complex
	SPR720	I/IIa	Inhibition of gyrase B/ParE
	SQ109	IIb/III	Inhibition of cell wall synthesis
	Sutezolid	IIb	Inhibition of protein synthesis
	TBA-7371	I//II	Inhibition of DprE1 enzyme
	TBI-166	I	Not fully elucidated
	TBI-223	I	Inhibition of protein synthesis
	TBAJ-876	I	Inhibition of F-ATP synthase

Table 2. New compounds in the pipeline for the treatment of TB (Working Group on

	Among these 22 drugs, 13 are new compounds including: BTZ-043, Delpazolid, GSK-3036656,
	Macozinone, OPC-167832, Q203, SQ109, SPR720, Sutezolid, TBAJ-876, TBA-7371, TBI-166 and
	TBI-223 (Table 2) (Working Group on New TB Drugs, 2021). Furthemore, six previously
	approved antibiotics are currently being tested for their application in association with MDR-
	TB treatment: clofazimine, levofloxacin, linezolid, moxifloxacin, rifampicin (high dose) and
	rifapentine.
	In the TB pipeline, several combinations of new and/or repurposed drugs are being checked
	in trials, either for drug-sensitive or drug-resistant TB. For drug-sensitive TB, most trials aim
	to shorten the current TB treatment. The RIFASHORT trial is based on increased doses of
	rifamycins (i.e rifampicin, rifampin or rifapentine) while the TRUNCATE-TB is for evaluation of
	the efficacy of 2-month regimens with combinations of multiples antibiotics (INH, PZA, EMB,
	RIF, linezolid, levofloxacin and BDQ)
	New TB
	Drugs, 2021).

Table 3 . miRNAs, targets and effects on genes involved in immunity against mycobacteria.

 3

	miRNA	Targets	Effects	References
	miRNA-21	IL-12	Th1 response Inhibition by targeting IL-12 production	(Z. Wu et al., 2012)
		Phosphofructokinase	Limitation of the host glycolysis and IL-	(Hackett et al.,
		muscle (PFK-M)	1β production	2020)
	miRNA-21-5p	Bcl-2, TLR-4	Attenuation of the secretion of IL-1β, IL-6, and TNF-α	(Zhao et al., 2019)
	miRNA-27	Ca 2+ channel, voltage-dependent, a2/D subunit 3	Downregulation of Ca 2+ signaling, inhibition of autophagosome formation	(F. Liu et al., 2018)
	miRNA-27b	Bcl-2-associated athanogene, TLR-2/MyD88 pathway	Pro-inflammatory cytokines decrease, NF-κB activation, apoptosis, ROS production	(Liang et al., 2018)
	miRNA-33	ATG5, ATG12, LC3B, and LAMP1	Autophagy inhibition and host lipid metabolism reprogramming	(Ouimet et al., 2016)
	miRNA-99b	TNF-α, TNF Receptor Superfamily Member 4	Suppression of cytokines overexpression (IL-6, IL-12, IL-1β)	(Y. Singh et al., 2013)

LIST OF TABLES

 Table 1 :
THESIS OBJECTIVES

Ethics statement

Buffy coats were obtained from anonymous healthy donors who signed a consent to donate their blood for research purposes.

Macrophages and cell lines

Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats by centrifugation with lymphocyte separation medium (Eurobio). CD14 + monocytes were purified by positive selection using microbeads coupled to an anti-CD14 antibody (Miltenyi Biotec) and magnetic columns. Monocytes were cultured at 37°C and 5% CO2 in RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Dutscher), and 2 mM Lglutamine (Gibco) (hereafter defined as complete medium) with macrophage colony stimulating factor (M-CSF, 20 ng/mL; Miltenyi Biotec). After six days of differentiation, the resulting macrophages were incubated in a buffer solution containing PBS, 2 mM ethylenediaminetetraacetic acid (EDTA) and 10% FBS, at 37°C and 5% CO2 for 15 minutes before being harvested and counted.

THP-1 cells derived from an acute monocytic leukaemia patient were cultured in complete medium. The cells are incubated at 37°C, with 5% CO2, and the culture medium was changed every 48 hours, before confluence was reached. THP-1 cells were differentiated into macrophages by treatment with phorbol 12-myristate 13-acetate (PMA) at 50 ng/mL for 48 hours.

Mouse embryonic fibroblasts (MEF) and Henrietta Lacks (HeLa, ATCC, CCL-2) cells were cultured in Dulbecco's Modified Essential Medium supplemented with L-glutamine (DMEM-

Determination of bacterial counts

Cells were lysed in water with 0.05% Triton X-100. Number of MTB was enumerated as previously described and plated on 7H11 [START_REF] Tailleux | Constrained Intracellular Survival of Mycobacterium tuberculosis in Human Dendritic Cells[END_REF]. CFUs were counted after three weeks at 37°C. L. monocytogenes was plated on BHI agar and CFUs were counted after 1 day at 37°C.

Measurement of cell death

Necrotic cell death was evaluated by staining cells with Live/Dead fixable violet dead cell stain kit (Invitrogen) as previously described [START_REF] Amaral | A major role for ferroptosis in Mycobacterium tuberculosis-induced cell death and tissue necrosis[END_REF]. Briefly, macrophages were incubated with Live/Dead staining solution (1:1000 diluted in PBS) at room temperature (RT) for 15 min in the dark. The staining reaction was stopped by washing the cells with PBS and 10% FBS. Cells were fixed with 4% paraformaldehyde (PAF) during 1 hour at RT and detached.

Analysis was performed using a CytoFLEX Flow Cytometer (Beckman Coulter, Brea, California).

More than 10,000 events per sample were recorded. The analysis was performed using the FlowJo software.

Compounds library for screening

Compounds library was obtained from Dante Rotili and Antonello Mai (Sapienza University, Rome), with each compound resuspended in DMSO. 384-well polypropylene plates (Greiner Bio-One) were prepared with compounds at final concentration of 10 μM in 0.5% DMSO (v/v).

High-content screening

Infected macrophages were plated at 2x10 4 cells/well in 384-well tissue culture plates, with each well containing different compound. Cells were fixed with 4% paraformaldehyde (PAF)

for 1 hour at RT and were strained with HCS CellMask Blue stain (HCS; Invitrogen Molecular Probes; 2µg/mL) and Hoechst 33342 (5µg/mL) in PBS for 30 minutes. Confocal images were acquired using the automated fluorescence microscope Opera Phenix High-Content Screening

Quantification and statistical analysis

Quantitative data were expressed as mean ± standard deviation (shown as error bar).

Statistical analyses were performed with Prism software (GraphPad Software Inc), using the t test and one-way analysis of variance (ANOVA) as indicated in the figure legends. Differences between groups were examined statistically as indicated (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p<0.0001). Results were considered statistically significant with a p-value < 0.05.

RESULTS

Supplemental