Seriam Realizados

A Seu Beto

Dona Suzane

Dona Gegé

Houem Rabah

Laurence Oumaima

Mary Fauvel

Aos Meus Amigos Bret

Natália, Angélica, Emmanuele Amanda Flora

Flávia Aburjaile

Luisa Rachid

Alberto Osorio

Thiago Baiano

Elma Lime Leite

Brenda Silva

Rosa Da Luz

Eric Guédon

Fillipe L R Do Carmo

Vasco Azevedo

Yves Le Loir

Dr Nadia Berkova
email: nadejda.berkova@inra.fr

Elma Lima Leite

Arthur Gautron

Martine Deplanche

Aurelie Nicolas

Jordane Ossemond

Minh Thu

David Gilot

Friedrich Goetz

Michael Otto

Av Antonio Carlos

Strain and cell type-specificity of host cell response to Staphylococcus aureus invasion

Keywords: Osteoblasts, Staphylococcus aureus, inflammasomes, caspase-1, IL-1β Staphylococcus aureus, invasion, intracellular persistence Staphylococcus aureus, Antibiotic Resistance, Bacteriocins, Biotechnology use of Bacteriocins

Staphylococcus aureus (S. aureus) is a highly adaptive and versatile Gram-positive bacterium that can cause a wide range of infectious diseases in humans or animals. In the early stages of infection, the interaction between S. aureus and the host cells causes inflammation, the process of which depends on several complex cellular mechanisms and a signaling cascade coordinated mainly by cytokines and their activation. The key factors determining the initiation of inflammation and its progression are pathogen/ microbe-associated molecular patterns (PAMPs/MAMPs), danger-associated molecular patterns (DAMPs), pattern recognition receptors (PRRs), innate and adaptive immunity. Inflammasomes, multi-protein signaling complexes that assemble after sensing PAMPs/ DAMPs, are key players of the innate immune response. Most inflammasomes are formed by an NLR receptor, an ASC adapter protein, and active caspase-1. Once assembled, inflammasomes act as activating platforms that promote the maturation of pro-inflammatory cytokines IL-1β and IL-18 in their active forms. Their role in different types of professional phagocytes during S. aureus infection has been extensively studied. Conversely, knowledge about the involvement of inflammasomes in non-professional phagocytes (e.g. epithelial cells, endothelial cells...) is very fragmented. This work aimed to study the role of inflammasome activation during S. aureus infection in non-occupational phagocytes. We adopted a model of S. aureus infection developed on osteoblastic cells (MG-63 line). Western blot and ELISA analyses were used to control caspase-1 activation and IL-1β maturation in the S. aureus MG-63 interaction. We generated MG-63 cells deleted from the caspase-1 gene (CASP1 -/-MG-63 cells) using the CRISPR/Cas9 approach. This allowed us to demonstrate the involvement of inflammasomes in the innate immune response to S. aureus by non-professional phagocytes. Unexpectedly, we have shown that S. aureus proliferates in CASP1 -/-MG-63 cells, suggesting that Casp1 (or Casp1-regulated phenomenon) is involved in the elimination of intracellular S. aureus. We found that the formation of the inflammasome in MG-63 cells is later than in professional phagocytes. Besides, the use of S. aureus strains (deletion mutants) expressing or not Phenol-Soluble Modulins (PSM) allowed us to determine the role of PSM in this trigger. Thus, we have demonstrated that S. aureus can activate inflammasome formation in non-professional phagocytes (MG-63) cells and that PSM is involved in this phenomenon.

II

Remerciements

Je remercie:

-Vasco Azevedo, mon directeur de thèse brésilien, pour l'opportunité qu'il m'a donnée, de son enseignement, et surtout, d'avoir cru en moi ; -Nadia Berkova, ma directrice de thèse Français; -Yves Le-Loir, pour toute son aide et pour l'accueil que j'ai reçu dans son institut de recherche ;

-Les membres du jury pour leur disponibilité à évaluer ce travail de thèse ; -Martine Deplanche pour ses enseignements et son amitié, merci beaucoup ; -Les équipes de recherche de l'INRA de Rennes, pour leur aide et leur disponibilité ; -Le programme de troisième cycle en génétique, pour tout son soutien dans la partie administrative de cette thèse de doctorat en cotutelle ; -L'École doctorale de l'Agrocampus Ouest -Rennes, pour tout son soutien dans la partie administrative de cette thèse de doctorat en cotutelle ; -Les organismes de financement CAPES et le Programme CAPES-COFECUB pour toute l'aide financière que j'ai reçue au Brésil et en France pendant la durée de cette thèse ;

-Tous les membres du laboratoire LGCM, en particulier à Fillipe pour le soutien ; -Mon cher frère Eldo; -Rafael Cabús pour tout le soutien nécessaire dans les moments difficiles et décisifs de ma vie. Tu as toujours cru en moi et m'a dit que mes rêves se réaliseraient; -Beto, Suzane, Gegé et Isabelle, je vous remercie pour le soutien; -À Houem Rabah, Oumaima, Laurence Fauvel, Mary Bret, merci beaucoup ! -Mes chers amis Flora, Natália, Angélica, Emmanuele, Amanda, Anne, Renata, Flávia Aburjaile, Rachid, Luisa Osorio, Alberto, Vinícius, Thiago Baiano et Daniel qui m'ont soutenue et encouragée de près ou de loin; -Rodolphe pour être fort et me soutenir toujours dans ces derniers moments difficiles.

Pour me dire toujours que tout est possible ; -Et surtout à Dieu, ma mère, Socorro, et mon père, Euclides, qui sont ma forteresse, merci pour votre soutien et votre amour inconditionnel à tout moment, je vous aime!

VI

List of figures

VIII

List of tables

Resumo

Staphylococcus aureus (S. aureus) é uma bactéria Gram-positiva altamente adaptativa e versátil que pode causar uma ampla gama de doenças infecciosas em humanos ou animais. Nos estágios iniciais da infecção, a interação entre S. aureus e as células hospedeiras causa inflamação, cujo processo depende de vários mecanismos celulares complexos e de uma cascata de sinalização coordenada principalmente pelas citocinas e sua ativação. Os principais fatores que determinam o início da inflamação e sua progressão são pathogen-/microbe-associated molecular patterns (PAMPs/ MAMPs), danger-associated molecular patterns (DAMPs), pattern recognition receptors (PRRs), e o sistema imune inato e adaptativo. Inflammassomas, complexos de sinalização multiprotéicos que se agrupam após a detecção de PAMPs/DAMPs, são fatores-chave da resposta imune inata. A maioria dos inflamassomas é formado por um receptor NLR, uma proteína adaptadora ASC e uma Caspase-1 ativa. Uma vez montados, os inflamassomas atuam como plataformas ativadoras que promovem a maturação das citocinas pró-inflamatórias IL-1β e IL-18 em suas formas ativas. Seu papel em diferentes tipos de fagócitos profissionais durante a infecção por S. aureus tem sido extensivamente estudado. Por outro lado, o conhecimento sobre o envolvimento de inflamassomas em fagócitos não profissionais (por exemplo, células epiteliais, células endoteliais ...) é muito fragmentado. O objetivo deste trabalho foi estudar o papel da ativação do inflamassoma durante a infecção por S. aureus em fagócitos não profissionais. Adotamos um modelo de infecção por S. aureus desenvolvido em células osteoblásticas . Análises de Western blot e ELISA foram usadas para controlar a ativação de caspase-1 e a maturação de IL-1β na interação de S. aureus/MG-63. Geramos células MG-63 deletadas do gene da caspase-1 (CASP1 -/ -MG-63) usando a abordagem CRISPR/Cas9. Isso nos permitiu demonstrar o envolvimento do inflamassoma na resposta imune inata a S. aureus por fagócitos não profissionais. Inesperadamente, mostramos que S. aureus prolifera nas células CASP1 -/ -MG-63, sugerindo que Casp1 (ou um fenômeno regulado pela Casp1) está envolvido na eliminação do S. aureus intracelular. Descobrimos que a formação do inflamassoma nas células MG-63 é mais tardia que nos fagócitos profissionais. Além disso, o uso de cepas de S. aureus (mutantes deletados) expressando ou não Phenol-Soluble Modulins (PSM) nos permitiu determinar o papel das PSMs como desencadeador. Assim, demonstramos que S. aureus é capaz de ativar a formação de inflamassomas em células não fagocíticas (MG-63) e que os PSMs estão envolvidos nesse fenômeno.

Palavras-chave: Osteoblástos, Staphylococcus aureus, inflammasomes, caspase-1, IL-1β

Résumé

Staphylococcus aureus (S. aureus) est une bactérie à Gram positif hautement adaptative et polyvalente qui peut causer un large éventail de maladies infectieuses chez l'homme ou l'animal. Lors des premières étapes de l'infection, l'interaction entre S. aureus et les cellules hôtes entraine une inflammation dont le processus dépend de plusieurs mécanismes cellulaires complexes et une cascade de signalisation coordonnée principalement par des cytokines et leur activation. Les facteurs clés déterminant l'initiation de l'inflammation et sa progression sont les pathogen-/microbe-associated molecular patterns (PAMPs/MAMPs), les danger-associated molecular patterns (DAMPs), les Pattern Recognition Receptors (PRRs), l'immunité innée et adaptative. Les inflammasomes, complexes de signalisation multiprotéines qui s'assemblent après la détection de PAMPs/DAMPs sont des facteurs clés de la réponse immunitaire innée. La plupart des inflammasomes sont composés d'un récepteur NLR, d'une protéine adaptatrice ASC et de la caspase-1 activée. Une fois assemblés, les inflammasomes agissent comme des plateformes activatrices qui favorisent la maturation des cytokines proinflammatoires IL-1β et IL-18 sous leurs formes actives. Leur rôle dans les différents types de phagocytes professionnels au cours de l'infection à S. aureus a été largement étudié. A l'inverse, les connaissances sur l'implication des inflammasomes dans les phagocytes nonprofessionnels (e.g. cellules épithéliales, endothéliales…) sont très parcellaires. L'objectif de ce travail était d'étudier le rôle de l'activation de l'inflammasome lors d'une infection à S. aureus sur des phagocytes non-professionnels. Nous avons adopté pour cela un modèle d'infection à S. aureus mis au point sur des cellules ostéoblastiques . Des analyses Western blot et ELISA ont été utilisées pour contrôler l'activation de Caspase-1 et la maturation de l'IL-1β lors de l'interaction S. aureus MG-63. Nous avons généré des cellules MG-63 délétées du gène caspase-1 (CASP1 -/-MG-63 cells) en utilisant l'approche CRISPR/Cas9. Ceci nous a permis de démontrer l'implication des inflammasomes dans la réponse immune innée à S. aureus par des phagocytes non-professionnels. De façon inattendue, nous avons montré que S. aureus prolifère dans les CASP1 -/-MG-63 cells, suggérant que Casp1 (ou les phénomènes régulés par Casp1) est impliquée dans l'élimination de S. aureus intracellulaire. Nous avons constaté que la formation de l'inflammasome dans les cellules MG-63 est cependant plus tardive que dans des phagocytes professionnels. De plus, l'utilisation de souches de S. aureus (mutants de délétion) exprimant ou non des Phenol Soluble Modulins (PSM) nous a permis de déterminer le rôle des PSM dans ce déclenchement. Ainsi, nous avons démontré que S. aureus est capable d'activer la formation d'inflammasome dans des cellules , phagocytes non professionnels et que les PSM sont impliqués dans ce phénomène.

Manuscript structure

This manuscript is subdivided into six major parts: I.

Chapter 1. Bibliographic Synthesis. A general introduction to the following topics:

General introduction / Introduction générale

General introduction

Staphylococcus aureus (S. aureus), is a Gram-positive bacterium capable of colonizing both human and animal hosts and have the potential to cause a wide range of diseases including pneumonia, bacteremia, sepsis, endocarditis, osteomyelitis, device-related infections, toxicshock syndrome, brain abscesses, mastitis among other diseases (FITZGERALD, 2012;[START_REF] Foster | Medical Microbiology[END_REF][START_REF] Wertheim | The role of nasal carriage in Staphylococcus aureus infections[END_REF]. S. aureus is a predominant cause of bone and joint infections (BJI), a disease associated with high morbidity and high health costs worldwide (DEL POZO; PATEL, 2009; TUCHSCHERR; GERACI; LÖFFLER, 2017; WRIGHT; NAIR, 2010; ZIMMERLI; TRAMPUZ; [START_REF] Zimmerli | Prosthetic-joint infections[END_REF]. S. aureus-induced BJI can evolve into chronic infection and become highly refractory to antibiotic treatment (LOWY, 1998). This is probably due to the several strategies that this bacterium has developed to invade host cells and to survive in the intracellular environment (EIFF; PETERS; PROCTOR, 2001; MONTGOMERY; DAVID; DAUM, 2015; TUCHSCHERR; GERACI; LÖFFLER, 2017).

In recent years, considerable progress has been made in our understanding of how pathogens are recognized by the innate immune system that forms the first line of defense against infection [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF][START_REF] Nicholson | The immune system[END_REF]. The invasion of foreign microorganisms induces a spectrum of inflammatory responses in the infected host (Fig. 1).

Generally, inflammation is classically described as an essential response of the host immune system that enables survival during infection or injury, and that maintains tissue homeostasis under a variety of noxious conditions (CHEN et al., 2017a;[START_REF] Cruvinel | Sistema imunitário: Parte I. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória[END_REF]MEDZHITOV, 2010a;[START_REF] Rankin | Beyond Host Defense: Emerging Functions of the Immune System in Regulating Complex Tissue Physiology[END_REF][START_REF] Rankin | Beyond Host Defense: Emerging Functions of the Immune System in Regulating Complex Tissue Physiology[END_REF].

The type of inflammatory response depends on the category of the inflammatory trigger. It was demonstrated that bacterial pathogens are recognized by receptors of the innate immune system, such as Toll-like receptors (TLRs), resulting in the induction of various inflammatory mediators such as inflammatory cytokines and chemokines, as well as prostaglandins, which act on target tissues [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF]CHEN et al., 2017a;[START_REF]Cytokine Profiling Plays a Crucial Role in Activating Immune System to Clear Infectious Pathogens[END_REF][START_REF] Turner | Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease[END_REF]. The acute inflammatory response is finished when the triggering agent is eliminated, the infection is cleared, and the damaged tissue is repaired. If this initial inflammatory response does not eliminate the pathogens, as in a case of chronic infections or unrepaired tissue damage, the adaptive immune system comes into play. The cells of the adaptive immune system translocate to the site of infection and begin to eliminate pathogens and damaged cells and tissue [START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF](MEDZHITOV, , 2010a)). However, in some chronic infections, inflammation can persist without adaptive immunity [START_REF] Cruvinel | Sistema imunitário: Parte I. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória[END_REF]. The innate response is characterized by being the first line of defense against infection in which leukocytes such as (e.g., neutrophils, monocytes, macrophages, etc.), are involved. These defense cells detect and attack other cells carrying pathogen-associated molecular patterns (PAMPs), and small proteins that signal pathogen invasion (i.e., cytokines and chemokines) or short peptides that directly attach to and restrict microbial pathogens. The cells of the adaptive immune system rely on T and B lymphocytes and proteins (i.e., antibodies) that detect and eliminate specific pathogens and also use cytokine/chemokine to signal and recruit other immune cells. Memory T and B lymphocytes have an essential role in immunity against pathogenic invasion. The complement system, along with natural killer cells and dendritic cells, straddles both innate and adaptive immunity. Adopted from [START_REF] Spiering | Primer on the Immune System[END_REF].

Thus, the innate immune response plays a pivotal role in the defense against pathogens and is initiated through the pattern recognition receptors (PRRs). PRRs recognize pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) and endogenous dangerassociated molecular pattern (DAMPs), leading to the activation of host defense pathways that result in the clearance of the infection [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF]JANG et al., 2015;KUMAR;KAWAI;[START_REF] Kumar | Pathogen Recognition by the Innate Immune System[END_REF][START_REF] Mogensen | Pathogen Recognition and Inflammatory Signaling in Innate Immune Defenses[END_REF][START_REF] Roh | Damage-Associated Molecular Patterns in Inflammatory Diseases[END_REF][START_REF] Roh | Damage-Associated Molecular Patterns in Inflammatory Diseases[END_REF] , 2002;[START_REF] Wilson | Structure and mechanism of interleukin-1 beta converting enzyme[END_REF].

Furthermore, inflammasome activation triggers pyroptosis, an inflammatory form of cell death [START_REF] Bergsbaken | Pyroptosis: host cell death and inflammation[END_REF]FINK;[START_REF] Bergsbaken | Pyroptosis: host cell death and inflammation[END_REF][START_REF] Yang | Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors[END_REF][START_REF] Yuan | Inflammatory caspase-related pyroptosis: mechanism, regulation and therapeutic potential for inflammatory bowel disease[END_REF]. The role of NLRP1, NLRP3, NLRC4, NLRP6, NLRP7, NLRP12 and the PYHIN proteins AIM2 and IFI16

in assembling inflammasome and controlling the innate immune response against various microbes including different bacteria and the involvement of various bacterial factors have been intensively explored during the past decade [START_REF] Caneparo | The Absent in Melanoma 2-Like Receptor IFN-Inducible Protein 16 as an Inflammasome Regulator in Systemic Lupus Erythematosus: The Dark Side of Sensing Microbes[END_REF]LIN;ZHANG, 2017;[START_REF] Sá | Inflammasomes and dermatology[END_REF][START_REF] Sá | Inflammasomes and dermatology[END_REF][START_REF] Vladimer | Inflammasomes and host defenses against bacterial infections[END_REF]. Studies have shown that these inflammasomes form in response to various stimuli and are also subject to negative regulatory mechanisms, which cause the production of inflammatory cytokines to occur in a controlled manner after the onset of the inflammatory response (NAIK; DIXIT, 2010).

The NLRP3 inflammasome is the best characterized NLR so far. NLRP3, together with proteins ASC and caspase-1, form a cytoplasmic oligomeric complex known as the NLRP3

Introduction générale

Staphylococcus aureus (S. aureus) est une bactérie à Gram positif capable de coloniser l'hôte humain ou animal et de provoquer un large panel d'infections allant de bénignes à potentiellement mortelles telles que pneumonie, septicémie, endocardite, ostéomyélite, infections associées aux dispositifs médicaux internes, syndrome de choc toxique, abcès, ou mammite entre autres maladies (FITZGERALD, 2012;[START_REF] Foster | Medical Microbiology[END_REF][START_REF] Wertheim | The role of nasal carriage in Staphylococcus aureus infections[END_REF] Au cours des dernières années, notre compréhension de la manière dont le système immunitaire inné reconnaît les agents pathogènes a considérablement avancé [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF][START_REF] Nicholson | The immune system[END_REF]. Une invasion de microorganismes étrangers induit un spectre de réponses inflammatoires chez l'hôte infecté (Fig. 1). L'inflammation est classiquement décrite comme une réponse essentielle de la réponse immunitaire de l'hôte qui permet la survie pendant une infection ou une blessure et maintient l'homéostasie tissulaire dans diverses conditions nocives [START_REF] Chen | Inflammatory responses and inflammation-associated diseases in organs[END_REF][START_REF] Cruvinel | Sistema imunitário: Parte I. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória[END_REF]MEDZHITOV, 2010a;[START_REF] Rankin | Beyond Host Defense: Emerging Functions of the Immune System in Regulating Complex Tissue Physiology[END_REF][START_REF] Rankin | Beyond Host Defense: Emerging Functions of the Immune System in Regulating Complex Tissue Physiology[END_REF].

Le type de réponse inflammatoire dépend de la catégorie du déclencheur inflammatoire. Il a été démontré que les bactéries pathogènes sont reconnues par les récepteurs du système immunitaire inné, tels que les Toll-like receptors (TLR), entraînant l'induction de divers médiateurs inflammatoires comme les cytokines et les chimiokines inflammatoires, ainsi que les prostaglandines, qui agissent sur les tissus cibles [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF][START_REF] Chen | Inflammatory responses and inflammation-associated diseases in organs[END_REF][START_REF]Cytokine Profiling Plays a Crucial Role in Activating Immune System to Clear Infectious Pathogens[END_REF][START_REF] Turner | Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease[END_REF] , 2008, 2010a). Cependant, dans certaines infections chroniques, l'inflammation peut persister sans immunité adaptative [START_REF] Cruvinel | Sistema imunitário: Parte I. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória[END_REF]. , 1990). In blood agar, S. aureus forms beta-hemolytic colonies due to the production of hemolysins (α-toxin, β-toxin, γ-toxin and δ-toxin) [START_REF] Blair | Factors Determining the Pathogenicity of Staphylococci[END_REF][START_REF] Dinges | Exotoxins of Staphylococcus aureus[END_REF][START_REF] Dinges | Exotoxins of Staphylococcus aureus[END_REF][START_REF] Dinges | Exotoxins of Staphylococcus aureus[END_REF].

The first fully sequenced and annotated genomes were published in 2001 by Hiramatsu's group, comparing the genomes of two methicillin-resistant strains, Mu50 and N315 [START_REF] Kuroda | Whole genome sequencing of meticillin-resistant Staphylococcus aureus[END_REF]. This opened the way to numerous other sequencing projects, which led to great insights into our understanding of this major pathogen. Genomic analyses of animal and human strains show that the whole genome is about 2.82 Mb in size. The genome of S. aureus contains 2.872 genes encoding 2.767 proteins. The GC content of the genome was found to be 32.7%. The core genome of S. aureus consists of about 80% of conserved genes between strains, encoding essential metabolic and regulatory functions, as well as surface proteins involved in adhesion and surface architecture [START_REF] Baba | Genome Sequence of Staphylococcus aureus Strain Newman and Comparative Analysis of Staphylococcal Genomes: Polymorphism and Evolution of Two Major Pathogenicity Islands[END_REF][START_REF] Gillaspy | The Staphylococcus aureus NCTC 8325 Genome. Gram-Positive Pathogens[END_REF][START_REF] Lindsay | Genomic variation and evolution of Staphylococcus aureus[END_REF][START_REF] Lindsay | Staphylococcus aureus: superbug, super genome[END_REF].

The other predicted genes make up an "accessory" genome, comprising a collection of mobile genetic elements (MGEs), including bacteriophages, chromosomal cassette and pathogenicity islands [START_REF] Kuroda | Whole genome sequencing of meticillin-resistant Staphylococcus aureus[END_REF][START_REF] Lindsay | Genomic variation and evolution of Staphylococcus aureus[END_REF][START_REF] Lindsay | Staphylococcus aureus: superbug, super genome[END_REF][START_REF] Lindsay | Understanding the rise of the superbug: investigation of the evolution and genomic variation of Staphylococcus aureus[END_REF]. MGEs encode most of the virulence factors of S. aureus. The exchange of virulence factors that are transported by MGEs from a strain to another is a driving force in S. aureus evolution. The difference in the gene content of S. aureus strains is due to deletions of genes and insertions by MGEs.

Some regions of the central genome show inter-strain variability. The allelic variation of structural and functional genes of S. aureus arises from the random substitution of nucleotides, followed by selection, recombination and horizontal gene transfers (CROSSLEY et al., 2009;[START_REF] Feng | Evolution and pathogenesis of Staphylococcus aureus : lessons learned from genotyping and comparative genomics[END_REF][START_REF] Lindsay | Genomic variation and evolution of Staphylococcus aureus[END_REF][START_REF] Lindsay | Staphylococcus aureus: superbug, super genome[END_REF]. In addition to the chromosome, S. aureus strains can also contain plasmid elements encoding for resistance and virulence determinants (CROSSLEY et al., 2009;[START_REF] Fitzgerald | Evolutionary genomics of Staphylococcus aureus: insights into the origin of methicillinresistant strains and the toxic shock syndrome epidemic[END_REF][START_REF] Lindsay | Genomic variation and evolution of Staphylococcus aureus[END_REF][START_REF] Lindsay | Genomic variation and evolution of Staphylococcus aureus[END_REF][START_REF] Lindsay | Staphylococcus aureus: superbug, super genome[END_REF][START_REF] Lindsay | Understanding the rise of the superbug: investigation of the evolution and genomic variation of Staphylococcus aureus[END_REF].

General pathogenesis and clinical diseases 1.3.1 Pathogenesis

The development of S. aureus infections consists of five different stages: bacterial colonization, followed by local infection, dissemination, metastatic infection and toxinosis (GNANAMANI;

HARIHARAN; PAUL-SATYASEELA, 2017). S. aureus is often found in healthy carriage in mammals and birds. It is a frequent inhabitant of human retronasal ways, in which it may be part of the human microbiota without causing disease in the host (OTTO, 2010a).

S. aureus is a commensal and opportunistic pathogen that causes a wide range of acute infections (septicemia, bacteremia, pneumonia, meningitis) and chronic diseases of bones, joints, and skin in humans, ranging from mild to life-threatening infections (CHAMBERS, 1997;FITZGERALD, 2012;LOWY, 1998). Osteomyelitis is an inflammatory disease that accompanies bone destruction. It is commonly caused by bacteria. However, infections occurring through fungi or other parasites have also been reported (GOMES; PEREIRA; [START_REF] Gomes | Osteomyelitis: an overview of antimicrobial therapy[END_REF][START_REF] Urs | Fungal osteomyelitis of maxillofacial bones: Rare presentation[END_REF].

S. aureus is the most common infectious causative organism of osteomyelitis with more than 70% of occurrences. In terms of prevalence, it is followed by Gram-negative bacilli,

Streptococcus pneumoniae and Streptococcus pyogenes (CHIAPPINI; MASTRANGELO;

LAZZERI, 2016). These bacteria adhere to the bone matrix leading to the formation of biofilms.

In these biofilms, pathogens undergo complex metabolic changes that make these bacteria less sensitive, both to the host immune response and to antibiotics. The complex nature of biofilm makes treatment difficult leading to long-term antibiotic therapy, and to high morbidity and elevated cure costs for patients and hospitals [START_REF] Craft | Methicillin-resistant Staphylococcus aureus (MRSA): antibiotic-resistance and the biofilm phenotype[END_REF].

S. aureus is also a major animal pathogen. In dairy animals, such as cows and small ruminants, mastitis remains a major concern worldwide that affects animal health, and leads to huge economic losses in the dairy industry due to veterinary treatments, premature culling, and the change in milk quality [START_REF] Lescourret | Predictive model of mastitis occurrence in the dairy cow[END_REF][START_REF] Lescourret | Predictive model of mastitis occurrence in the dairy cow[END_REF][START_REF] Lescourret | Predictive model of mastitis occurrence in the dairy cow[END_REF]PETON;LE LOIR, 2014;[START_REF] Rainard | Knowledge gaps and research priorities in Staphylococcus aureus mastitis control[END_REF]. Although viruses, fungi or protozoa can cause mastitis, the most common causes are bacteria (AMIN; HAMOUDA; [START_REF] Amin | PCR assays for detecting major pathogens of mastitis in milk samples[END_REF][START_REF] Deb | Trends in diagnosis and control of bovine mastitis: a review[END_REF].

Escherichia coli is often associated with acute mastitis in bovine. In contrast, S. aureus most commonly causes subclinical mastitis that tends to become chronic in cows [START_REF] Günther | Differentiating Staphylococcus aureus from Escherichia coli mastitis: S. aureus triggers unbalanced immune-dampening and host cell invasion immediately after udder infection[END_REF][START_REF] Sears | Management and treatment of staphylococcal mastitis[END_REF][START_REF] Sears | Management and treatment of staphylococcal mastitis[END_REF][START_REF] Younis | Meta-Analysis of Transcriptional Responses to Mastitis-Causing Escherichia coli[END_REF][START_REF] Younis | Meta-Analysis of Transcriptional Responses to Mastitis-Causing Escherichia coli[END_REF][START_REF] Younis | Meta-Analysis of Transcriptional Responses to Mastitis-Causing Escherichia coli[END_REF].

Subclinical mastitis accounts for about 80% of all economic losses associated with mastitis, related to reduced milk production and quality, as well as treatment and prevention costs (PETROVSKI; TRAJCEV; [START_REF] Petrovski | A review of the factors affecting the costs of bovine mastitis[END_REF]. Massive antibiotic use in veterinary medicine is regarded as a major problem in terms of the emergence and spread of antibiotic-resistant strains [START_REF] Fair | Antibiotics and Bacterial Resistance in the 21st Century[END_REF][START_REF] Fair | Antibiotics and Bacterial Resistance in the 21st Century[END_REF]LI;[START_REF] Li | Bacteria antibiotic resistance: New challenges and opportunities for implant-associated orthopedic infections[END_REF]VENTOLA, 2015a). Prevention (e.g. antimastitis vaccine) is by far the best strategy. However, most vaccines tested to date do not allow the development of an efficient and protective defense against S. aureus mastitis [START_REF] Wallemacq | CD40 triggering induces strong cytotoxic T lymphocyte responses to heat-killed Staphylococcus aureus immunization in mice: a new vaccine strategy for staphylococcal mastitis[END_REF].

In human medicine, S. aureus causes huge problems such as nosocomial infections, notably because some strains are resistant to a wide variety of antimicrobials. Penicillin was discovered by Alexander Fleming in 1928 and was first administered to infected patients as a chemotherapeutic agent in 1941. The resistance to penicillin in S. aureus rapidly appeared in 1942, followed by more general mechanisms of resistance against the family of beta-lactams (CHAIN et al., 1940;FLEMING, 1929a;[START_REF] Fletcher | First clinical use of penicillin[END_REF][START_REF] Rammelkamp | Resistance of Staphylococcus aureus to the Action of Penicillin[END_REF][START_REF] Rammelkamp | Resistance of Staphylococcus aureus to the Action of Penicillin[END_REF]VENTOLA, 2015a). The resistance of S. aureus to beta-lactams and other types of antibiotics has contributed to a decrease in the number of effective drugs against this microorganism (LEVY; MARSHALL, 2004).

Virulence factors

The importance and success of S. aureus as a pathogen relies on the combination of toxinsmediated virulence, invasiveness and antimicrobial resistance (LE LOIR;BARON;[START_REF] Le Loir | Staphylococcus aureus and food poisoning[END_REF], as well as cell-cell interaction, immune response evasion, and tissue damage [START_REF] Feng | Evolution and pathogenesis of Staphylococcus aureus : lessons learned from genotyping and comparative genomics[END_REF][START_REF] Gordon | Pathogenesis of Methicillin-Resistant Staphylococcus aureus Infection[END_REF][START_REF] Gordon | Pathogenesis of Methicillin-Resistant Staphylococcus aureus Infection[END_REF]. The pathogenicity of this bacterium is due to both the diversity of its virulence factors and its ability to express its virulence in various host environments [START_REF] Peacock | Virulent combinations of adhesin and toxin genes in natural populations of Staphylococcus aureus[END_REF].

The repertoire of S. aureus virulence factors is extensive and comprises secreted and structural products, including membrane proteins, that direct the process of pathogenesis [START_REF] Gordon | Pathogenesis of Methicillin-Resistant Staphylococcus aureus Infection[END_REF][START_REF] Gordon | Pathogenesis of Methicillin-Resistant Staphylococcus aureus Infection[END_REF]. Due to the impact of this bacterium on human and animal health, the structure and function of its virulence factors, its interaction with the host and the molecular mechanisms involved in resistance to antibiotics have been extensively studied [START_REF] Hecker | A proteomic view of cell physiology and virulence of Staphylococcus aureus[END_REF][START_REF] Lindsay | Genomic variation and evolution of Staphylococcus aureus[END_REF][START_REF] Lindsay | Staphylococcus aureus: superbug, super genome[END_REF].

Based on their mechanism of action and role in pathogenesis, staphylococcal virulence factors are classified as represented in [START_REF] Wann | The fibronectin-binding MSCRAMM FnbpA of Staphylococcus aureus is a bifunctional protein that also binds to fibrinogen[END_REF].

The fibronectin binding proteins FnBPA and FnBPB of S. aureus are responsible for the Fn adhesion process into the host cell surface [START_REF] Foster | Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus[END_REF][START_REF] Foster | Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus[END_REF]HÖÖK, 1998;JOSSE;LAURENT;DIOT, 2017). Binding to Fn plays a major role in bacteria-cell interactions, but S. aureus also produces components able to bind to Fg and collagen (Hienz et al, 1996).

The binding to Fg is mainly mediated by Clumping factor A and B (ClfA) and (ClfB) proteins, which allow bacterial adhesion to blood plasma and biomaterials exposed to human or animal blood [START_REF] Foster | Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus[END_REF]HÖÖK, 1998).

All these factors allow S. aureus to bind to the components of the extracellular matrix, facilitating the infection of several cell types, such as epithelial and immunological cells (OTTO, 2010a;[START_REF] Pietrocola | Staphylococcus aureus Manipulates Innate Immunity through Own and Host-Expressed Proteases[END_REF]. S. aureus also produces a variety of other secreted virulence factors including toxins (leucocidins, haemolysins, exfoliative toxins, enterotoxins, (TSST-1) -Toxic shock syndrome toxin -1), enzymes, and a group of factors encompassing the phenol-soluble modulins (PSM) (GNANAMANI; HARIHARAN; PAUL-SATYASEELA, 2017; OTTO, 2014a). PSMs may play an important role in the different stages of pathogenesis, including adhesion, invasion, persistence, evasion, and death of host cells (GORDON; [START_REF] Gordon | Pathogenesis of Methicillin-Resistant Staphylococcus aureus Infection[END_REF][START_REF] Otto | Phenol-soluble modulins[END_REF].

The expression of virulence factors is tightly controlled in S. aureus. Following the establishment of the colonization, S. aureus represses the expression of surface proteins associated with adhesion and internalization and produces toxins and other secreted virulence factors responsible for bacterial dispersion and, consequently, the symptoms of infections [START_REF] Novick | Autoinduction and signal transduction in the regulation of staphylococcal virulence[END_REF][START_REF] Novick | Autoinduction and signal transduction in the regulation of staphylococcal virulence[END_REF][START_REF] Novick | Quorum Sensing in Staphylococci[END_REF]. The different virulent factors expressed are controlled by a definite locus named the accessory gene regulator (agr) and the staphylococcal accessory regulator (sar) [START_REF] Jarraud | Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease[END_REF].

The agr system is formed by a two-component system, which is responsible for the regulation of several virulence factors associated with the infectious processes (NOVICK; [START_REF] Novick | Quorum Sensing in Staphylococci[END_REF][START_REF] Otto | Quorum-sensing control in Staphylococci -a target for antimicrobial drug therapy[END_REF]. The agr operon is composed of four genes (agrA, agrB, agrC and agrD)

that respectively produce AgrA, AgrB, AgrC and the auto-inducible peptide (AIP) (Fig. 2).

Virulence expression is based on a quorum-sensing mechanism. During bacterial growth, AIP accumulates in the surrounding medium and when AIP concentration reaches a given threshold, it induces a shift of expression resulting in a repression of the genes encoding surface proteins and an induction of genes encoding secreted proteins (toxins). AIP, which is processed and secreted by AgrB, binds to the membrane receptor formed by AgrC, which phosphorylates or dephosphorylates AgrA. AgrA functions as a signal transducer of the AIP binding to the ArgC receptor, activating the expression of the agr operon and the RNAIII gene.

The latter expresses the RNAIII, a regulator of several genes related to virulence in S. aureus OTTO, 2015;[START_REF] Novick | Autoinduction and signal transduction in the regulation of staphylococcal virulence[END_REF][START_REF] Novick | Autoinduction and signal transduction in the regulation of staphylococcal virulence[END_REF][START_REF] Novick | Quorum Sensing in Staphylococci[END_REF]. Cytotoxins and enzymes can be seen as a means to convert host molecules into nutrients for bacterial growth or to cleave antibiotic molecules (OTTO, 2014a). Hemolysins (α, β, γ, and δ), nucleases, proteases, lipases, hyaluronidase, and collagenase can indeed provide nutrients for the bacterium from the host material. Besides, enzymes such as β-lactamases are responsible for inhibiting the activity of antibiotics derived from β-lactam rings. Among the toxins produced by S. aureus, superantigens are potent and non-specific immune response inducers [START_REF] Makhlin | Staphylococcus aureus ArcR Controls Expression of the Arginine Deiminase Operon[END_REF][START_REF] Tokajian | Toxins and Antibiotic Resistance in Staphylococcus aureus Isolated from a Major Hospital in Lebanon[END_REF]. Also referred to as pyrogenic toxins, they include staphylococcal enterotoxins (more than 25 SEs have been described so far) and toxic shock syndrome toxin-1 (TSST-1) [START_REF] Choi | Interaction of Staphylococcus aureus toxin "superantigens" with human T cells[END_REF][START_REF] Dinges | Exotoxins of Staphylococcus aureus[END_REF][START_REF] Dinges | Exotoxins of Staphylococcus aureus[END_REF][START_REF] Dinges | Exotoxins of Staphylococcus aureus[END_REF][START_REF] Krakauer | Staphylococcal Superantigens: Pyrogenic Toxins Induce Toxic Shock[END_REF][START_REF] Marrack | The staphylococcal enterotoxins and their relatives[END_REF][START_REF] Marrack | The staphylococcal enterotoxins and their relatives[END_REF]. Quorum sensing system agr encodes gene products that ultimately promote further transcription of agr locus in a process called autoactivation. According to (NOVICK; GEISINGER, 2008).

(LE;

Phenol-soluble modulins (PSMs)

In 1999, Seymour Klebanoff described the so-called Phenol-soluble modulins (PSM), a group of molecules contained in the supernatant of Staphylococcus epidermidis, isolated by size exclusion chromatography with the use of a solvent containing phenol (MEHLIN; HEADLEY; [START_REF] Mehlin | An inflammatory polypeptide complex from Staphylococcus epidermidis: isolation and characterization[END_REF][START_REF] Otto | Quorum-sensing control in Staphylococci -a target for antimicrobial drug therapy[END_REF]. PSMs are part of a family of short amphipathic, α-helical peptides, which are produced by various staphylococcal strains and which have great importance in the virulence process of S. aureus (Fig. 3) [START_REF] Gonzalez | Phenol soluble modulin (PSM) variants of community-associated methicillin-resistant Staphylococcus aureus (MRSA) captured using mass spectrometry-based molecular networking[END_REF][START_REF] Otto | Phenol-soluble modulins[END_REF]PESCHEL;[START_REF] Peschel | Phenol-soluble modulins and staphylococcal infection[END_REF]. PSMs participate in the formation of biolfims. PSMs may be antimicrobial agents, they also act to induce cell lysis, cytokine production through the FPR2 receptor, and participate in the S. aureus dispersion process.

PSMs were described as a proinflammatory "complex" of seven PSMs, including the shortest α type with approximately 20-25 amino acids in length (four PSMα1-PSMα4 peptides), the longest β type with approximately 43-45 amino acids (PSMβ1 and PSMβ2) and δ-toxin peptides ranging in length from 25 to 26 amino acids [START_REF] Cheung | Phenol-soluble modulins -critical determinants of staphylococcal virulence[END_REF][START_REF] Cheung | Functional characteristics of the Staphylococcus aureus δ-toxin allelic variant G10[END_REF][START_REF] Mehlin | An inflammatory polypeptide complex from Staphylococcus epidermidis: isolation and characterization[END_REF][START_REF] Mehlin | An inflammatory polypeptide complex from Staphylococcus epidermidis: isolation and characterization[END_REF][START_REF] Mehlin | An inflammatory polypeptide complex from Staphylococcus epidermidis: isolation and characterization[END_REF]. The PSM-encoding genes scattered at different locations in Likewise several other major staphylococcal exotoxins, PSMs are positively regulated by agr, the global regulatory quorum sensing system [START_REF] Gomes-Fernandes | Accessory gene regulator (Agr) functionality in Staphylococcus aureus derived from lower respiratory tract infections[END_REF][START_REF] Queck | RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus[END_REF][START_REF] Xu | The Agr Quorum Sensing System Represses Persister Formation through Regulation of Phenol Soluble Modulins in Staphylococcus aureus[END_REF]. The PSMs are agr-regulated through AgrA, not by RNAIII, which modulates several other virulence genes in S. aureus [START_REF] Marroquin | MroQ Is a Novel Abi-Domain Protein That Influences Virulence Gene Expression in Staphylococcus aureus via Modulation of Agr Activity[END_REF]PESCHEL;[START_REF] Peschel | Phenol-soluble modulins and staphylococcal infection[END_REF][START_REF] Tan | Therapeutic Targeting of the Staphylococcus aureus Accessory Gene Regulator (agr) System[END_REF]. PSMs have a pronounced ability to lyse human leukocytes and other cell types, to stimulate an inflammatory response and the formation of biofilm [START_REF] Cheung | Phenol-soluble modulins -critical determinants of staphylococcal virulence[END_REF][START_REF] Cheung | Functional characteristics of the Staphylococcus aureus δ-toxin allelic variant G10[END_REF]PESCHEL;[START_REF] Peschel | Phenol-soluble modulins and staphylococcal infection[END_REF][START_REF] Towle | Solution Structures of Phenol-Soluble Modulins α1, α3, and β2, Virulence Factors from Staphylococcus aureus[END_REF]. aureus infection, in addition to elevating leukocyte death in vitro [START_REF] Wang | Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA[END_REF]. The dissemination capacity of S. aureus and S. epidermidis, associated with biofilm formation, is directly related to the production of PSMs (LE; OTTO, 2015;[START_REF] Otto | Staphylococcus epidermidis: a major player in bacterial sepsis[END_REF][START_REF] Queck | RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus[END_REF][START_REF] Tsompanidou | Distinct Roles of Phenol-Soluble Modulins in Spreading of Staphylococcus aureus on Wet Surfaces[END_REF][START_REF] Wang | Staphylococcus epidermidis surfactant peptides promote biofilm maturation and dissemination of biofilm-associated infection in mice[END_REF].

The ability to induce cellular lysis is the most relevant feature of these PSMs [START_REF] Cheung | Phenol-soluble modulins -critical determinants of staphylococcal virulence[END_REF]. The action of the PSMs is not specific and is independent of receptors. 2012;[START_REF] Chu | Staphylococcus aureus Phenol-Soluble Modulins α1-α3 Act as Novel Toll-Like Receptor (TLR) 4 Antagonists to Inhibit HMGB1/TLR4/NF-κB Signaling Pathway[END_REF][START_REF] Da | Phenol-Soluble Modulin Toxins of Staphylococcus haemolyticus[END_REF][START_REF] Otto | Staphylococcus epidermidis: a major player in bacterial sepsis[END_REF].

Among other activities, PSMs induce neutrophil chemotaxis, oxygen radical production, intracellular calcium influx, and release of proinflammatory cytokines such as Interleukin-8 (IL-8), a member of the CXC subfamily of chemoattractant cytokines [START_REF] Henkels | IL-8-induced neutrophil chemotaxis is mediated by Janus kinase 3 (JAK3)[END_REF][START_REF] Parker | Innate Immune Signaling Activated by MDR Bacteria in the Airway[END_REF][START_REF] Wang | Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA[END_REF]. The ability of the PSMs to induce an inflammatory response classify them as pathogen-associated molecular patterns (PAMPs), which are a group of microbial molecules that alert the host organism when a pathogen infection occurs [START_REF] Bianchi | PAMPs and alarmins: all we need to know about danger[END_REF][START_REF] Cheung | Phenol-soluble modulins -critical determinants of staphylococcal virulence[END_REF]. In addition, PSMs have been shown to be capable to decreasie the expression of proinflammatory cytokines such as IL-32, IL-6 and IL-8 (DEPLANCHE et al., 2016).

2. The inflammasome, a primordial complex of innate immunity

Mechanisms of the inflammatory response

The immune system is formed by a series of effector mechanisms which can destroy a variety of invading pathogenic organisms belonging to four main groups fungi, bacteria, parasites, and viruses [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF][START_REF] Mesquita Júnior | Sistema imunitário -parte II: fundamentos da resposta imunológica mediada por linfócitos T e B[END_REF][START_REF] Nicholson | The immune system[END_REF].

The immune system is composed of two types of responses: antigen-specific adaptive immune response and the innate immune response recognizing microbial associated molecular patterns (MAMP's) (RYU, 2017). Innate immune response described as the organism's first line of defense is of primary importance in the immediate recognition and elimination of invasive microorganisms (ESPINOSA; RIVERA, 2016). The innate immune response consists basically of physical and chemical barriers, reactive oxygen species (ROS), innate immune cells, and soluble mediators such as the complement system, innate antibodies, and associated cytokines and recognizes pathogen-associated molecular patterns (PAMPs) [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF][START_REF] Cruvinel | Sistema imunitário: Parte I. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória[END_REF].

PAMPs are recognized by pattern recognition receptors (PRRs), expressed primarily in cells of the innate immune system (KUMAR; KAWAI; [START_REF] Kumar | Pathogen Recognition by the Innate Immune System[END_REF][START_REF] Mogensen | Pathogen Recognition and Inflammatory Signaling in Innate Immune Defenses[END_REF]. PRRs can also recognize host molecules that contain damage-associated molecular patterns (DAMPs), these molecules are released when a pathogenic organism is able to multiply in the host as a result of the infection process or as a result of a tissue injury (sterile inflammation), these microorganisms are usually absent from the extracellular environment (GRAZIOLI; [START_REF] Grazioli | Mitochondrial Damage-Associated Molecular Patterns: From Inflammatory Signaling to Human Diseases[END_REF][START_REF] Land | The Role of Damage-Associated Molecular Patterns (DAMPs) in Human Diseases: Part II: DAMPs as diagnostics, prognostics and therapeutics in clinical medicine[END_REF][START_REF] Rani | Damage-associated molecular patterns (DAMPs) released after burn are associated with inflammation and monocyte activation[END_REF]. To initiate inflammation, coordinated action between PAMPs/MAMPs and DAMPs is required. Inflammation is induced by both the recognition of foreign molecules and by cell damage [START_REF] Di | The therapeutic potential of modifying inflammasomes and NOD-like receptors[END_REF][START_REF] Gasteiger | Cellular Innate Immunity: An Old Game with New Players[END_REF][START_REF] Roh | Damage-Associated Molecular Patterns in Inflammatory Diseases[END_REF][START_REF] Roh | Damage-Associated Molecular Patterns in Inflammatory Diseases[END_REF].

The inflammatory pathway is composed of inducers, sensors, inflammatory mediators and target tissues in which mediators act (MEDZHITOV, 2010a). In the last decades, two types of inflammatory conditions have been identified: acute and chronic reaction (MEDZHITOV, 2010a;ZHOU;HONG;[START_REF] Zhou | Triptolide Attenuates Inflammatory Response in Membranous Glomerulo-Nephritis Rat via Downregulation of NF-κB Signaling Pathway[END_REF]. The acute inflammatory response is caused by infection or tissue injury leading to the migration of neutrophils and monocytes/macrophages from the blood to the tissue [START_REF] Chu | Staphylococcus aureus Phenol-Soluble Modulins α1-α3 Act as Novel Toll-Like Receptor (TLR) 4 Antagonists to Inhibit HMGB1/TLR4/NF-κB Signaling Pathway[END_REF][START_REF] Cruvinel | Sistema imunitário: Parte I. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória[END_REF][START_REF] Selders | An overview of the role of neutrophils in innate immunity, inflammation and host-biomaterial integration[END_REF]TAKEUCHI;[START_REF] Takeuchi | Pattern Recognition Receptors and Inflammation[END_REF]. This inflammatory response involves receptors (PRRs) located on the surface of resident macrophages or mast cells, and their activation leads to the production of inflammatory mediators such as cytokines and chemokines (CHEN et al., 2017a;[START_REF] Cruvinel | Sistema imunitário: Parte I. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória[END_REF].

During the inflammatory response, leukocytes are activated and induce the release of toxic substances contained in their granules like reactive oxygen species (ROS) and reactive nitrogen species (RNS) [START_REF] Chertov | Leukocyte granule proteins mobilize innate host defenses and adaptive immune responses[END_REF][START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF]NGUYEN;[START_REF] Nguyen | Neutrophils to the ROScue: Mechanisms of NADPH Oxidase Activation and Bacterial Resistance[END_REF][START_REF] Nguyen | Neutrophils to the ROScue: Mechanisms of NADPH Oxidase Activation and Bacterial Resistance[END_REF]. When pathogens can escape from the innate immune system, the adaptive system comes into action [START_REF] Gasteiger | Cellular Innate Immunity: An Old Game with New Players[END_REF][START_REF] Nicholson | The immune system[END_REF]. To maintain homeostasis, cell death acts continuously as a type of inflammatory response (ARBOGAST; GROS, 2018). Four types of cell death are known: apoptosis and pyroptosis are related to the action of caspases, whereas autophagy and necrosis act as an alarm system that indicates that the cell can no longer cope with aggression (DELOU; BIASOLI; [START_REF] Delou | The Complex Link between Apoptosis and Autophagy: a Promising New Role for RB[END_REF][START_REF] Fan | The cellular decision between apoptosis and autophagy[END_REF][START_REF] Fan | The cellular decision between apoptosis and autophagy[END_REF][START_REF] Yang | Programmed cell death and its role in inflammation[END_REF]. Thus, the error in the elimination of the infectious agent by the immune system can lead to a persistent inflammatory process inducing a chronic inflammatory process contributing to a variety of chronic inflammatory diseases (CHEN et al., 2017a;[START_REF] Cruvinel | Sistema imunitário: Parte I. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória[END_REF][START_REF] Ferrero-Miliani | Chronic inflammation: importance of NOD2 and NALP3 in interleukin-1? generation[END_REF].

Inducers of the inflammatory response

Inflammatory inducers are endogenous or exogenous signals that initiate an inflammatory response through immune system receptors , such as Toll-like receptors (TLRs), leading to the production of mediators such as interleukins and chemokines that act on target tissues [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF]MEDZHITOV, 2010a).

Exogenous inducers may be of microbial origin or not, such as toxic compounds and foreign bodies, allergens, and may be classified in PAMPs and virulence factors such as pore-forming exotoxins produced by Gram-positive bacteria, which are detected by the NLRP3 inflammasome (NACHT -leucine-rich repeat-and pyrin-domain-containing protein) (GREANEY; LEPPLA; [START_REF] Greaney | Bacterial Exotoxins and the Inflammasome[END_REF][START_REF] Latz | The inflammasomes: mechanisms of activation and function[END_REF][START_REF] Mariathasan | Cryopyrin activates the inflammasome in response to toxins and ATP[END_REF][START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF]. These exogenous inducers are not restricted to pathogens, but may be generated from an alternative detection pathway, through components induced by damaged tissues or cell death [START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF]. Endogenous inducers result from stressed, damaged, or malfunctioning tissues involved in acute inflammatory responses and tissue injury (CHEN et al., 2017a;[START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF]. Another class of endogenous inducers related to chronic inflammatory responses in particular includes uric acid crystals and oxidized lipoproteins (MEDZHITOV, 2008).

Mediators of the inflammatory response

Mediators of the inflammatory response are either secreted by the cells or are derived from plasma proteins, and may circulate in the blood plasma as inactive precursors and have higher concentrations during the acute phase of the inflammatory process [START_REF] Abdulkhaleq | The crucial roles of inflammatory mediators in inflammation: A review[END_REF][START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF]. These mediators can be classified according to their biochemical characteristics into seven groups: amines and vasoactive peptides, fragments derived from complement components, lipid mediators, cytokines, chemokines and proteolytic enzymes [START_REF] Cruvinel | Sistema imunitário: Parte I. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória[END_REF][START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF]. Several mediators have a direct effect on the target tissues, acting, for example, in the recruitment of leukocytes and by inducing the production of other mediators (CHEN et al., 2017a;MEDZHITOV, 2010b).

The production of inflammatory mediators, such as cytokines, plays a crucial role, which is regulated by anti-inflammatory signals. Cytokines are the main mediators of the inflammatory response and are small proteins secreted by cells involved in the regulation of inflammation, tissue repair and hematopoiesis [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF].

Cytokines act in various biological processes, and are involved in conserved signaling pathways responsible for controlling various types of cellular response, such as apoptosis and cell differentiation [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF]CHEN et al., 2017a). There are over fifty cytokines organized into several classes, which are classified into groups: Interleukins, tumor necrosis factors (TNF), interferons (IFN), colony stimulating factors (CSF), transforming growth factors (TGF) and chemokines [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF]. Among the proinflammatory cytokines are:

TNF, IL-1, IL-12, IL-18 and IFN-γ; and among the anti-inflammatory cytokines are IL-4, IL-10, IL-13 and TGF-β (CARRILLO et al., 2017). However, the family of interleukin (IL) -1 ligands and receptors stands out among cytokines due to the important role they play in acute and chronic inflammations (DINARELLO, 2009a(DINARELLO, , 2018a)).

IL-1β

2.4.1 The maturation of IL-1β

The concept of interleukin (IL) appeared in 1979 and has defined a protein capable of acting as a communication signal between different populations of leukocytes (A. DINARELLO, 2017;DINARELLO, 2009a). IL-1 family members are responsible for severe attacks in the context of systemic or local inflammation and contribute for many chronic diseases (CHEN et al., 2017a).

Inflammatory processes related to a dysregulated production of IL-1 are involved in the pathophysiology of several common diseases such as atherosclerosis, osteoarthritis, metabolic syndromes and type 2 diabetes (type 2 diabetes or type 2 diabetes mellitus) (A.

DINARELLO, 2017). However, numerous biological properties of the IL-1 family are still nonspecific (DINARELLO, 2018a).

Currently, there are 11 members of the IL-1 family (IL-1α, IL-1β, IL-1Rα, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, and IL-IL-38) (GUAN; ZHANG, 2017), and 10 members of the IL-1 family of receptors (IL-1R1 to ILR10) [START_REF] Boraschi | The family of the interleukin-1 receptors[END_REF]. Among the IL-1 family, IL-1β, also known as IL-1F2, is one of the most characterized. It is considered as a key pro-inflammatory cytokine, mainly produced by myeloid cells, inducing the physiological, hematological, metabolic and immunological characteristics of the host after infection, trauma or immunological activation (DINARELLO, 2018a).

The gene structure, expressional regulation, and function of IL-1β are evolutionarily well conserved [START_REF] Palomo | The interleukin (IL)-1 cytokine family -Balance between agonists and antagonists in inflammatory diseases[END_REF], and gene encoding it is not constitutively expressed (DINARELLO, 2018a;[START_REF] Yin | Inflammasomes are Differentially Expressed in Cardiovascular and other Tissues[END_REF][START_REF] Zhu | Dissemination of an Enterococcus Inc18-Like vanA Plasmid Associated with Vancomycin-Resistant Staphylococcus aureus[END_REF][START_REF] Zhu | Cutting Edge: Distinct Regulatory Mechanisms Control Proinflammatory Cytokines IL-18 and IL-1β[END_REF]. Thus, in response to endogenous or exogenous inflammatory agents, such as Toll-like receptor (TLRs) ligands, which are responsible for the recognition of a host of PAMPs, IL-1β is synthesized as a procytokine (pro-IL-1β), having a molecular weight of 31-kDa. Pro-IL-1β is then cleaved by caspase-1 at two sites related to the sequence: Asp27-Gly28 (site 1) and Asp116-Ala117 (site 2) with a molecular weight of 17.5-kDa, resulting in mature IL-1β generation [START_REF] Afonina | Proteolytic Processing of Interleukin-1 Family Cytokines: Variations on a Common Theme[END_REF][START_REF] Amarante-Mendes | Pattern Recognition Receptors and the Host Cell Death Molecular Machinery[END_REF][START_REF] Cameron | Amino acid sequence analysis of human interleukin 1 (IL-1). Evidence for biochemically distinct forms of IL-1[END_REF][START_REF] Cerretti | Molecular cloning of the interleukin-1 beta converting enzyme[END_REF]DINARELLO, 2018a;[START_REF] Guarda | Regulation of inflammasome activity[END_REF][START_REF] Guarda | Regulation of inflammasome activity[END_REF][START_REF] Kwak | Intracellular interleukin (IL)-1 family cytokine processing enzyme[END_REF]SCHRODER;TSCHOPP, 2010). Pro-IL-1β accumulates in the cytosol [START_REF] Semino | Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways[END_REF][START_REF] Stoffels | ATP-Induced IL-1Î 2 Specific Secretion: True Under Stringent Conditions[END_REF].

IL-1β maturation can occur through two possible levels of activation: One induced by NF-κBmediated leading to pro-IL-1β expression and the second option by of the proteolytic cleavage of caspase-1 and subsequent maturation of pro-IL-1β (NAIK; [START_REF] Naik | Modulation of Inflammasome Activity for the Treatment of Auto-inflammatory Disorders[END_REF][START_REF] Yang | Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors[END_REF].

Studies indicate that caspase-8 might be able to cleave pro-IL-1β during immune responses (LATZ; XIAO; STUTZ, 2013b).

The cleavage and thus maturation of IL-1β also appear to take place out of the cell and could involve other proteases that can cleave IL-1β but are present in the extracellular inflammatory environment [START_REF] Giuliani | The P2X7 Receptor-Interleukin-1 Liaison[END_REF]LOPEZ-CASTEJON;BROUGH, 2011;[START_REF] Stehlik | Multiple interleukin-1β-converting enzymes contribute to inflammatory arthritis[END_REF].

Extracellular processing of pro-IL-1β is not novel and other proteases derived from neutrophils and mast cells have been implicated to be present in pro-inflammatory environments [START_REF] Afonina | Proteolytic Processing of Interleukin-1 Family Cytokines: Variations on a Common Theme[END_REF][START_REF] Bent | Interleukin-1 Beta-A Friend or Foe in Malignancies[END_REF][START_REF] Dautova | Calcium phosphate particles stimulate interleukin-1β release from human vascular smooth muscle cells: A role for spleen tyrosine kinase and exosome release[END_REF]. Several studies have reported that exosomes were involved in the transfer of fully functional cytokines and growth factors [START_REF] Cossetti | Extracellular vesicles from neural stem cells transfer IFN-γ via Ifngr1 to activate Stat1 signaling in target cells[END_REF][START_REF] Dautova | Calcium phosphate particles stimulate interleukin-1β release from human vascular smooth muscle cells: A role for spleen tyrosine kinase and exosome release[END_REF][START_REF] Webber | Cancer exosomes trigger fibroblast to myofibroblast differentiation[END_REF].

Excretion of IL-1β

The excretion process of IL-1β is not fully understood. Several secretory pathways have been proposed: exocytosis of granules derived from lysosomes; the release of exosomes derived from multi-vesicular bodies and the release of microparticles from the plasma membrane [START_REF] Andrei | The Secretory Route of the Leaderless Protein Interleukin 1β Involves Exocytosis of Endolysosome-related Vesicles[END_REF][START_REF] Di | The therapeutic potential of modifying inflammasomes and NOD-like receptors[END_REF].

IL-1β secretion is also strongly associated with autophagy, an unconventional process (DANIELS; BROUGH, 2017). This process is independent of the endoplasmic reticulum and the Golgi apparatus and involves the autodigestion function of the cells by autophagy, which leads to the release of cellular content (DANIELS; [START_REF] Daniels | Unconventional Pathways of Secretion Contribute to Inflammation[END_REF][START_REF] Kuroda | Whole genome sequencing of meticillin-resistant Staphylococcus aureus[END_REF].

The role of autophagy in IL-β secretion is complex. Several studies suggest a tonic inhibition of IL-1β secretion both by the degradation of pro-IL-1β and by the negative regulation of inflammasome NLRP3 [START_REF] Harris | Autophagy Controls IL-1β Secretion by Targeting Pro-IL-1β for Degradation[END_REF]. However, additional research proposes that mature IL-1β is actively packaged in vesicles and secreted after autophagy stimulation [START_REF] Dupont | Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β: Autophagy-based unconventional secretory pathway[END_REF][START_REF] Guan | Recent Advances: The Imbalance of Cytokines in the Pathogenesis of Inflammatory Bowel Disease[END_REF]ZHANG, 2017).

The regulation of IL-1β

IL-1β participates in most events involved in the activation and regulation of inflammatory response (CHEN et al., 2017a). IL-1β can stimulate other cytokines such as IL-6, TNF-α, IL-1α, granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte-colony stimulating factor (G-CSF) [START_REF] Bhattacharya | Dual Role of GM-CSF as a Pro-Inflammatory and a Regulatory Cytokine: Implications for Immune Therapy[END_REF]DINARELLO, 2018a;[START_REF] Söderquist | Granulocyte colony-stimulating factor (G-CSF) and interleukin (IL)-8 in sera from patients with Staphylococcus aureus septicemia[END_REF]. It is also implicated in the release of neutrophils (polymorphonuclear cells), activation of lymphocytes, differentiation of Th17 cells and activation of dendritic cells [START_REF] Bent | Interleukin-1 Beta-A Friend or Foe in Malignancies[END_REF][START_REF] Costa | Recent advances on the crosstalk between neutrophils and B or T lymphocytes[END_REF][START_REF] Pelletier | Evidence for a cross-talk between human neutrophils and Th17 cells[END_REF].

IL-1β also plays a role in the secretion of vascular endothelial growth factor (VEGF), reactive oxygen species (ROS) and reactive nitrogen species (RNS) [START_REF] Papiewska-Pająk | Vascular endothelial growth factor-D modulates oxidant-antioxidant balance of human vascular endothelial cells[END_REF][START_REF] Roberts | Toxicological and pathophysiological roles of reactive oxygen and nitrogen species[END_REF]. IL-1β is an essential cytokine for the host immune system as it also participates in the activation and differentiation of T cells, B cells and natural killer (NK) cells (CHEN;KANG;[START_REF] Chen | The independence of and associations among apoptosis, autophagy, and necrosis[END_REF][START_REF] Cruvinel | Sistema imunitário: Parte I. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória[END_REF][START_REF]Cytokine Profiling Plays a Crucial Role in Activating Immune System to Clear Infectious Pathogens[END_REF].

This proinflammatory mediator is generated at lesion sites and coordinates the recruitment of leukocytes to neutralize and phagocyte pathogens, being one of the first cytokines to be secreted during the early stages of inflammation (CHEN;KANG;[START_REF] Chen | The independence of and associations among apoptosis, autophagy, and necrosis[END_REF][START_REF] Cruvinel | Sistema imunitário: Parte I. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória[END_REF]SCHRODER;TSCHOPP, 2010).

Furthermore, IL-1β, IL-6 and tumor necrosis factor α (TNFα) are considered the most important cytokines of inflammation in bone physiology and pathology [START_REF] Kwan Tat | IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology[END_REF][START_REF] Romas | Involvement of receptor activator of NFκB ligand and tumor necrosis factor-α in bone destruction in rheumatoid arthritis[END_REF][START_REF] Romas | Involvement of receptor activator of NFκB ligand and tumor necrosis factor-α in bone destruction in rheumatoid arthritis[END_REF][START_REF] Romas | Involvement of receptor activator of NFκB ligand and tumor necrosis factor-α in bone destruction in rheumatoid arthritis[END_REF]. The balance of cell activity, such as osteoblasts that are located on the bone surface and are derived from mesenchymal progenitors (BLAIR, 1998), and osteoclasts that are multinucleated cells derived from the myeloid lineage [START_REF] Blair | Osteoclastic bone resorption by a polarized vacuolar proton pump[END_REF], has been reported as essential for the maintenance of bone homeostasis and its renewal. Any imbalance in the relative levels of activity of these two cell types may induce bone pathology (HENDERSON;[START_REF] Henderson | Hard labour: bacterial infection of the skeleton[END_REF]. S. aureus infection can cause such imbalance, mediated in part by the inflammatory response [START_REF] Rasigade | PSMs of hypervirulent Staphylococcus aureus act as intracellular toxins that kill infected osteoblasts[END_REF].

Osteoblasts and osteoclasts interact permanently. The osteoblasts can identify different danger signs and modulate the differentiation and activity of osteoclasts (HENDERSON;[START_REF] Henderson | Hard labour: bacterial infection of the skeleton[END_REF][START_REF] Matsuo | Osteoclast-osteoblast communication[END_REF][START_REF] Matsuo | Osteoclast-osteoblast communication[END_REF]. A key interaction in the regulation of osteoclastogenesis occurs between the receptor activator of nuclear factor kappa B (RANK) expressed by osteoclast precursors and its ligand-expressed RANKL by osteoblasts (LIU; ZHANG, 2015; LORENZO; HOROWITZ; [START_REF] Lorenzo | Osteoimmunology: Interactions of the Bone and Immune System[END_REF][START_REF] Roggia | Up-regulation of TNF-producing T cells in the bone marrow: A key mechanism by which estrogen deficiency induces bone loss in vivo[END_REF][START_REF] Romas | Involvement of receptor activator of NFκB ligand and tumor necrosis factor-α in bone destruction in rheumatoid arthritis[END_REF][START_REF] Romas | Involvement of receptor activator of NFκB ligand and tumor necrosis factor-α in bone destruction in rheumatoid arthritis[END_REF][START_REF] Romas | Involvement of receptor activator of NFκB ligand and tumor necrosis factor-α in bone destruction in rheumatoid arthritis[END_REF].

In this context, IL-1β stands out as a strong stimulator in vitro and in vivo of bone resorption through the regulation of RANKL that stimulates osteoclastogenesis [START_REF] Luo | TNF-α and RANKL promote osteoclastogenesis by upregulating RANK via the NF-κB pathway[END_REF][START_REF] Ruscitti | The role of IL-1β in the bone loss during rheumatic diseases[END_REF]. Thus, IL1β plays a predominant role in the regulation of bone remodeling, and that any disturbance of the expression levels of this cytokine may lead to an imbalance in bone remodeling homeostasis in favor of osteoclastogenesis and bone resorption activity and bone mineralization and production by osteoblasts, eventually leading to the destruction of bone tissue (DINARELLO, 2009b;[START_REF] Rasigade | PSMs of hypervirulent Staphylococcus aureus act as intracellular toxins that kill infected osteoblasts[END_REF][START_REF] Yang | Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors[END_REF].

Other cytokines

IL-1α or (IL-1F1), like IL-1β, was identified in 1974, both bind to the same IL-1 receptor (IL-1R)

and are potent proinflammatory cytokines that are constitutively expressed in many cell types (DINARELLO, 2010). However, little is known about this cytokine in comparison to IL-1β. IL-1α, unlike IL-1β, is also active in its precursor form and is present intracellularly in healthy tissues [START_REF] Dinarello | IL-1: discoveries, controversies and future directions[END_REF](DINARELLO, , 2018a;;[START_REF] Werman | The precursor form of IL-1 is an intracrine proinflammatory activator of transcription[END_REF]. Depending on the activator, IL-1α secretion is dependent on inflammation (DI PAOLO; SHAYAKHMETOV, 2016).

IL-1α processing induces proteolytic cleavage of pro-IL-1α in mature IL-1α. However, this cleavage is dependent on calpains which are Ca2-dependent proteases present on the cytosolic side of the cell membrane (CARRUTH; DEMCZUK; [START_REF] Carruth | Involvement of a calpain-like protease in the processing of the murine interleukin 1 alpha precursor[END_REF][START_REF] Fettelschoss | Inflammasome activation and IL-1 target IL-1 for secretion as opposed to surface expression[END_REF][START_REF] Tapia | The three cytokines IL-1β, IL-18, and IL-1α share related but distinct secretory routes[END_REF]. Another study demonstrated that IL-1α surface expression is independent of caspase-1, whereas mature IL-1α secretion requires the activation of the inflammasome and caspase-1 [START_REF] Fettelschoss | Inflammasome activation and IL-1 target IL-1 for secretion as opposed to surface expression[END_REF][START_REF] Gross | Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1[END_REF]. According to (DINARELLO, 2010), intracellular IL-1α is also associated with chromatin playing a dynamic role in the nucleus of living cells [START_REF] Dinarello | IL-1: discoveries, controversies and future directions[END_REF]. During the apoptotic process, this intracellular cytokine is concentrated in dense nuclear foci and is not released along with the cytoplasmic content but remains inactive. However, the precursor of IL-1a is released from cells that undergo necrosis by becoming active [START_REF] Chen | Identification of a key pathway required for the sterile inflammatory response triggered by dying cells[END_REF][START_REF] Cohen | Differential release of chromatin-bound IL-1 discriminates between necrotic and apoptotic cell death by the ability to induce sterile inflammation[END_REF].

IL-18 (IL-1F4 or previously IFN-gamma inducing factor) was first described in 1989 and is In addition to IL-1β and IL-18, caspase is also able to regulate the activity of two other cytokines: IL-33 and IL-37. IL-33, also called IL-1F11, is a pro-inflammatory cytokine that has been recently added to the IL-1 family [START_REF] Afonina | Proteolytic Processing of Interleukin-1 Family Cytokines: Variations on a Common Theme[END_REF][START_REF] Cherry | A novel IL-1 family cytokine, IL-33, potently activates human eosinophils[END_REF]. It is a constitutive intracellular cytokine that is produced as pro-IL-33 and, after undergoing cleavage, is secreted into the extracellular medium as mature IL-33 (YANG et al., 2015). This cleavage occurs via proteases such as cathepsin G and elastase type and independently of caspase-1 [START_REF] Lefrançais | IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G[END_REF][START_REF] Talabot-Ayer | Interleukin-33 is biologically active independently of caspase-1 cleavage[END_REF]. However, studies have also shown that IL-33 can also be cleaved by Caspase-1 in vitro (CAYROL; [START_REF] Girard-Guyonvarc'h | Unopposed IL-18 signaling leads to severe TLR9-induced macrophage activation syndrome in mice[END_REF][START_REF] Talabot-Ayer | Interleukin-33 is biologically active independently of caspase-1 cleavage[END_REF].

IL-33 is expressed at high levels in the nucleus of various cell types of the immune system, including non-professional phagocytes such as endothelial, epithelial and fibroblast cells in humans and mice (CAYROL; [START_REF] Girard-Guyonvarc'h | Unopposed IL-18 signaling leads to severe TLR9-induced macrophage activation syndrome in mice[END_REF]CHEN;KANG;[START_REF] Chen | The independence of and associations among apoptosis, autophagy, and necrosis[END_REF][START_REF] Sokol | The Chemokine System in Innate Immunity[END_REF][START_REF] Sokol | The Chemokine System in Innate Immunity[END_REF]. IL-33 is structurally related to IL-1b and IL-18, responsible for activating Th1/Th17 lymphocytes; however, it is biological functions mainly result in the production of IL-5 andIL-13 (DINARELLO, 2010, 2018b;[START_REF] Oboki | IL-33 is a crucial amplifier of innate rather than acquired immunity[END_REF].

IL-37 or IL-1F7 is a recently described cytokine belonging to the IL-1 family which exhibits anti- The innate immune system is a sophisticated system, which relies on its ability to detect invading microbes, tissue damage or stress through evolutionarily conserved receptors [START_REF] Janeway | Approaching the Asymptote? Evolution and Revolution in Immunology[END_REF]MEDZHITOV;[START_REF] Medzhitov | Innate Immunity[END_REF]. It has a large number of well-conserved microbial structures, which are known as pathogen-associated molecular patterns (PAMPs), recognized by pattern recognition receptors (PRRs) [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF]JANG et al., 2015;[START_REF] Mogensen | Pathogen Recognition and Inflammatory Signaling in Innate Immune Defenses[END_REF]. However, these receptors are less numerous than those of the adaptive immune system. The recognition of PAMPs by a PRR activates a signaling pathway leading to an inflammatory response that induces secretion of cytokines and chemokines and, consequently, the activation of an adaptive immune response [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF]JANG et al., 2015;[START_REF] Mogensen | Pathogen Recognition and Inflammatory Signaling in Innate Immune Defenses[END_REF]. PRRs are expressed primarily by cells of the innate immune system such as macrophages, monocytes, dendritic cells, and neutrophils. However, other cell types are able to express PRRs example of epithelial cells (JANG et al., 2015).

Historically, the theory that cells of the immune system can detect and respond to all molecular patterns of pathogens has been developed by Charles Janeway [START_REF] Janeway | Approaching the Asymptote? Evolution and Revolution in Immunology[END_REF]. However, this model failed to explain how the immune system could distinguish between commensal and pathogenic bacteria. Thus, Matzinger proposed an alternative theory about the innate immune system that said that the system did not distinguish between what is proper (cells of the organism) and what is not itself (agents foreign to the organism), but rather respond to the signs of danger that are emitted by the body's cells, inducing an inflammatory response in response to these danger signals [START_REF] Matzinger | Danger, and the Extended Family[END_REF][START_REF] Pradeu | The danger theory: 20 years later[END_REF][START_REF] Pradeu | The danger theory: 20 years later[END_REF]. TLRs can be classified according to their cellular location, for example the TLRs located on the plasma membrane are TLR 1,2,[START_REF] Del Pozo | Infection associated with prosthetic Joints[END_REF]5,6,[START_REF] Strowig | Inflammasomes in health and disease[END_REF] whereas the TLRs located in the endosomes are TLR 3, 7, 8, 9 and 13 (KAWAI;[START_REF] Kawai | The roles of TLRs, RLRs and NLRs in pathogen recognition[END_REF]. Broadly, TLRs can detect signs of danger such as peptides, lipopeptides, glycopeptides, glycolipids and nucleic acids (KAWAI;

AKIRA , 2009). NLR receptors, along with TLRs, cooperate to recognize and respond to pathogens and activate pro and anti-inflammatory mechanisms [START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF].

CLRs recognize specific polysaccharides and glycopeptides [START_REF] Artigas | Glycopeptides as Targets for Dendritic Cells: Exploring MUC1 Glycopeptides Binding Profile toward Macrophage Galactose-Type Lectin (MGL) Orthologs[END_REF][START_REF] Wolfert | Adaptive immune activation: glycosylation does matter[END_REF][START_REF] Wolfert | Adaptive immune activation: glycosylation does matter[END_REF]. RLRs are cytoplasmic receptors and respond primarily to the presence of RNA viruses and together with the NLRs are cytosolic proteins that probe intracellular microbial molecules and danger signs [START_REF] Raymond | Microbial recognition and danger signals in sepsis and trauma[END_REF].

NOD-like receptors (NLRs)

NLRs are a family of receptors located on the cytosol that recognize PAMPs. They are pattern recognition receptors that stand out as one of the major receptors involved in regulating the innate immune response. NLRs cooperate along with TLRs and act on the regulation of inflammatory and apoptotic responses, being found in professional phagocytes such as macrophages and dendritic cells and non-professional phagocytes such as epithelial cells (JANG et al., 2015).

The NLRs can be characterized by three structural domains:a leucine-rich repeat (LRR); a

The Caspase-1 protein

In 1980, caspase-1 was initially discovered to play its role in the maturation of pro-caspase-1 that exists in the inactive form called (proform or zymogen) named ICE (Interleukin-1 converting enzyme or transformant IL-1 enzyme) due to its activity of pro-IL-1β processing in its mature form IL-1β (AFONINA et al., 2015). However, the mechanism involved in its , 2011). These results show that the re-expression of the non-cleaved form of caspase-1 in macrophages that were deficient for the caspase-1 gene is able to induce cell death, but with reduced IL-1β secretion (BROZ; MONACK, 2011). Also, interestingly, caspase-1 is able to regulate anti-inflammatory signaling pathways, for example its catalytic activity is responsible for cleaving and activating IL-37 (DINARELLO, 2018a;[START_REF] Wang | Neutrophils in tissue injury and repair[END_REF].

Non-canonical activation of inflammatory caspases

Caspase-8 (Cysteinyl aspartic acid-protease 8) is an early protease of the extrinsic apoptosis pathway, a form of programmed cell death [START_REF] Elmore | Apoptosis: A Review of Programmed Cell Death[END_REF][START_REF] Masumoto | ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells[END_REF].

Caspase-8 is implicated in a non-canonical type of activation and is directly involved in the cleavage and activation of IL-1β, inducing inflammatory responses [START_REF] Ketelut-Carneiro | A Dectin-1-Caspase-8 Pathway Licenses Canonical Caspase-1 Inflammasome Activation and Interleukin-1β Release in Response to a Pathogenic Fungus[END_REF]LATZ;XIAO;STUTZ, 2013a).The assembly of the non-canonical caspase-8 inflammasome occurs via ASC protein, without interaction with cytosolic receptors [START_REF] Chung | NLRP3 regulates a non-canonical platform for caspase-8 activation during epithelial cell apoptosis[END_REF]LATZ;XIAO;STUTZ, 2013b;[START_REF] Masumoto | ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells[END_REF]. However, the interaction between caspase-8 and ASC occurs via their DED (Death effector domain) and PYD domains [START_REF] Fu | Cryo-EM Structure of Caspase-8 Tandem DED Filament Reveals Assembly and Regulation Mechanisms of the Death-Inducing Signaling Complex[END_REF].

Caspase-11 (non-canonical) is composed of an N-terminal and C-terminal CARD and can activate the NLRP3 inflammasome by directly binding intracellular lipopolysaccharide (LPS) to the CARD domain in macrophages infected with Escherichia coli, Vibrio cholera, Legionella pneumophila, Salmonella typhimurium and other Gram-negative bacteria that enter the cytosol (murine caspase-11, caspase-4 and caspase-5 are orthologs of caspase-11 in humans) [START_REF] Jozala | Aqueous Two-Phase Micellar System for Nisin Extraction in the Presence of Electrolytes[END_REF][START_REF] Munday | Molecular cloning and pro-apoptotic activity of ICErelII and ICErelIII, members of the ICE/CED-3 family of cysteine proteases[END_REF][START_REF] Yi | Regulatory Roles of the Caspase-11 Non-Canonical Inflammasome in Inflammatory Diseases[END_REF]. Caspase-11 can also activate the NLRP3 inflammasome by reducing intracellular potassium levels, causing the release of ATP and leading to the maturation of IL-1β and IL-18, in addition to playing an important role in pyroptosis via the direct cleavage of gasdermin D (RÜHL; BROZ, 2015). Caspase-11 and caspase-1 act in concert in the inflammatory process of the host (YI, 2018).

The ASC protein

The ASC adapter protein is a 22 KDa protein composed of (179-195) amino acids, which is composed of an N-terminal PYD domain that regulates its self-association and interactions with the NLRP3 protein and a C-terminal CARD domain. Its main function is the activation of caspase-1 [START_REF] Masumoto | ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells[END_REF], thus, as a function of its concentration, the ASC protein modulates the activation of pro-caspase-1 and maturation of IL-1β (STEHLIK, 2009). This protein plays an essential role in the activation of all inflammasomes, being implicated in the secretion of IL-1β, IL-18, and IFN-γ (KUMAR;KAWAI;[START_REF] Kumar | Pathogen Recognition by the Innate Immune System[END_REF].

Studies have shown that, in the structure of the NLRP3 and AIM2 inflammasomes, their PYD domain is able to induce a prion-like polymerization of the PYD domain of ASC, being confirmed through visualization by cytometry and immunofluorescence analyses [START_REF] Cai | Prion-like Polymerization Underlies Signal Transduction in Antiviral Immune Defense and Inflammasome Activation[END_REF][START_REF] Dick | ASC filament formation serves as a signal amplification mechanism for inflammasomes[END_REF][START_REF] Rühl | Caspase-11 activates a canonical NLRP3 inflammasome by promoting K(+) efflux[END_REF][START_REF] Rühl | Caspase-11 activates a canonical NLRP3 inflammasome by promoting K(+) efflux[END_REF]. The exposed ASC filaments cause caspase-1 recruitment via CARD-CARD interaction [START_REF] Dick | ASC filament formation serves as a signal amplification mechanism for inflammasomes[END_REF]. Recently, studies have shown that the formation of ASC specks can occur due to the accumulation of cells dying by pyroptosis in the microenvironment [START_REF] Stutz | ASC Speck Formation as a Readout for Inflammasome Activation[END_REF]. According to Broz, P. ( 2011) the formation of ASC specks is independent of the activity of caspase-1, however it is necessary that oligomerization of ASC in large stable insoluble aggregates occurs that are released to the extracellular medium via pyroptosis leading to activation of inflammasomes (BROZ; [START_REF] Broz | Molecular mechanisms of inflammasome activation during microbial infections: Mechanisms of inflammasome activation[END_REF][START_REF] Dick | ASC filament formation serves as a signal amplification mechanism for inflammasomes[END_REF].

The NLRP1 inflammasome

The NLRP1 inflammasome was the first member of the NLR family to be described, however, it is the least studied [START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF]. The NLRP1 gene is unique in humans whereas in murines there are three homologous genes: Nlrp1a, Nlpr1b and Nlrp1c, which are highly NLRP1b was identified as inducing the secretion of IL-1β and IL-18 and killing rat macrophage cells in response to Bacillus anthracis lethal toxin (LeTx) (Fig. 4) [START_REF] Newman | Susceptibility to Anthrax Lethal Toxin-Induced Rat Death Is Controlled by a Single Chromosome 10 Locus That Includes rNlrp1[END_REF]. LeTx is a bipartite toxin composed of an antigen-binding protective (PA) subunit and a part of the catalytic lethal factor (LF) [START_REF] Kushner | A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen[END_REF]. LF is a zinc metalloprotease that cleaves and inactivates all mitogen activated protein kinases (MAPKs) (AGRAWAL; [START_REF] Agrawal | Anthrax lethal toxin: a weapon of multisystem destruction[END_REF][START_REF] Newman | Susceptibility to Anthrax Lethal Toxin-Induced Rat Death Is Controlled by a Single Chromosome 10 Locus That Includes rNlrp1[END_REF][START_REF] Ranson | Regulation and Sensing of Inflammasomes and Their Impact on Intestinal Health[END_REF][START_REF] Ranson | Regulation and Sensing of Inflammasomes and Their Impact on Intestinal Health[END_REF][START_REF] Ranson | Regulation and Sensing of Inflammasomes and Their Impact on Intestinal Health[END_REF].

Thus, NLRP1 is associated with the activations of pyroptosis and caspases-2 and 9 in the complex called apoptosome. The apoptosome is a protein complex formed by the association of cytochrome C to the Apaf-1 protein and pro-caspase-9 during the intrinsic apoptosis signaling pathway (BRATTON; SALVESEN, 2010).

In addition, human NLRP1 was shown to recognize muramyl-dipeptide (MDP), a peptideoglycan composite, it may induce changes in conformation leading to the activation of Caspase-1 (Fig.

The NLRP3 inflammasome

The inflammasome NLRP3 (also known as cryopyrin and NALP3) is the most extensively characterized member of the NLR family due to the diversity of its activation stimuli and its association with a wide range of inflammatory pathologies (LATZ; XIAO; STUTZ, 2013a). The NLRP3 inflammasome contains a structure of 3 separate domains; an N-terminal pyrin domain (PYD), a nucleotide-binding and oligomerization domain (NATCH) and a C-terminal leucinerich repeat (LRR). However, unlike other NLRs, the NLRP3 inflammasome does not have (CARD), thus requiring the adapter protein (ASC) to bind to pro-caspase-1 (Fig. 6) [START_REF] Bauernfeind | Inflammasomes: current understanding and open questions[END_REF][START_REF] Duncan | Neisseria gonorrhoeae activates the proteinase cathepsin B to mediate the signaling activities of the NLRP3 and ASC-containing inflammasome[END_REF][START_REF] Gross | Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence[END_REF][START_REF] Ichinohe | Influenza virus activates inflammasomes via its intracellular M2 ion channel[END_REF][START_REF] Ichinohe | Influenza virus activates inflammasomes via its intracellular M2 ion channel[END_REF][START_REF] Ichinohe | Influenza virus activates inflammasomes via its intracellular M2 ion channel[END_REF][START_REF] Joly | Cutting edge: Candida albicans hyphae formation triggers activation of the Nlrp3 inflammasome[END_REF][START_REF] Kanneganti | Critical role for Cryopyrin/Nalp3 in activation of caspase-1 in response to viral infection and double-stranded RNA[END_REF][START_REF] Mariathasan | Cryopyrin activates the inflammasome in response to toxins and ATP[END_REF][START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Muruve | The inflammasome recognizes cytosolic microbial and host DNA and triggers an innate immune response[END_REF].

(
However, the activation medium of the NLRP3 inflammasome is still controversial due to the large number of substances that can trigger its formation (Table 2). The most accepted theory The canonical pathway requires 2 steps: priming and activation. A first signal called "Priming" is initiated when a PAMPs or DAMPs bind to a TLR receptor through (LPS), or after signaling via endogenous cytokine receptors such as IL-1R or through tumor necrosis factor (TNF-alpha) (Fig. 8) (GRAZIOLI; [START_REF] Grazioli | Mitochondrial Damage-Associated Molecular Patterns: From Inflammatory Signaling to Human Diseases[END_REF][START_REF] Mogensen | Pathogen Recognition and Inflammatory Signaling in Innate Immune Defenses[END_REF]. This signal is required for the transcription, translation, and unubiquitination of NLRP3 to induce pro-IL-1β expression via activation of the transcription factor (NF-κB) [START_REF] Kinoshita | NLRP3 Mediates NF-κB Activation and Cytokine Induction in Microbially Induced and Sterile Inflammation[END_REF][START_REF] Liu | NF-κB signaling in inflammation[END_REF][START_REF] Qiao | TLR-induced NF-κB activation regulates NLRP3 expression in murine macrophages[END_REF]. This priming signal allows activating K63-specific deubiquitinase (BRCC3), a JAMM domain-containing Zn2+ metalloprotease leading to the unsubiquitination of NLRP3, a step necessary for its activation (HE; HARA; [START_REF] He | Mechanism and Regulation of NLRP3 Inflammasome Activation[END_REF][START_REF] Py | Deubiquitination of NLRP3 by BRCC3 critically regulates inflammasome activity[END_REF].

The non-canonical pathway of the NLRP3 inflammasome activation," which depends on caspase-11 in mice (human orthologs are caspase 4 and caspase 5), has been described to be pivotal in the maintenance of intestinal immune homeostasis [START_REF] Kayagaki | Noncanonical inflammasome activation by intracellular LPS independent of TLR4[END_REF][START_REF] Pellegrini | Canonical and Non-Canonical Activation of NLRP3 Inflammasome at the Crossroad between Immune Tolerance and Intestinal Inflammation[END_REF]. Activation of the non-canonical pathway of caspase-11 in response to gram-negative bacteria was explained when it was discovered in recent years that LPS, which is a component of the outer membrane of gram-negative bacteria, delivered intracellularly by transfection, electroporation or by bacteria that reach the cytoplasm signals, independent of TLR4, binding directly to caspase-11 [START_REF] Broz | Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1[END_REF][START_REF] Kayagaki | Noncanonical inflammasome activation by intracellular LPS independent of TLR4[END_REF]RATHINAM;VANAJA;FITZGERALD, 2012). Since its discovery, the unconventional canonical pathway of inflammasome activation has been the subject of intense research.

The third mechanism of activation for the NLRP3 inflammasome in human monocytes is described as an "alternative pathway" and is similar to the canonical pathway (GAIDT; HORNUNG, 2017). This pathway depends on the binding of LPS via TLR4. However, contrary , 1994).

Followed by other studies about the importance of K + efflux in the activation of the NLRP3 inflammasome [START_REF] Giuliani | The P2X7 Receptor-Interleukin-1 Liaison[END_REF][START_REF] Muñoz-Planillo | K+ efflux is the Common Trigger of NLRP3 inflammasome Activation by Bacterial Toxins and Particulate Matter[END_REF][START_REF] Piccini | ATP is released by monocytes stimulated with pathogen-sensing receptor ligands and induces IL-1β and IL-18 secretion in an autocrine way[END_REF].

This efflux is induced in response to various stimuli such as ATP, nigericin and several extracellular crystals, all known to decrease cytosolic K + levels through the P2X7 receptor [START_REF] Giuliani | The P2X7 Receptor-Interleukin-1 Liaison[END_REF]. These pore channels promote potassium efflux, resulting in low concentrations of intracellular K + , causing conformational changes in NLRP3 thereby activating the NLRP3 inflammasome (HE; HARA; NÚÑEZ, 2016; [START_REF] Muñoz-Planillo | K+ efflux is the Common Trigger of NLRP3 inflammasome Activation by Bacterial Toxins and Particulate Matter[END_REF]. Thus, the drop in K + concentration in the cytosol is proposed as a common trigger for the activation of the NLRP3 inflammasome (HE; HARA; NÚÑEZ, 2016; [START_REF] Muñoz-Planillo | K+ efflux is the Common Trigger of NLRP3 inflammasome Activation by Bacterial Toxins and Particulate Matter[END_REF].

Particles such as alum or silica are phagocytosed by cells of the immune system and activate the NLRP3 inflammasome through the degradation of the phagolysosomal membrane causing the release of Cathepsin B, a protease that is able to bind directly to the NLRP3 receptor and induce the activation of inflammasome (LATZ; XIAO; STUTZ, 2013a). Furthermore, this particulate material may trigger a K + efflux, which is dependent on phagocytosis and is required for the activation of the NLRP3 inflammasome [START_REF] Muñoz-Planillo | K+ efflux is the Common Trigger of NLRP3 inflammasome Activation by Bacterial Toxins and Particulate Matter[END_REF][START_REF] Yang | Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors[END_REF].

The production of ROS in the cellular cytoplasm of macrophages and monocytes occurs via a mitochondrial dysfunction caused due to cellular stress in response to a stimulus such as AIM2 is able to detect from 80 base pairs of virus DNA such as mouse cytomegalovirus to cytosolic bacteria such as Francisella tularensis and Listeria monocytogenes, acting against these pathogens and promoting cell death in order to restrict bacterial growth (RATHINAM;

FITZGERALD, 2016; RATHINAM; VANAJA; FITZGERALD, 2012).

The NLRC4 inflammasome

The NLRC4 inflammasome or ICE-protease activating factor (IPAF) is formed by the NLRC4 protein and caspase-1 (DI VIRGILIO, 2013; DUNCAN; CANNA, 2018). NLRC4 is composed of an N-terminal CARD domain interacting directly with caspase-1. However, although NLRC4 possesses a CARD domain, ASC is required to amplify the platform's operation and its activation (Fig. 9) [START_REF] Lage | Emerging Concepts about NAIP/NLRC4 Inflammasomes[END_REF][START_REF] Vance | The NAIP/NLRC4 inflammasomes[END_REF].

Bacterial PAMPs are responsible for the formation of the NLRC4 inflammasome which is activated via NAIP (NLR family-apoptosis inhibitory protein) which binds to specific bacterial ligands and is co-assembled with NLRC4 [START_REF] Zhao | The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus[END_REF]. However, other bacteria such as Salmonella typhimurium, Legionella pneumophila, Shigella flexneri and Pseudomonas aeruginosa, are also known activators of this inflammasome (BROZ; MONACK, 2011).

In NAIP1 mice it is bound to the bacterial secretory system type III (T3SS), protein needle, NAIP2 to T3SS basal protein, and NAIP5 and NAIP6 bacterial flagellin, which is its most characterized activator [START_REF] Bauernfeind | Inflammasomes: current understanding and open questions[END_REF][START_REF] Miao | Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1β via Ipaf[END_REF][START_REF] Rayamajhi | Cutting Edge: Mouse NAIP1 Detects the Type III Secretion System Needle Protein[END_REF][START_REF] Ren | Flagellin-Deficient Legionella Mutants Evade Caspase-1-and Naip5-Mediated Macrophage Immunity[END_REF][START_REF] Zhao | The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus[END_REF]. However, in humans, NAIP1 was only found bound to the T3SS protein needle [START_REF] Yang | Sequence determinants of specific pattern-recognition of bacterial ligands by the NAIP-NLRC4 inflammasome[END_REF]. Thus, after the junction of NAIP and its ligand, oligomerization with NLRC4 and ASC can be accomplished by initiating the assembly of the inflammasome [START_REF] Vance | The NAIP/NLRC4 inflammasomes[END_REF].

Others inflammasomes

The NLRP6 inflammasome is implicated in the homeostasis of the intestinal flora and digestive tract of the host (DI VIRGILIO, 2013; OKUMURA; TAKEDA, 2017). The NLRP6 receptor expressed in cells of the immune system or epithelial cells is able to form inflammasome [START_REF] Elinav | NLRP6 Inflammasome Regulates Colonic Microbial Ecology and Risk for Colitis[END_REF][START_REF] Lei | The interplay between the intestinal microbiota and the immune system[END_REF]NAIR;[START_REF] Lei | The interplay between the intestinal microbiota and the immune system[END_REF]. In the intestinal epithelium, NLRP6, plays a protective role in preserving the intestinal flora, but on the other hand can play a detrimental role by aggravating bacterial infections [START_REF] Di | The therapeutic potential of modifying inflammasomes and NOD-like receptors[END_REF][START_REF] Yin | The protective roles of NLRP 6 in intestinal epithelial cells[END_REF].

In a study by Elinav and colleagues (2011), it was shown that the absence of NLRP6 in mice causes a change in the composition of their microbiota, leading to an increase bacterial phyla Bacteroidetes (Prevotellaceae) and TM7, and a decrease of Lactobacillus ssp. [START_REF] Elinav | NLRP6 Inflammasome Regulates Colonic Microbial Ecology and Risk for Colitis[END_REF]. This activity is suggestive of the role of this inflammasome in the regulation of the microbiota.Furthermore, NLRP6/KO mice, when compared to wild-type mice, were shown to secrete less IL-18 and were more likely to develop tumors in a chemically induced DSS (Dextran-sodium sulfate) model of colitis, (CHEN et al., 2017a;[START_REF] Elinav | NLRP6 Inflammasome Regulates Colonic Microbial Ecology and Risk for Colitis[END_REF][START_REF] Mao | The Role of NLRP3 and IL-1β in the Pathogenesis of Inflammatory Bowel Disease[END_REF].

The NLRP7 inflammasome is responsible for the maturation and release of IL-1β and IL-18 after stimulation by microbial lipopeptides, being the cause of septic shock in mice (DI [START_REF] Di | The therapeutic potential of modifying inflammasomes and NOD-like receptors[END_REF][START_REF] Khare | An NLRP7-Containing Inflammasome Mediates Recognition of Microbial Lipopeptides in Human Macrophages[END_REF]. NLRP7, ASC and caspase-1 form the complex molecule, however, the mechanism of action of this inflammasome does not involve the production of ROS, but the cathepsin B and K + efflux pathways [START_REF] Di | The therapeutic potential of modifying inflammasomes and NOD-like receptors[END_REF][START_REF] Radian | NLRP7 and related inflammasome activating pattern recognition receptors and their function in host defense and disease[END_REF].

The NLRP12 inflammasome participates in the recognition of the bacterium Yersinia pestis and promotes the release of IL-1β and IL-18, as well as the secretion of IFN-γ by the activation of lymphocytes, being also associated with the inhibition of NF-kB [START_REF] Silveira | NLRP12 negatively regulates proinflammatory cytokine production and host defense against Brucella abortus[END_REF][START_REF] Vladimer | Inflammasomes and host defenses against bacterial infections[END_REF]. The interaction with the adapter protein ASC is required for the assembly the NLRP12 inflammasome, supporting the inclusion of NLRP12 as part of the inflammasome family (DI VIRGILIO, 2013). In humans, autoimmune diseases such as vitiligo, characterized as a chronic inflammatory disease in which melanocytes are destroyed, leading to skin depigmentation, rheumatoid arthritis, psoriasis, and autoimmune thyroiditis are related to a variant of the NLRP1 protein coding gene, thus the inflammasome plays an important role in regulating the adaptive immune response [START_REF] Ciraci | Physiology and Pathology of Autoinflammation: NOD like Receptors in Autoinflammation and Autoimmunity[END_REF][START_REF] Marie | Inflammasome activation and vitiligo/nonsegmental vitiligo progression[END_REF].

Cryopyrinopathies, also known as periodic syndromes associated with cryopyrin (CAPS), are inflammasomopathies caused by autosomal dominant mutations in the gene encoding NLRP3. To date, frequent episodes of generalized inflammation occur without evidence of an apparent stimulus, even because the host's own immune system functions as a trigger for the pathological process. Thus, understanding how inflammasome works and its regulation process is fundamental for the development of adequate therapeutic strategies focused on the resolution of inflammation.

Chapter 2. Problem statement of the project thesis

Advances on several research fronts have significantly broadened our knowledge about the inflammation triggers and modulation of the host inflammatory response. The recent discovery of the inflammasomes protein complex has been of great importance in the fields of immunology and medicine. Such importance is due to both the identification and characterization of the molecular mechanisms that link the cell stimulation (by pathogens, physical or chemical lesions) to the activation of early immune cell responses in the host.

Concerning the inflammation process by bacterial pathogens, Staphylococcus aureus is a major bacterial human pathogen that colonizes the nares and skin and frequently invades the soft tissues and bloodstreams. S. aureus has numerous mechanisms to evade and subvert the immune system, causing a variety of invasive and superficial infections [START_REF] Tong | Staphylococcus aureus Infections: epidemiology, pathophysiology, clinical manifestations, and management[END_REF]. This is aureus infection can trigger the local and systemic production of these three cytokines through host PRRs [START_REF] Rasigade | PSMs of hypervirulent Staphylococcus aureus act as intracellular toxins that kill infected osteoblasts[END_REF]. The increased production of these cytokines then leads to an imbalance in the homeostasis of bone turnover in favor of the activity of osteoclastogenesis and bone resorption (FENG;[START_REF] Feng | Disorders of bone remodeling[END_REF][START_REF] Peeters | Teicoplanin-based antimicrobial therapy in Staphylococcus aureus bone and joint infection: tolerance, efficacy and experience with subcutaneous administration[END_REF][START_REF] Rasigade | PSMs of hypervirulent Staphylococcus aureus act as intracellular toxins that kill infected osteoblasts[END_REF].

Studies have shown that the invasion and persistence of S. aureus in host cells described as "non-professional phagocytes" is documented in many cell types, including epithelial cells, endothelial cells and keratinocytes [START_REF] Kintarak | Internalization of Staphylococcus aureus by Human Keratinocytes[END_REF][START_REF] Rasigade | PSMs of hypervirulent Staphylococcus aureus act as intracellular toxins that kill infected osteoblasts[END_REF].

However, the interactions between osteoblastic cells and S. aureus remains comparatively understudied. In this context, we hypothesized that persistent infection by S. aureus involves the transcriptional reprogramming of osteoblastic cells to tolerate the asymptomatic transport of intracellular bacteria, leading to altered immune response, more specifically to the modification of the NLRP3 inflammasome activation and subsequent processing of the cytokine pro-inflammatory IL-1β.

Therefore, to analyze the activation of the inflammasome, the activation of pro-caspase-1 that undergo proteolytic processing to produce 2 subunits that dimerize to form the active caspase-1 needs to be monitored. Furthermore, monitoring the active caspase-1 (comprising the p20 and p10 subunits) and their effect on IL-1β induction is essential. To demonstrate that IL-1β is indeed activated by the inflammasome were required the generation of a deletion mutant Cas1 - /-MG-63 using the CRISPR/Cas9 gene editing system. Thus, this work was divided into two parts:

 In the first part of the study, a model is required to allow investigating the involvement of the NLRP3 inflammasome in S. aureus infection. The knockout caspase-1 gene in MG-63 cells will be through CRISPR/cas9 gene editing approach. Subsequently, the activation of caspase-1 in wild type (WT) MG-63 and mutant cell line CASP1 -/-MG-63 cells will be monitored (Fig. 10-A).

 In the second part of the study, the production of a soluble active form of IL-1β will be monitored in WT MG-63 vs CASP1 -/-MG-63 cells in an in vitro cellular model of chronic infection using long-term infections (from 6 h to 11 days) of osteoblasts with different strains of S. aureus (Fig. 10-B). Pertinence du projet de Thèse [START_REF] Rasigade | PSMs of hypervirulent Staphylococcus aureus act as intracellular toxins that kill infected osteoblasts[END_REF]. La production accrue de ces cytokines entraîne alors un déséquilibre dans l'homéostasie du remodelage osseux au profit de l'activité de l'ostéoclastogenèse et de la résorption osseuse (FENG;[START_REF] Feng | Disorders of bone remodeling[END_REF][START_REF] Peeters | Teicoplanin-based antimicrobial therapy in Staphylococcus aureus bone and joint infection: tolerance, efficacy and experience with subcutaneous administration[END_REF][START_REF] Rasigade | PSMs of hypervirulent Staphylococcus aureus act as intracellular toxins that kill infected osteoblasts[END_REF].

Des études ont montré que l'invasion et la persistance de S. aureus dans les cellules hôtes décrites comme des "phagocytes non professionnels" sont documentées dans de nombreux types de cellules, notamment les cellules épithéliales, les cellules endothéliales et les kératinocytes [START_REF] Kintarak | Internalization of Staphylococcus aureus by Human Keratinocytes[END_REF][START_REF] Rasigade | PSMs of hypervirulent Staphylococcus aureus act as intracellular toxins that kill infected osteoblasts[END_REF]. Cependant, les interactions

Introduction

Staphylococcus aureus is a versatile Gram-positive bacterium and an opportunistic pathogen able to cause a wide range of infections in both humans (Chambers, 1997;Fitzgerald, 2012;Lowy, 1998), and animals (Peton and Le Loir, 2014). It has long been considered a strictly extracellular pathogen but it is now also regarded as a facultative intracellular pathogen [START_REF] Fraunholz | Intracellular staphylococcus aureus: Live-in and let die[END_REF]. The mechanisms leading to internalization and underlying the intracellular persistence of S. aureus are mostly carried out using one strain in interaction with one model cell line, representative of a given cell type or tissue.

S. aureus uses a plethora of virulence factors to accommodate a diversity of niches in its various hosts. The array of virulence factors varies from a strain to another and, therefore, S.

aureus strains do not all interact with the host cells in the same fashion and they do not all induce the same cellular response. Similarly, not all host cells respond in a same way to interaction with S. aureus. This can be due to cell types (professional and non-professional phagocytes). However, this can also be observed with different cell lines for a given cell type.

Although these variations are more and more documented in the literature, as far as we know, they have not been reviewed yet. In this review, we address the variability of strain-specific traits as well as that of the host cells leading to a wide range of cell responses as outcomes of S. aureus-host cell interactions. We particularly focus on reports that experimentally addressed these issues.

S. aureus, a versatile opportunistic pathogen

S. aureus strains greatly vary in terms of virulence factors, which explains their ability to live as commensal bacteria, or adopt a pathogenic lifestyle and induce manifestations of disease as extracellular bacteria (in impetigo, abscesses, furuncles, septicemia, necrotizing pneumonia), as biofilm formers (catheter-induced infective endocarditis, atherosclerosis), or as intracellular pathogens. In the latter case, regarded as an immune-evasive strategy, S.

aureus survives within its host cells and escapes detection by professional phagocytes, which leads to persistent infections.

S. aureus strains also greatly vary at the genotypic level with regard to their host origin. This was shown through Pulsed-field Gel Electrophoresis (PFGE) analysis [START_REF] Alves | Molecular characterisation of Staphylococcus aureus strains isolated from small and large ruminants reveals a host rather than tissue specificity[END_REF][START_REF] Hennekinne | Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macro-restriction fragments[END_REF], or Multilocus sequence typing (MLST) approach, with some clonal complexes that are mostly found associated to a given host. Although all clonal complexes (CC) are found in human host, CC1, CC5 and CC130 are predominant in cattle, CC133 in small ruminants, CC1, CC5, CC9 and CC97 in swine, CC8 and CC9 in horse, and CC5 in poultry [START_REF] Guinane | Evolutionary Genomics of Staphylococcus aureus Reveals Insights into the Origin and Molecular Basis of Ruminant Host Adaptation[END_REF][START_REF] Pantosti | Methicillin-Resistant Staphylococcus aureus Associated with Animals and Its Relevance to Human Health[END_REF]. These host-specific traits are also retrieved at the genomic level [START_REF] Ben Zakour | Genome-Wide Analysis of Ruminant Staphylococcus aureus Reveals Diversification of the Core Genome[END_REF], and at the molecular level. Some S. aureus virulence factors that are indeed specifically present or preferentially active on their target in a given host as demonstrated e.g. for bovine variant of von Willebrand factor-binding protein [START_REF] Viana | Adaptation of Staphylococcus aureus to ruminant and equine hosts involves SaPI-carried variants of von Willebrand factorbinding protein[END_REF], equine variant of staphylococcal complement inhibitor (SCIN) [START_REF] De Jong | Identification of a staphylococcal complement inhibitor with broad host specificity in equid Staphylococcus aureus strains[END_REF], or ovine variants of staphylococcal enterotoxin type C [START_REF] Marr | Characterization of novel type C staphylococcal enterotoxins: biological and evolutionary implications[END_REF].

S. aureus adhesion and internalization

Host cells invasion (i.e. adhesion onto and internalization into host cells) is a strategy used by certain bacterial microorganisms to evade the immune system [START_REF] Alexander | Factors influencing the internalization of Staphylococcus aureus and impacts on the course of infections in humans[END_REF].

Bacterial adhesion to the host cell surface is the first step of the host cell invasion reviewed in [START_REF] Sinha | Mechanism and consequences of invasion of endothelial cells by Staphylococcus aureus[END_REF]. S. aureus expresses an array of specific molecules, adhesins, which allow the bacterium to adhere to the matrix of host cells [START_REF] Clarke | Surface adhesins of Staphylococcus aureus[END_REF].

These adhesion factors are either covalently linked to the S. aureus envelope and called microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) or secreted in the surrounding medium and called secretable expanded repertoire adhesive molecules (SERAM) (Foster and Höök, 1998). S aureus uses MSCRAMMs to adhere to the cell, directly or via bridging ligands, i.e. cellular eukaryotic proteins with affinity for both host cell receptors and MSCRAMMs [START_REF] Alexander | Factors influencing the internalization of Staphylococcus aureus and impacts on the course of infections in humans[END_REF][START_REF] Dziewanowska | Staphylococcal Fibronectin Binding Protein Interacts with Heat Shock Protein 60 and Integrins: Role in Internalization by Epithelial Cells[END_REF].

Fibronectin-binding proteins (FnBPs) are reportedly the most important factor for host cell invasion and are therefore extensively studied for their role in staphylococcal infection [START_REF] Alexander | Factors influencing the internalization of Staphylococcus aureus and impacts on the course of infections in humans[END_REF]. Their action is mediated via fibronectin bridging between host integrin α5β1 and staphylococcal surface proteins, FnBPs A and B [START_REF] Dziewanowska | Fibronectin Binding Protein and Host Cell Tyrosine Kinase Are Required for Internalization of <em>Staphylococcus aureus</em> by Epithelial Cells[END_REF][START_REF] Kintarak | Internalization of Staphylococcus aureus by Human Keratinocytes[END_REF][START_REF] Sinha | Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells[END_REF]. In human osteoblasts and bovine mammary epithelial cells, fibronectin can bind via the α5β1 integrin leading to the fixation of S. aureus and favoring its internalization by non-professional cells [START_REF] Ahmed | Staphylococcus aureus fibronectin binding proteins are essential for internalization by osteoblasts but do not account for differences in intracellular levels of bacteria[END_REF][START_REF] Dziewanowska | Fibronectin Binding Protein and Host Cell Tyrosine Kinase Are Required for Internalization of <em>Staphylococcus aureus</em> by Epithelial Cells[END_REF].

Bacterial invasion followed by the infection process in various cell types have in common that the uptake mechanism requires an intact cytoskeleton [START_REF] Menzies | Internalization of Staphylococcus aureus by Endothelial Cells Induces Apoptosis[END_REF]. During the internalization of S. aureus, the involvement of cytoskeletal elements was demonstrated, such as actin microfilaments [START_REF] Ellington | Mechanisms of Staphylococcus aureus invasion of cultured osteoblasts[END_REF][START_REF] Jevon | Mechanisms of internalization of Staphylococcus aureus by cultured human osteoblasts[END_REF]. The integrin-linked kinase (ILK) activity plays an important role in the internalization of S. aureus in epithelial cells [START_REF] Wang | Integrin-linked kinase is an essential link between integrins and uptake of bacterial pathogens by epithelial cells[END_REF]. The subsequent activation of ILK occurs after interaction with the cytoplasmic domains of β integrins that occurs due to the interaction among α5β1 and the cytoskeleton [START_REF] Wang | Integrin-linked kinase is an essential link between integrins and uptake of bacterial pathogens by epithelial cells[END_REF].

Moreover, studies have shown that rearrangement focal adhesion leads to the remodeling of the actin cytoskeleton by the loss of focal adhesion kinase (FAK), including the adaptor protein paxillin and vinculin. The rearrangement is accompanied by a centripetal movement of S.

aureus on the host cell surface [START_REF] Fraunholz | Intracellular staphylococcus aureus: Live-in and let die[END_REF][START_REF] Schröder | Live cell imaging of phagosome maturation in Staphylococcus aureus infected human endothelial cells: small colony variants are able to survive in lysosomes[END_REF]. Besides, the focal adhesion protein tensin, vinculin, and zyxin are recruited to the site of bacterial uptake [START_REF] Agerer | Cellular invasion by Staphylococcus aureus reveals a functional link between focal adhesion kinase and cortactin in integrin-mediated internalisation[END_REF]Wright and Nair, 2010). Figure 1-A shows the mechanisms involved in the invasion of S. aureus into non-professional phagocytes cells.

It has been shown that when FnBPs interact directly with human heat shock protein 60 (Hsp60)

on the membranes of human and bovine epithelial cells, the internalization efficiency is maximal [START_REF] Dziewanowska | Staphylococcal Fibronectin Binding Protein Interacts with Heat Shock Protein 60 and Integrins: Role in Internalization by Epithelial Cells[END_REF]. Hirschhausen and colleagues (2010) demonstrated that S. aureus autolysin (Atl) was identified to function as adhesin/invasin with the heat shock protein, Hsc70 as the direct cell receptor [START_REF] Hirschhausen | A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor: Atl-mediated staphylococcal internalization[END_REF]. The Alt-dependent internalization involves direct interaction with Hsc70 or an indirect interaction through the Fn connected to integrin α5β1 [START_REF] Hirschhausen | A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor: Atl-mediated staphylococcal internalization[END_REF].

The extracellular adherence protein (Eap) is a multifunctional protein consisting of 4 to 6 tandem-repeat domains that stimulate the adherence of staphylococci to fibroblasts and endothelial cells [START_REF] Fraunholz | Intracellular staphylococcus aureus: Live-in and let die[END_REF][START_REF] Hussain | Insertional Inactivation of eap in Staphylococcus aureus Strain Newman Confers Reduced Staphylococcal Binding to Fibroblasts[END_REF][START_REF] Palma | Adherence of Staphylococcus aureus is enhanced by an endogenous secreted protein with broad binding activity[END_REF]. However, the host cell receptor is not yet known. Besides, Weidenmaier and colleagues (2004) have

shown that wall teichoic acid (WTA) plays an important role in nasal colonization by S. aureus and that under certain conditions are important in adherence to endothelial cells. However, their host cell-binding partners have not yet been identified, but there is evidence that a scavenger receptor family is involved in WTA binding [START_REF] Weidenmaier | Role of teichoic acids in Staphylococcus aureus nasal colonization, a major risk factor in nosocomial infections[END_REF][START_REF] Weidenmaier | Lack of wall teichoic acids in Staphylococcus aureus leads to reduced interactions with endothelial cells and to attenuated virulence in a rabbit model of endocarditis[END_REF][START_REF] Weidenmaier | Differential roles of sortase-anchored surface proteins and wall teichoic acid in Staphylococcus aureus nasal colonization[END_REF]. interaction is the bacterial adhesion onto the cell surface, whose efficiency drives the internalization rate of the strains [START_REF] Haggar | Extracellular adherence protein from Staphylococcus aureus enhances internalization into eukaryotic cells[END_REF]. Various cell types can indeed internalize S. aureus and infected cells can serve as reservoir and vehicle for the systemic dissemination of S. aureus. Once internalized, S. aureus capacity to adopt a small colony variant phenotype is also dramatically important in its persistence within the host cells. Of course, these differences in terms of interaction capacities result in variations in terms of host cell response and infection outcome.

5. Phenotypic modifications also alter the S. aureus-host cell interaction S. aureus small colony variants (SCVs) are linked to bacterial persistence (Lowy, 1998). The intracellular persistence of S. aureus reviewed by [START_REF] Garzoni | Staphylococcus aureus: new evidence for intracellular persistence[END_REF][START_REF] Sendi | Staphylococcus aureus as an intracellular pathogen: the role of small colony variants[END_REF], has been described for a variety of host cells and pathogen strains. The relationship between S. aureus SCVs and persistent infection was first reported in a small clinical series in 1995 [START_REF] Proctor | Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus[END_REF]. Once internalized, S. aureus can adopt a SCV phenotype is characterized changes in transcriptome and proteome, which are phenotypically associated with slow growth rates, reduced metabolic activity, increased resistance to antibiotics and anti-bacterial host strategies such as the oxidative burst. SCV is often nonhemolytic, non-cytotoxic [START_REF] Proctor | Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus[END_REF]Tuchscherr et al., 2011Tuchscherr et al., , 2017)). S. aureus SCVs were found to produce fewer lytic enzymes, thereby allowing them to persist within the host cells [START_REF] Proctor | Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections[END_REF]. Additionally, SCVs of S. aureus even have been shown to survive and grow within host cell phagosomes [START_REF] Schröder | Live cell imaging of phagosome maturation in Staphylococcus aureus infected human endothelial cells: small colony variants are able to survive in lysosomes[END_REF]Tuchscherr et al., 2011), and their extended intracellular survival is probably involved in the chronicity of some S. aureus infections.

Besides, SCV also largely avoid the activation of the host immune system [START_REF] Tuchscherr | Staphylococcus aureus Small-Colony Variants Are Adapted Phenotypes for Intracellular Persistence[END_REF](Tuchscherr et al., , 2011)). Wild-type S. aureus induces the production of IL-1β, IL-6, and IL-12 and of tissue remodeling factors, which dramatically differs from immune responses to SCVs, characterized by the induction of TLR2 signaling [START_REF] Ou | Reduced Innate Immune Response to a Staphylococcus aureus Small Colony Variant Compared to Its Wild-Type Parent Strain[END_REF].

SCV phenotype results from various point mutations in genes involved in heme, or menadione biosynthesis, in oxidative phosphorylation, in cytochrome c assembly or thymidylate synthase (thyA), which often result in an altered electron transport chain [reviewed in [START_REF] Proctor | Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections[END_REF]]. These point mutations are reversible and account for reversion to the wild-type phenotype. Transposable elements may also be involved in SCV phenotype, which is also reversible in this case [START_REF] Kleinert | Influence of IS 256 on Genome Variability and Formation of Small-Colony Variants in Staphylococcus aureus[END_REF]. SCV phenotype switching may thus result from multiple pathways. As of yet, strain-specific behavior in terms of the ability to adopt an SCV phenotype has not been described in the literature. Although it is an important feature in S.

aureus-host cell interactions, with consequences on the cell response, one cannot conclude about variability concerning SCV phenotype.

In veterinary medicine, S. aureus is notably responsible for mastitis in dairy ruminants and strain-specific features were also identified, which had differential effects on bovine mammary epithelial cells (bMEC). The bMEC are the most abundant cell type of lactating parenchyma and they not only express the full repertoire of TLRs but are also highly immune competent.

When challenged with the Gram-negative pathogen Escherichia coli (E. coli), bMEC strongly expresses a wide range of immune factors including cytokines and chemokines, membrane protecting factors and bactericidal components. This vigorous activation is driven by the TLR signaling activation triggered by the E. coli LPS. TLR signaling activates the NF-κB complex of transcription factors, which in turn, activate immune gene expression. This strongly contrasts with S. aureus intramammary infections, which fail to activate any TLR signaling in bMEC and, hence, elicit only a weak immune response [START_REF] Petzl | Pathogen-specific responses in the bovine udder. Models and immunoprophylactic concepts[END_REF]. However, S. aureus can efficiently colonize the mammary epithelium and numerous strain-specific features have been identified in the first steps of the intramammary infection. Notably, the capabilities of S. aureus to adhere to and to internalize into bMEC strongly vary in a strain-dependent fashion (Bouchard et al., 2013;Peton et al., 2014).

Some S. aureus strains can reproducibly induce subclinical and persistent mastitis whether other strains induce clinical and non-persistent mastitis in experimental animal models. These clinical outcomes correlate with genetic features such as lesser toxin production and better adhesion to bMEC in vitro for the strains that induce mild mastitis [START_REF] Maréchal | Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis[END_REF]Peton et al., 2014). The host immune response is also different, with a stronger inflammatory response in the early steps of the infection for non-persistent strains, and differential expression of cytokines in the curse of the infection [START_REF] Pereyra | Immune response of Staphylococcus aureus strains in a mouse mastitis model is linked to adaptive capacity and genotypic profiles[END_REF].

Strain-specific ability to adhere and internalize

A comparative study of 120 S. aureus strains isolated from bovine mastitis milk samples belonging to lineages CC97, CC151 and ST136, showed no significant differences between the lineages in the ability to adhere to or internalize within bMEC although there were significant differences between individual isolates. In most cases, adherence to bMEC was correlated with the ability to bind bovine fibronectin, although isolates from CC151 could not bind bovine fibronectin in vitro, but adhered to bMEC in a fibronectin-independent manner [START_REF] Budd | Lineage associated expression of virulence traits in bovineadapted Staphylococcus aureus[END_REF]. Adhesion to and internalization into bMEC were also compared in RF122

and Newbould 305, two bovine mastitis S. aureus strains, and were correlated to genome analysis. Newbould 305 had better adhesion and internalization rates than RF122 and this correlated well with a longer fibronectin-binding protein (FnBP) compared to RF122 FnBP, as predicted from genome sequence [START_REF] Bouchard | Genome Sequence of Staphylococcus aureus Newbould 305, a Strain Associated with Mild Bovine Mastitis[END_REF]Peton et al., 2014).

Strain-specific cytotoxicity

Directly after host cell invasion, the bacteria can induce strong inflammatory effects and cytotoxic. Once internalized into the host cells, S. aureus cytotoxicity appears to be characteristic of individual strains rather than a general feature. [START_REF] Krut | Strain-specific association of cytotoxic activity and virulence of clinical Staphylococcus aureus isolates[END_REF] observed that among 23 randomly collected clinical S. aureus isolates, a majority were killed inside keratinocytes and fibroblasts. This shows that the uptake of S. aureus is a means of cellautonomous host defense for non-professional phagocytes. However, seven independent isolates survived intracellularly and induced significant cytotoxicity for host cells. Their results

suggest that these characteristics are stable and genetically determined [START_REF] Krut | Strain-specific association of cytotoxic activity and virulence of clinical Staphylococcus aureus isolates[END_REF].

Invasion of human keratinocytes by S. aureus and such S. aureus intracellular in human keratinocytes was shown to be haemolysin-independent and followed by features of apoptotic and necrotic cell death [START_REF] Mempel | Invasion of human keratinocytes by Staphylococcus aureus and intracellular bacterial persistence represent haemolysin-independent virulence mechanisms that are followed by features of necrotic and apoptotic keratinocyte cell death[END_REF]. Such cytotoxic strains can escape degradation by the endolysosomal pathway. These dramatic differences in terms of cytotoxic and noncytotoxic strains may partly explain the inter-strain differences in terms of dissemination of S.

aureus infection.

The internalization of S. aureus in osteoblasts cells leads to the secretion of cytokines, chemokines and growth factors that induce the activation of the immune system [START_REF] Turner | Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease[END_REF]. It has been demonstrated that the interaction between S. aureus and osteoblastic cells increase the production of IL-6, CCL2 (MCP-1), CCL3 (MIP-1α) and CCL5 (RANTES), as well as up-regulated of CXCL8 (IL-8) and CXCL2 (MIP-2α) [START_REF] Dapunt | The macrophage inflammatory proteins MIP1α (CCL3) and MIP2α (CXCL2) in implant-associated osteomyelitis: linking inflammation to bone degradation[END_REF][START_REF] Josse | Staphylococcus aureus vs. Osteoblast: Relationship and Consequences in Osteomyelitis[END_REF][START_REF] Wright | Regulation of chemokine gene expression and secretion in Staphylococcus aureus-infected osteoblasts[END_REF]. Besides, primary osteoblasts showed increased expression of the CCL5 and CXCL11 gene compared to the corresponding cell line after internalization with S. aureus 6850 and SH100, demonstrating that the internalization process induces a different response in primary cells and cell lines [START_REF] Strobel | Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain[END_REF].

The internalization of S. aureus by endothelial cells induces the expression of cytokines, chemokines and adhesion molecules such as IL-6, IL-8, MCP-1, RANTES and ICAM-1 [START_REF] Grundmeier | Staphylococcal Strains Vary Greatly in Their Ability to Induce an Inflammatory Response in Endothelial Cells[END_REF][START_REF] Strindhall | Variations among clinical isolates of Staphylococcus aureus to induce expression of E-selectin and ICAM-1 in human endothelial cells[END_REF][START_REF] Tekstra | Infection of human endothelial cells with Staphylococcus aureus induces the production of monocyte chemotactic protein-1 (MCP-1) and monocyte chemotaxis[END_REF] as well as genes associated with cell cycle, signaling and homeostasis [START_REF] Grundmeier | Staphylococcal Strains Vary Greatly in Their Ability to Induce an Inflammatory Response in Endothelial Cells[END_REF]. These findings suggest that in addition to inducing the inflammatory process, internalization also induces cellular events.

The prevention or activation of cell death may be a critical factor in the outcome of an infection and was well observed as a response to infection by several pathogens. It has been reported

that pathogens are capable of modulating apoptosis and thus starting an infection in several types of host cells like osteoblasts, endothelial, and epithelial cells [START_REF] Bayles | Intracellular Staphylococcus aureus escapes the endosome and induces apoptosis in epithelial cells[END_REF][START_REF] Fraunholz | Intracellular staphylococcus aureus: Live-in and let die[END_REF][START_REF] Haslinger-Loffler | Multiple virulence factors are required for Staphylococcus aureus-induced apoptosis in endothelial cells[END_REF][START_REF] Kubica | A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages[END_REF]Lamkanfi and Dixit, 2014). Cell death of non-prophylactic phagocytes induces activation of caspase-2 due to the efflux of potassium caused by aerolysin or α-toxin [START_REF] Imre | Caspase-2 is an initiator caspase responsible for pore-forming toxin-mediated apoptosis: Caspase-2 is responsible for PFTmediated apoptosis[END_REF]. The induction of apoptosis in S. aureus-infected osteoblasts is associated with tumor necrosis factor-related ligation inducing ligand (TRAIL) release [START_REF] Alexander | Factors influencing the internalization of Staphylococcus aureus and impacts on the course of infections in humans[END_REF]. TRAIL interacts with death receptor 4 (DR4), also known as TRAIL receptor 1 (TRAILR1) and death receptor 5 (DR5) [START_REF] Young | Causative agents of osteomyelitis induce death domain-containing TNF-related apoptosis-inducing ligand receptor expression on osteoblasts[END_REF]. Subsequently, TRAIL induces the activation of the intrinsic pathway of apoptosis through caspase-9 and the extrinsic pathway through caspase-8, which leads to activation of caspase-3 and subsequently to apoptosis (Figure 2) [START_REF] Alexander | Factors influencing the internalization of Staphylococcus aureus and impacts on the course of infections in humans[END_REF][START_REF] Claro | Staphylococcus aureus protein A binds to osteoblasts and triggers signals that weaken bone in osteomyelitis[END_REF][START_REF] Josse | Staphylococcus aureus vs. Osteoblast: Relationship and Consequences in Osteomyelitis[END_REF]Josse et al., , 2017)).

Cytotoxicity induced by S. aureus extracellular vesicles

Secretion is an important component of bacterial pathogenesis once the delivery of molecules and virulence factors contribute to the process of pathogen infection. Growing interest has been given to the study of extracellular vesicles (EVs), nanoparticles (30-150 nm) of lipid bilayer that are secreted by any living cell and carry bioactive compounds such as proteins, metabolites and nucleic acids, able to modulate the metabolism and physiology of local or distant targets cells, participating in pathogenesis and immunomodulation [START_REF] Brown | Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi[END_REF].

Various virulence factors have been identified being carried by EVs from different S. aureus strains, such as superantigens, toxins, coagulases, adhesins and other related proteins [START_REF] Askarian | Staphylococcus aureus Membrane-Derived Vesicles Promote Bacterial Virulence and Confer Protective Immunity in Murine Infection Models[END_REF][START_REF] Gurung | Staphylococcus aureus Produces Membrane-Derived Vesicles That Induce Host Cell Death[END_REF][START_REF] Jeon | Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells[END_REF][START_REF] Lee | Gram-positive bacteria produce membrane vesicles: Proteomics-based characterization of Staphylococcus aureus-derived membrane vesicles[END_REF][START_REF] Tartaglia | Staphylococcus aureus Extracellular Vesicles Elicit an Immunostimulatory Response in vivo on the Murine Mammary Gland[END_REF]. Interestingly, cytotoxicity of EVs towards host cells varies according to EVs proteome and cell line type studied.

Since virulence factors vary from a strain to another, so can the cargo of EVs released, affecting cytotoxicity and host cell response. It was demonstrated that the presence of αhemolysin in EVs is directly related to cell death, and EVs from α-hemolysin-negative strains have very low or no cytotoxic effect to different cell types [START_REF] Hong | An Important Role of α-Hemolysin in Extracellular Vesicles on the Development of Atopic Dermatitis Induced by Staphylococcus aureus[END_REF][START_REF] Thay | Staphylococcus aureus α-Toxin-Dependent Induction of Host Cell Death by Membrane-Derived Vesicles[END_REF].

As another example, EVs from M060 S. aureus strain containing exfoliative toxin A (ETA) were highly cytotoxic towards HEp-2 cells, contrary to EVs from three other S. aureus isolates that lack the same protein [START_REF] Jeon | Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells[END_REF]. Moreover, both intact and disrupted M060 EVs show the same cytotoxic at low concentrations towards HaCaT cells, while disrupted ATCC 25923 S. aureus strain EVs are four times less cytotoxic than intact EVs [START_REF] Kwon | Inhibitory effects of thymol on the cytotoxicity and inflammatory responses induced by Staphylococcus aureus extracellular vesicles in cultured keratinocytes[END_REF]. On the other hand, exposure of HaCaT cells to EVs from ATCC 25923 induces a stronger immune response than that of M060 EVs [START_REF] Kwon | Inhibitory effects of thymol on the cytotoxicity and inflammatory responses induced by Staphylococcus aureus extracellular vesicles in cultured keratinocytes[END_REF]. In a study by [START_REF] Tartaglia | Staphylococcus aureus Extracellular Vesicles Elicit an Immunostimulatory Response in vivo on the Murine Mammary Gland[END_REF], EVs secreted by S. aureus N305 strain were not cytotoxic to PS and MAC-T cells but induced the expression of cytokines in vitro, and promoted tissue inflammation, deterioration, and local cytokine production, in vivo [START_REF] Tartaglia | Staphylococcus aureus Extracellular Vesicles Elicit an Immunostimulatory Response in vivo on the Murine Mammary Gland[END_REF]. In some cases, EVs-associated molecules showed to be more efficient than extracts or soluble proteins to the induction of immune response and cell cytotoxicity [START_REF] Hong | An Important Role of α-Hemolysin in Extracellular Vesicles on the Development of Atopic Dermatitis Induced by Staphylococcus aureus[END_REF][START_REF] Kim | Staphylococcus aureus -derived extracellular vesicles induce monocyte recruitment by activating human dermal microvascular endothelial cells in vitro[END_REF]. These findings suggest that EVs cargo and integrity can influence host cell response and that even non-cytotoxic EVs can promote immunomodulation.

Different cell types also influence host-EVs interaction and consequently cytotoxicity and host cell responses. EVs from Staphylococcus aureus subsp. aureus Rosenbach MSSA476 induced extensive cell death to human neutrophils and THP1 cells, while caused minimal cytotoxicity to keratinocytes (HaCaT) at the same concentrations [START_REF] Askarian | Staphylococcus aureus Membrane-Derived Vesicles Promote Bacterial Virulence and Confer Protective Immunity in Murine Infection Models[END_REF]. This finding could be explained due to the difference between these cell types. Again in another study, S. aureus JE2 EVs showed to be less cytotoxic to airway epithelial cells (A549) than to erythrocytes and neutrophil-like HL60 cells [START_REF] Wang | Release of Staphylococcus aureus extracellular vesicles and their application as a vaccine platform[END_REF]. EVs can also induce production of cell adhesion molecules such as E-selectin, ICAM1 and VCAM1, and consequently, recruitment of monocytes (THP1) [START_REF] Kim | Staphylococcus aureus -derived extracellular vesicles induce monocyte recruitment by activating human dermal microvascular endothelial cells in vitro[END_REF], contributing to the infiltration of immune cells. Cell response can also differ between cell types, for example, after exposure to 14458 S. aureus ATCC EVs, alveolar macrophages produced TNF-α and IL-6, while A549 cells produced only IL-6 [START_REF] Kim | Staphylococcus aureusderived extracellular vesicles induce neutrophilic pulmonary inflammation via both Th1 and Th17 cell responses[END_REF]. Table 1 shows some examples of strainspecific EVs cytotoxicity. Altogether, this data shows that EVs can interact with and modulate the immune response of host cells, suggesting that EVs can play an important role in staphylococcal pathogenesis.

Impact on the host cell cycle

We previously demonstrated that culture supernatant compounds of some S. aureus strains are able to induce a G2/M phase transition delay in HeLa cells (Alekseeva et al., 2013). Phenolsoluble modulin α (PSMα) peptides are responsible for this host cell cycle alteration, which also correlated with a decrease in the defensin genes expression suggesting a diminution of antibacterial functions of epithelial cells. PSM production is strain-dependent and, by testing the supernatant of S. aureus human clinical isolates, we found that the degree of G2/M phase transition delay correlated with PSMα1 production (Deplanche et al., 2015). Similarly, the staphylococcal enterotoxin-like protein type O, an S. aureus superantigen, is also able to induce such phase transition delay. Aside from these PSM and SElO, also known as cyclomodulins [START_REF] El-Aouar Filho | Heterogeneous Family of Cyclomodulins: Smart Weapons That Allow Bacteria to Hijack the Eukaryotic Cell Cycle and Promote Infections[END_REF], the "lipoprotein-like" proteins (lpl) in S. aureus contribute to internalization into non-professional antigen-presenting cells such as keratinocytes (Nguyen et al., 2016). Lpl1, a representative of the lpl cluster, also caused G2/M phase transition delay. As lpl genes are carried by strain-specific genomic islands (νSaα) [START_REF] Baba | Genome Sequence of Staphylococcus aureus Strain Newman and Comparative Analysis of Staphylococcal Genomes: Polymorphism and Evolution of Two Major Pathogenicity Islands[END_REF], this Lpl associated phenotype is also strain-dependent. This mechanism might give an advantage to some strains in fostering an infection.

10. Impact on the innate immune response and on the inflammatory response Some bacterial traits are associated with a higher inflammatory response (Chen et al., 2018).

PBMC response in terms of IL-17 and IL-19 interleukins differs with the origin of the S. aureus strains. PBMC of a given individual will respond to interaction with the individual's endogenous strain by an increased production of IL-19 (an IL-17 suppressor), which may be regarded as a tolerogenic response. Contrarily, PBMC interaction with an exogenous strain (not carried by that individual) will induce a strong IL-17 response, which may facilitate S. aureus clearance.

IL-17 is indeed essential for protection against extracellular pathogens at mucosal surfaces [START_REF] Reiss-Mandel | Staphylococcus aureus Colonization Induces Strain-Specific Suppression of Interleukin-17[END_REF].

Non-proteinous surface components, like wall teichoic acid, may also play a role in the differential cell response. High WTA producer strains were shown to be more virulent in a mouse abscess model and induce stronger T cell proliferation and interferon gamma (IFNγ)

production by human T cells [START_REF] Wanner | Wall teichoic acids mediate increased virulence in Staphylococcus aureus[END_REF].

Impact on the epigenetic modifications in host cells

Epigenetic modifications as a consequence of host-pathogen interaction is of growing interest to understand and decipher how some opportunistic pathogens are rapidly cleared from the host body whereas others can evade the immune surveillance and cause recurrent and chronic infection. Epigenetic control mechanisms comprise DNA methylation, histone modification and the activity of non-coding RNAs. S. aureus induced epigenetic modifications in host cells is still poorly documented [START_REF] Modak | Epigenetic response in mice mastitis: Role of histone H3 acetylation and microRNA(s) in the regulation of host inflammatory gene expression during Staphylococcus aureus infection[END_REF]. [START_REF] Modak | Epigenetic response in mice mastitis: Role of histone H3 acetylation and microRNA(s) in the regulation of host inflammatory gene expression during Staphylococcus aureus infection[END_REF] investigated the inflammatory response in mice mammary tissue and the epigenetic modifications induced by two strains of S. aureus, isolated from field samples (cow mastitis milk) when used in experimental intramammary infection. Their results showed strain specific hyperacetylation at histone H3K9 and H3K14 residues. Transcriptome analysis in mammary tissue in the infected mice revealed a set of upregulated genes that significantly correlated with the promoter specific, histone H3K14 acetylation. Small RNA sequencing revealed several differentially expressed miRNAs in the infected mice mammary tissue. One of 2 S. aureus strains activated the NF-κB signaling leading to severe inflammation and induction of immune surveillance, which could possibly lead to rapid clearance of S. aureus strain. The other strain repressed the inflammatory response, which might result in a long-term infection of the host mammary tissue [START_REF] Modak | Epigenetic response in mice mastitis: Role of histone H3 acetylation and microRNA(s) in the regulation of host inflammatory gene expression during Staphylococcus aureus infection[END_REF].

Cell types and S. aureus invasion

The capacity for intracellular survival within professional phagocytes (monocytes, macrophages, and even neutrophils) is a critical factor facilitating the dissemination of S.

aureus in the host [START_REF] Garzoni | Staphylococcus aureus: new evidence for intracellular persistence[END_REF][START_REF] Kubica | A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages[END_REF]. The interactions between S. aureus and phagocytes have been extensively studied in recent years and focused mainly on neutrophils and macrophages [START_REF] Kubica | A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages[END_REF][START_REF] Lacoma | Investigating intracellular persistence of Staphylococcus aureus within a murine alveolar macrophage cell line[END_REF][START_REF] Nagl | Phagocytosis and killing of bacteria by professional phagocytes and dendritic cells[END_REF][START_REF] Rigby | Neutrophils in innate host defense against Staphylococcus aureus infections[END_REF][START_REF] Spaan | Neutrophils Versus Staphylococcus aureus : A Biological Tug of War[END_REF][START_REF] Tranchemontagne | Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages[END_REF], and recently on the mechanism of response of dendritic cells (DCs), which are cells that can influence immune responses promoting effector functions of other phagocytes [START_REF] Schindler | Dendritic cells are central coordinators of the host immune response to Staphylococcus aureus bloodstream infection[END_REF], in addition to activate the innate immune response [START_REF] Chan | Innate Immune Memory Contributes to Host Defense against Recurrent Skin and Skin Structure Infections Caused by Methicillin-Resistant Staphylococcus aureus[END_REF]. Bone marrow-derived dendritic cells (BMDCs) were able to kill S. aureus, however, certain strains of S. aureus were shown to have the ability to subvert DC death by autophagy (O' Keeffe et al., 2015).

It has been demonstrated that strains of S. aureus can subvert neutrophils in addition to being able to persist within macrophages derived from human monocytes suggesting that these phagocytic cells provide an intracellular niche that facilitates the dissemination of S. aureus in vivo, due to its mobility [START_REF] Derby | STUDIES ON BACTERIEMIA: V. THE EFFECT OF SIMULTANEOUS LEUKOPENIA AND RETICULOENDOTHELIAL BLOCKADE ON THE EARLY BLOOD STREAM CLEARANCE OF STAPHYLOCOCCI[END_REF][START_REF] Kubica | A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages[END_REF].

This mechanism of S. aureus dissemination is also seen in traditional intracellular bacteria, such as Mycobacterium tuberculosis and Listeria monocytogenes [START_REF] Drevets | Dissemination of <em>Listeria monocytogenes</em>by Infected Phagocytes[END_REF][START_REF] Schlesinger | Entry of Mycobacterium tuberculosis into mononuclear phagocytes[END_REF]. Besides, evidence exists that several strains of S. aureus can invade and persist within non-professional phagocytic cells such as endothelial, epithelial, osteoblasts, keratinocytes and fibroblasts [START_REF] Ellington | Mechanisms of Staphylococcus aureus invasion of cultured osteoblasts[END_REF][START_REF] Löffler | Staphylococcus aureus persistence in nonprofessional phagocytes[END_REF][START_REF] Sinha | Mechanism and consequences of invasion of endothelial cells by Staphylococcus aureus[END_REF][START_REF] Usui | Conspicuous Ingestion of Staphylococcus aureus Organisms by Murine Fibroblasts In Vitro[END_REF]. A pioneering study demonstrated the ability of endothelial cells to encompass S.

aureus [START_REF] Ogawa | Bacterial adherence to human endothelial cells in vitro[END_REF]. Besides, S. aureus can adhere and invade cultured osteoblasts [START_REF] Hudson | Internalization ofStaphylococcus aureusby cultured osteoblasts[END_REF]. Primary endothelial cells and epithelial cells are known to absorb a large number of bacteria and can ingest S. aureus, as well as osteoblasts and fibroblasts cells [START_REF] Löffler | Staphylococcus aureus persistence in nonprofessional phagocytes[END_REF]. These differences between cell types (primary cells and cell lines)

regarding bacterial degradation and host inflammatory processes have been revised [START_REF] Seidl | Divergent Responses of Different Endothelial Cell Types to Infection with Candida albicans and Staphylococcus aureus[END_REF]Tuchscherr et al., 2011).

A recent study demonstrated that most clinical isolates of S. aureus on endothelial cells have an invasive phenotype and the pro-inflammatory and cytotoxic effects depend on of invasion properties of the infectious strains [START_REF] Strobel | Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain[END_REF]. This study showed the analysis of the uptake of S. aureus 6850, SH1000 and Cowan1 into different host cell types like epithelial, endothelial, fibroblasts and osteoblasts have differences in intracellular bacterial number after the infection process [START_REF] Strobel | Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain[END_REF]. Besides, keratinocytes cells can ingest S. aureus but showed reduced levels if intracellular S. aureus in comparison with epithelial cells and endothelial cells.

S. aureus strains 6850 shown uptake different between primary cell and osteoblast cell line, with a low level of intracellular bacterium after infection in primary osteoblasts compared with an osteoblast cell line that presented a greater capacity of bacterial uptake [START_REF] Strobel | Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain[END_REF], suggesting differences in bacterial invasiveness. In addition, the intracellular persistence of S. aureus inside osteoblasts is also supported by the presence of SCVs. Clinical strains of S. aureus can persist within osteoblasts for several weeks. Persistence induces bacterial phenotypic diversity, including SCV phenotypes, accompanied by changes in virulence factor expression [START_REF] Tuchscherr | Clinical S. aureus Isolates Vary in Their Virulence to Promote Adaptation to the Host[END_REF].

Conclusion

We have begun to appreciate many general aspects of the inflammatory process in immune cells and their expert contributions to homeostasis and disease. However, we are still far from predicting how information collected by recognizing a particular agent (e.g. S. aureus) will be decoded by the immune system. It has been shown in this review that S. aureus can exert various effects on eukaryotic host cells. This pathogen has developed many strategies for adhering, invading and persisting in intracellular localization for long periods. The host cell response is highly complex, as both the selection of bacterial strains and host cell types play an important role in different cell responses. Although much progress has been made in understanding the molecular biology of this pathogen, much is still unknown. There are many examples where both professional and nonprofessional phagocytes contribute to homeostasis, but an interesting question is: How do these two cell populations communicate to ensure that inflammation is attenuated. The role in coupling innate and adaptive immunity via antigen presentation has unmasked several receptors, as well as signal transduction pathways and/or membrane trafficking routes that are only partially understood. Taken together, this shows that these speculative ideas are subject to experimental testing, which we hope will be the subject of further investigation. show that S. aureus-infected osteoblast-like MG-63 but not caspase-1 knockout CASP1 -/-MG-63 cells, which were generated using CRISPR/Cas9 technology, activate the inflammasome as monitored by the release of mature IL-1β. The effect was strain-dependent and significantly smaller in a natural mutant of the Agr quorum-sensing system of S. aureus as well as isogenic mutants in the Agr-controlled genes encoding phenol-soluble modulins. Furthermore, we found that the lack of caspase-1 in CASP1 -/-MG-63 cells impair the defense functions of osteoblasts, as bacterial clearance was drastically decreased in CASP1 -/-MG-63 compared to wild-type MG-63 cells. These data provide insight into the mechanism of S. aureus BJI and establish a crucial role of caspase-1 in bacterial clearance by osteoblasts.

Key words: Osteoblasts, Staphylococcus aureus, inflammasomes, caspase-1, IL-1β

Introduction

Staphylococcus aureus is a highly adaptive and versatile Gram-positive bacterium that has major importance for human and animal health [START_REF] Liu | Molecular pathogenesis of Staphylococcus aureus infection[END_REF]. S. aureus causes diseases ranging from relatively minor skin infections to life-threatening infections such as bacteremia, pneumonia, meningitis, endocarditis, and sepsis (2). S. aureus is also the main cause of bone and joint infections (BJI), particularly in the presence of orthopedic devices [START_REF] Kahl | Clinical Significance and Pathogenesis of Staphylococcal Small Colony Variants in Persistent Infections[END_REF]. Despite adequate antimicrobial therapy, BJI is linked to a high percentage of relapse, often leading to the development of chronic disease demanding particularly challenging treatments [START_REF] Del Pozo | Infection associated with prosthetic Joints[END_REF]. While research into treatment options has been focused mostly on antibacterial strategies, it is widely believed that the treatment of recurrent S. aureus infections would also benefit considerably from reinforcement of host defenses (5), and a deeper understanding of the immune response is crucial for the development of new anti-infective strategies.

The innate immune response plays a crucial role in the defense against pathogens and is initiated through pattern recognition receptors (PRRs), which recognize microbial pathogenassociated molecular patterns (PAMPs) and endogenous danger-associated molecular patterns (6). This leads to the activation of host defense pathways that results in the clearance of infections. The innate immune response against microbes involves a major inflammatory pathway known as the activation of inflammasomes, multi-protein signaling complexes that assemble after recognition of danger signals and/or pathogens [START_REF] Schroder | The inflammasomes[END_REF]. Inflammasomes consist of a family of cytosolic receptors called NLRs (nucleotide-binding domain and leucine-rich repeats containing receptors), PYHIN protein family and enzymatic components, most notably caspase-1 and less frequently caspase-11 [START_REF] Broz | Inflammasomes: mechanism of assembly, regulation and signalling[END_REF]. Caspase-1 is synthesized in cells as an inactive precursor of 45 kDa and forms two subunits of 20 and 10 kDa after inflammasome activation [START_REF] Lamkanfi | Mechanisms and functions of inflammasomes[END_REF]. Most inflammasomes also use an adaptor molecule known as ASC (apoptosis-associated speck-like protein) [START_REF] Mariathasan | Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf[END_REF].

After stimulation with pathogens or damage-associated molecular patterns, inflammasome assembly leads to the autocatalytic cleavage of caspase-1, processing of pro-IL-1β and pro-IL-18 into their mature active forms and their secretion from cells [START_REF] Strowig | Inflammasomes in health and disease[END_REF].

Furthermore, inflammasome activation may trigger pyroptosis, an inflammatory form of cell death. The role of different nuclear receptors, as well as the PYHIN proteins AIM2 and IFI16

in assembling inflammasomes and controlling the innate immune response against various microbes, has been intensively explored [START_REF] Higa | Lose the battle to win the war: bacterial strategies for evading host inflammasome activation[END_REF][START_REF] Vladimer | Inflammasomes and host defenses against bacterial infections[END_REF]. Special attention has been paid to the investigation of the role of inflammasomes during infection with intracellular pathogens, like Salmonella typhimurium, Legionella pneumophila, Listeria monocytogenes and other Gramnegative bacteria [START_REF] Amer | Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf[END_REF][START_REF] Maltez | Inflammasomes coordinate pyroptosis and natural killer cell cytotoxicity to clear infection by a ubiquitous environmental bacterium[END_REF][START_REF] Morales | A type I IFN-dependent DNA damage response regulates the genetic program and inflammasome activation in macrophages[END_REF][START_REF] Zaki | Salmonella exploits NLRP12dependent innate immune signaling to suppress host defenses during infection[END_REF]. Some studies were also performed investigating the role of inflammasomes in phagocytes and epithelial cells during infection with the Gram-positive pathogen S. aureus [START_REF] Kremserova | Frontline Science: Staphylococcus aureus promotes receptorinteracting protein kinase 3-and protease-dependent production of IL-1β in human neutrophils[END_REF][START_REF] Ma | LncRNA XIST mediates bovine mammary epithelial cell inflammatory response via NF-κB/NLRP3 inflammasome pathway[END_REF]. However, an involvement of inflammasomes and processed IL-1β, which plays a crucial role in bone homeostasis [START_REF] Ruscitti | The role of IL-1β in the bone loss during rheumatic diseases[END_REF], in S. aureus-associated BJI has not yet been demonstrated.

In the present work, we discovered that in response to S. aureus infection, osteoblast-like wildtype (WT) MG-63 cells activate inflammasomes in contrast to caspase-1 knockout CASP1 -/- MG-63 cells, which we generated using CRISPR/Cas9 technology. Inflammasome activation was monitored by caspase-1 activation and the release of mature soluble IL-1β. Mutant S.

aureus strains were employed, demonstrating a key contribution of phenol-soluble modulins (PSMs) in triggering the release of soluble IL-1β, which is strongly implicated in the pathology of BJI [START_REF] Ruscitti | The role of IL-1β in the bone loss during rheumatic diseases[END_REF][START_REF] Lee | IL-1 plays an important role in the bone metabolism under physiological conditions[END_REF]. We demonstrated that in addition to the key role of caspase-1 in the release of pro-inflammatory cytokines in infected non-phagocytic cells, caspase-1 is implicated in cell defense mechanisms like as a restriction of the intracellular replication of bacteria that is critical in the induction of both innate and adaptive immune processes. Supernatants of ThP-1+LPS+ATP (6 h) were diluted 1:5, the result presented in Fig. 1C is multiplied by the dilution factor. In contrast, IL-1β was not detected 2 h post-treatment, while only 4 pg/ml of IL-1β was detected in MG-63 cells 6 h after the exposure to LPS+ATP.

Deletion of caspase-1 gene in human osteoblasts MG-63 using the CRISPR/Cas9

gene editing system.

It has been shown that stimulation is required for IL-1β transcription and protein expression by MG-63 cells [START_REF] Deplanche | Staphylococcus aureus induces DNA damage in host cell[END_REF]. Since MG-63 cells are commonly used as a model to study BJI [START_REF] Richter | Induction of macrophage function in human THP1 cells Is associated with rewiring of MAPK signaling and activation of MAP3K7 (TAK1) protein kinase[END_REF] we used MG-63 cells for CRISPR/Cas9-mediated CASP1 deletion to study the involvement of inflammasomes in S. aureus infection of osteoblasts. There are 6 isoforms of caspase-1 encoded by the CASP1 gene, located on chromosome 11 [START_REF] Mali | RNAguided human genome engineering via Cas9[END_REF]. Caspase-1 alpha and beta are the main transcripts in human cells. To generate CASP1 deletion MG-63 cells we used a previously validated sgRNA ATTGACTCCGTTATTCCGAA [START_REF] Schmid-Burgk | Caspase-4 mediates non-canonical activation of the NLRP3 inflammasome in human myeloid cells[END_REF] (Fig. 2A).

Based on sequences analysis, the sgRNA is predicted to target four Caspase-1 isoforms including alpha and beta. Following CRISPR lentiviral infection and puromycine selection, individual clones, which were identified as a result of limiting dilution of puromycin-treated cells, were tested by Western blotting for the lack of the 45-kDa band corresponding to pro-caspase-1. Finally, a deletion clone (CASP1 -/-MG-63, G2 clone) showing CRISPR-induced genomic alteration (Fig. 2B) was selected for further analysis. Western blot analysis of WT MG-63 vs CASP1 -/-MG-63 cells confirmed the lack of pro-caspase-1 in CASP1 -/-MG-63 cells (Fig. 3A).

Inflammasomes involvement in caspase-1 dependent IL-1β release by S. aureus infected MG-63 osteoblasts

Activation of the NLRP3 inflammasome involves the oligomerization of the adaptor protein ASC (apoptosis-associated speck-like protein containing A CARD) into speck-like aggregates [START_REF] Sanjana | Improved vectors and genome-wide libraries for CRISPR screening[END_REF]. This structure recruits and activates pro-caspase-1, which promotes the maturation of IL 1β and IL-18.

To ensure that the deletion of CASP1 does not impair upstream events of inflammasome formation, the level of NLRP3 protein expression and the formation of ASC specks was showed an MFI increase from 7900 to 14000 (Fig. 3B).

Taking into account that both types of cells contain NLRP3 (Fig. 3B), while pro-caspase-1 is present only in WT MG-63 cells (Fig. 3A) we next determined whether an exposure of both cell lines to S. aureus SA113 leads to the formation of ASC specks, since their formation does not require the presence of pro-caspase-1. Consequently, we analyzed immunofluorescence staining for the adaptor ASC in WT and CASP1 -/-MG-63 cells. As shown in Fig. 3C,S. aureus infection triggers assembly of the ASC specks in the cytosols of WT as well as CASP1-/-MG-

cells (green).

Previously it was reported that the transcripts of IL-1β and IL-18 were observed after an exposure of mice osteoblasts to S. aureus, however the secretion of IL-1β and IL-18 was not detected despite the presence of active caspase-1 [START_REF] Gilot | A non-coding function of TYRP1 mRNA promotes melanoma growth[END_REF].

To understand whether S. aureus activates inflammasomes in human osteoblast-like MG-63 cells, we measured IL-1β production in WT and CASP1 -/-MG-63 cells. The presence of NLRP3

and the lack of pro-caspase-1 in CASP1 -/-MG-63 cells allows these cells to be used as a control for the estimation of NLRP3 inflammasome activity in MG-63 cells by the measurement of IL-1β, which is processed due to caspase-1. The assessment of IL-1β in WT and CASP1 -/- MG-63 cells exposed to inflammasomes activators LPS+ATP (Fig. 3D) as well as to S. aureus LAC (Fig. 3E) revealed the lack of IL-1β production in CASP1 -/-MG-63 in contrast to WT MG-63 cells.

To clarify the possible role of human osteoblasts in the production of IL-1β, we analyzed the kinetics (from 2 to 11 days) of IL-1 β production by WT and CASP1 -/-MG-63 cells exposed to S. aureus SA113. As demonstrated in Fig. 4A, the level of IL-1β increased from day 2 to 11.

In contrast, IL-1β was not detected in supernatants from infected CASP1 -/-MG-63 cells during the tested period.

To verify whether IL-1β release by infected osteoblasts was strain-dependent, we analyzed the kinetics of IL-1 β release by MG-63 exposed to different S. aureus strains: LAC (USA300), MW2 (USA 400) and SA113. As shown in Fig. 4B, all strains induce IL-1β release; however, MW2 and LAC induced significantly higher levels of IL-1β than SA113. IL-1β was not detected in the supernatants of MG-63 cells exposed to killed bacteria of any of the three strains, suggesting that factors associated with viable bacteria are involved in inflammasome activation.

2.4. Pivotal role of S. aureus PSM toxins in stimulation of IL-1β release by infected osteoblasts.

The quorum-sensing system in S. aureus known as the accessory gene regulator (Agr)

regulates the expression of many virulence factors. Agr regulates main S. aureus toxins, among which regulation of PSMs (PSMα 1 to 4, PSMβ 1 and 2, and δ-toxin sometimes called PSMƔ) is exceptionally direct and strict [START_REF] Joo | The isolation and analysis of phenol-soluble modulins of Staphylococcus epidermidis[END_REF], [START_REF] Wang | Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA[END_REF]. It has previously been shown that PSMs stimulate the production of inflammatory cytokines by infected keratinocytes [START_REF] Mauthe | WIPI-1 positive autophagosome-like vesicles entrap athogenic Staphylococcus aureus for lysosomal degradation[END_REF]. Furthermore, our results on strain dependence of IL-1β stimulation in osteoblasts suggested an involvement of Agr, as the low levels of IL-1β in cells infected with strain SA113 as opposed to those infected with strains MW2 and LAC correlate with levels of Agr functionality in those strains.

SA113 is functionally Agr-defective, while MW2 and LAC show high Agr activity. These considerations prompted us to investigate the role of PSMs in IL-1β production by infected osteoblasts.

First, we analyzed LAC (USA300) wild-type and its isogenic mutant LACΔpsmαβhld (S. aureus strain lacking PSMα, PSMβ and δ-toxin) for their ability to stimulate the release of IL-1β. As shown in Fig. 5A, the level of IL-1β was significantly decreased in the supernatants of WT MG-63 cells exposed to LAC Δpsmαβhld compared to WT MG-63 cells exposed to LAC wild-type on days 5 and 9 post-infection. To further investigate which PSM were involved in the simulation of IL-1β release, we used the PSM deletion strain LACΔpsmαβhld and complemented strains, expressing either the four PSMα peptides (LACΔpsmαβhld-pTXΔα1-4), the two PSMβ peptides (LACΔpsmαβhld-pTXΔβ1-2), or the δ-toxin (LACΔpsmαβhld-pTXΔhld) and monitored IL-1β levels in cell exposed to those strains up to 9 days postinfection. As shown in Fig. 5B, there was a significant decrease in the IL-1β released by cells exposed to the LACΔpsmαβhld strain compared to LAC. Exposure to complemented mutants demonstrated that the release was partially restored when the strains were complemented with PSMα (LACΔpsmαβhld-pTXΔα1-4), PSMβ (LACΔpsmαβhld pTXΔβ1-2) or δ-toxin (LACΔpsmαβhld-pTXΔhld). However, the difference was statistically significant only when strains were complemented with PSMβ or, at earlier time points, δ-toxin (LACΔpsmαβhld-pTXΔhld) (Fig. 5).

S. aureus clearance by osteoblasts depends on caspase-1

It was reported that caspase-1 is essential for the restriction of intracellular pathogen replication in professional phagocytes [START_REF] Amer | Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf[END_REF], however the role of caspase-1 in bacterial clearance in non-professional phagocytes, which include osteoblasts, has not been investigated. Therefore, we analyzed whether the lack of caspase-1 in CASP1 -/-MG-63 cells is correlated with a failure to control the intracellular replication of S. aureus bacteria. The quantity of intracellular bacteria during S. aureus infection of WT and CASP1 -/-MG-63 cells was determined after gentamicin treatment of infected cells as described in Material and Methods. According to CFU determination bacterial internalization was not impaired in CASP1 - /-MG-63 cells, as we observed equivalent levels of internalized bacteria in WT MG-63 cells at 2 h post-infection at ~ 10 2 CFU/10 5 host cells. Six and 24 hours post-infection significantly higher numbers of viable bacteria were recovered from CASP1 -/-MG-63 cells compared to WT MG-63 cells (Fig. 6A). These results suggest that the lack of caspase-1 impairs the ability of osteoblasts to restrict S. aureus growth. These findings were further confirmed using confocal microscopy. As shown in Fig. 6B,6 h post-infection a considerably higher number of intracellular S. aureus bacteria (red) was observed in CASP1 -/-MG-63 cells than WT MG-63 cells, while assembly of the ASC specks was observed in both cell types (green).

Discussion

Despite antibiotic therapy associated with complex surgical procedures, Staphylococcus aureus BJI is particularly difficult to treat because bacteria employ multiple approaches such as the formation of biofilm and small-colony variants as well as the invasion and intraosteoblastic persistence, leading to chronicity and relapse [START_REF] Alekseeva | Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency[END_REF]- [START_REF] Bouchard | Inhibition of Staphylococcus aureus invasion into bovine mammary epithelial cells by contact with live Lactobacillus casei[END_REF]. We aimed to investigate the contributions of internalized S. aureus on alteration of host defense events such as inflammasomes activation and bacterial clearance in infected osteoblasts.

Activation of inflammasomes during infection have been extensively characterized in

professional phagocytes [START_REF] Berkova | Aspergillus fumigatus conidia inhibit tumour necrosis factor-or staurosporine-induced apoptosis in epithelial cells[END_REF]- [START_REF] Kazemzadeh-Narbat | Drug release and bone growth studies of antimicrobial peptide-loaded calcium phosphate coating on titanium[END_REF]. The implication of inflammasomes in response of S. aureus infected osteoblasts, which play a pivotal role in BJI, was not demonstrated. Bone homeostasis is kept by an equilibrium between bone resorption and bone formation [START_REF] Ruscitti | The role of IL-1β in the bone loss during rheumatic diseases[END_REF]. Osteoblasts synthesize various bone matrix proteins that are involved in bone formation, as well as regulate the maturation of osteoclasts responsible for bone resorption. IL-1β, a proinflammatory cytokine, plays a pivotal role in bone formation and bone resorption [START_REF] Lee | IL-1 plays an important role in the bone metabolism under physiological conditions[END_REF]. IL-1β is mainly expressed by monocytes, macrophages, and dendritic cells [START_REF] Masumoto | Pyrin N-terminal homology domain-and caspase recruitment domain-dependent oligomerization of ASC[END_REF]- [START_REF] Queck | RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus[END_REF], and is processed into a soluble mature form by the inflammasomes compound, active caspase-1. In the present work we focused on the monitoring of soluble IL-1β produced by infected osteoblasts.

We demonstrated that human osteoblast-like MG-63 cells release a soluble IL-1β starting from Moreover, a low level of IL-1β is produced at the beginning of the infection, thus the monitoring of IL-1β depends from the sensitivity of the detection method. Rising IL-1β production in S.

aureus-infected MG-63 cells in contrast to its lack in infected CASP1 -/-MG-63 cells demonstrated caspase-1 dependent mechanisms of IL-1β production by human osteoblasts.

Our data corroborate observations of others that demonstrated caspase-1 depending mechanism of IL-1 beta processing and its release by mice macrophages during infection [START_REF] Tuchscherr | Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection[END_REF].

The absence of IL-1β production by cells exposed to heat-killed bacteria indicates that factors associated with viable bacteria are involved in the activation of NLRP3 inflammasome.

Employment of deletion and complemented PSMs mutants demonstrated a pivotal role of S.

aureus toxins PSMs in inflammasomes related IL-1β production by infected osteoblastic cells.

We would like to highlight that IL-1β production by infected osteoblasts appears to be specifically dependent on PSM betas among PSMs. This is noteworthy, as we don't know any specific phenotypes attributable to those PSMs so far. In addition to PSMs the action of other factors cannot be excluded since complemented mutants did not restore entirely the level of secreted IL-1β.

The role of caspase-1 in the production of inflammatory cytokines has been extensively investigated [START_REF] Marriott | Bacterial infection of osteoblasts induces interleukin-1beta and interleukin-18 transcription but not protein synthesis[END_REF], [START_REF] Vuong | Investigational drugs to treat methicillin-resistant Staphylococcus aureus[END_REF], while much less attention has been paid to its impact on other immune processes. Nevertheless, recent investigations point out that additionally to the processing of IL-1β and IL-18 caspase-1 regulates unconventional protein secretion [START_REF] Broz | Inflammasomes: intracellular detection of extracellular bacteria[END_REF], activates lipid metabolic pathway [START_REF] Lamkanfi | The Inflammasomes[END_REF], restricts pathogens replication in professional phagocytes [START_REF] Miller | Inflammasome-mediated production of IL-1beta is required for neutrophil recruitment against Staphylococcus aureus in vivo[END_REF], [START_REF] Shimada | Staphylococcus aureus evades lysozyme-based peptidoglycan digestion that links phagocytosis, inflammasome activation, and IL-1beta secretion[END_REF] and induces pyroptosis, proinflammatory cell death [START_REF] Chang | Dectin-2 is a primary receptor for NLRP3 inflammasome activation in dendritic cell response to Histoplasma capsulatum[END_REF]. Our investigation of host defense event during S. aureus infection of non-professional phagocytes, such as the control of intracellular bacterial proliferation, reveals a drastic increase in the proliferation of internalized bacteria in osteoblastic CASP1 -/-MG-63 cells. This finding is in keeping with the previous reports demonstrating the correlation of the lack of caspase-1 activation with a failure to restrict the replication of S. aureus inside of phagocytic cells [START_REF] Franchi | NLRC4-driven production of IL-1β discriminates between pathogenic and commensal bacteria and promotes host intestinal defense[END_REF], [START_REF] Lima-Junior | Inflammasome-derived IL-1β production induces nitric oxide-mediated resistance to Leishmania[END_REF]. Indeed, it has been demonstrated that during S. aureus infection of professional phagocytes NLRP3 inflammasomes and caspase-1 regulate phagosomes acidification that is essential for the activation of the hydrolytic enzymes required for killing of internalized bacteria [START_REF] Franchi | NLRC4-driven production of IL-1β discriminates between pathogenic and commensal bacteria and promotes host intestinal defense[END_REF], [START_REF] Lopez-Castejon | Understanding the mechanism of IL-1β secretion[END_REF].

Moreover, it was suggested that early activation of NLRP3 [START_REF] Franchi | NLRC4-driven production of IL-1β discriminates between pathogenic and commensal bacteria and promotes host intestinal defense[END_REF], [START_REF] Lopez-Castejon | Understanding the mechanism of IL-1β secretion[END_REF] inflammasomes is triggered by events, such as production of ROS that are associated with the phagocytic process in macrophages [START_REF] Franchi | NLRC4-driven production of IL-1β discriminates between pathogenic and commensal bacteria and promotes host intestinal defense[END_REF]. Recently, we demonstrated that S. aureus induce ROS in WT MG-63 cells [START_REF] Grahames | Pharmacological characterization of ATP-and LPS-induced IL-1beta release in human monocytes[END_REF]. We hypothesize that S. aureus-induced ROS play the similar role in inflammasome activation in non-phagocytic cells.

Collectively, our results demonstrate that human osteoblast-like MG-63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and processing of IL-1β, the master cytokine of inflammation [START_REF] Spano | PMA withdrawal in PMA-treated monocytic THP1 cells and subsequent retinoic acid stimulation, modulate induction of apoptosis and appearance of dendritic cells[END_REF]. The outcome of the infection depends on the balance between the host immune response and the action of main S. aureus virulence factors, PSMs, whose production may differ among the S. aureus strains, as we already observed in our previous work [START_REF] Raupach | Caspase-1-mediated activation of interleukin-1beta (IL-1beta) and IL-18 contributes to innate immune defenses against >Salmonella enterica</i> serovar Typhimurium infection[END_REF]. Besides our observations of inflammasomes activation, we demonstrated that CASP1 -/-MG-63 cells were unable to restrict the intracellular replication of S. aureus. It points out that the active caspase-1 prevent exacerbated intracellular replication of S. aureus in non-professional phagocytes in addition to professional phagocytes, suggesting the universal pivotal role of caspase-1 in pathogens clearance independently from the type of cells. Our results raise the fascinating possibility, that pathogens, which inhibit caspase-1 activation, do so not only to govern the generation of inflammatory cytokines but also to impede the bacterial replication in infected osteoblasts that are critical for the defense response during BJI. These findings deserve further investigation to elucidate the ability of pathogens to inhibit inflammasomes activation and disturb the function of phagocytic vesicles in osteoblasts in order to evade host cells defense response. Current findings redefine our understanding of the role of osteoblasts in BJI, suggesting that osteoblasts are not passive bystanders, but active players in host defenses against S. aureus infection.

4. Materials and Methods:

Maintenance of eukaryotic cells lines

The human osteoblast-like MG-63 (LGC Standards, Teddington, UK) cells and human monocyte ThP1 cells (ATCC TIB-202) were cultured in DMEM and RPMI 1640 respectively, supplemented with 10% fetal calf serum (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2 as described [START_REF] Grahames | Pharmacological characterization of ATP-and LPS-induced IL-1beta release in human monocytes[END_REF]. Trypsin/EDTA (Sigma) was used for cell subculturing to release adherent cells. In order to differentiate ThP1 cells into macrophages, cells were treated with 20 nM phorbol 12-myristate 13-acetate (PMA) for 48 h [START_REF] Sollberger | Caspase-1: the inflammasome and beyond[END_REF].

4.4.

Deletion of the caspase-1 gene in human osteoblast-like MG-63 cells using the CRISPR/Cas9 gene editing system.

The caspase-1 gene in MG-63 cells was deleted using CRISPR/Cas9 technology [START_REF] Keller | Active caspase-1 is a regulator of unconventional protein secretion[END_REF]. The sgRNA sequence (ATTGACTCCGTTATTCCGAA), which as previously published targets the CASP1 gene [START_REF] Schmid-Burgk | Caspase-4 mediates non-canonical activation of the NLRP3 inflammasome in human myeloid cells[END_REF], was cloned into the lentiCRISPR v2 (Addgene #52961) [START_REF] Gurcel | Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival[END_REF]. Deletion was achieved via lentiviral infection of MG-63 cells. Lentiviral production has been performed as recommended (http://tronolab.epfl.ch) and previously described [START_REF] Akhter | Caspase-7 activation by the Nlrc4/Ipaf inflammasome restricts Legionella pneumophila infection[END_REF]. After antibiotic selection

[puromycine (3 days, 1µg/mL)], cells were seeded in 96-well plates at 0.3 cells/well for singlecell clonal expansion. Single-cell clones were tested by Western blot analysis for the presence of the 45-kDa band corresponding to pro-caspase-1. Clones that did not present 45-kDa bands were recloned in 96-well plates (10 and 30 cells per plate). Afterward, single-cell clones were transferred in 6-wells plate and grown to 80% of confluence. Again, the clones that lacked 45-kDa bands (Western Blot) were selected and grown in 25-ml flasks. CRISPR-induced genomic modifications were verified by nucleotide sequence analysis, and the lack of caspase-1 was confirmed by Western blot analysis.

4.5.

S. aureus strains description

The methicillin-resistant strains S. aureus MW2 (USA400), LAC (USA300) wild type, the isogenic mutant LAC ∆psmαβhld, which lacks the psmα and psmβ operons and in which translation of the hld gene is abolished by mutation of the start codon, LAC (pTX∆16) which carries the control plasmid, the deletion mutant LAC∆psmαβhld (pTX∆16) and the complemented strains expressing either the four PSMα peptides (LAC∆psmαβhld-pTX∆α1-4), the two PSMβ peptides (LAC∆psmαβhld-pTX∆β1-2), or the δ-toxin (LAC∆psmαβhld-pTX∆hld)

were obtained from the Laboratory of Bacteriology, NIH, USA [START_REF] Master | Mycobacterium tuberculosis prevents inflammasome activation[END_REF][START_REF] Fernandes-Alnemri | The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation[END_REF]. The pTX∆16 plasmids were derived from plasmid pTX15 with the deletion of the xylR repressor gene. The tetracycline-resistant strains harboring plasmid pTX∆16 were grown in BHI containing 12.5 µg/ml of tetracycline. S. aureus SA113 and SA113 (pctuf-mCherry) strain which carries plasmid-encoded mCherry (red fluorescence) fused with the propeptide of lipase for fluorescence enhancement were obtained from the Laboratory of Microbial Genetics, University of Tübingen [START_REF] Sokolovska | Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function[END_REF]. Aliquots from overnight cultures on BHI broth were diluted (1:50) in DMEM. The growth curves of mutants were similar to that of the wild type. Strains were grown at 37°C under anaerobic conditions until cultures reached an optical density of 0.6 at 600 nm, corresponding to 10 8 CFU/ml (Colony Forming Units). CFU was determined after plating a bacterial suspension on BHI agar followed by overnight incubation as described ( 64).

4.6.

Cell culture infection 2.5x10 5 of either MG-63, CASP1 -/-MG-63, or ThP1 cells were seeded in the wells of 12-well plates. ThP1 were treated with PMA as described above before treatment. Cells then were exposed to S. aureus strains at MOI 50:1. An MOI value of 50:1 was selected in order to prevent the induction of host cell death. Bacterial concentrations were estimated spectrophotometrically and were confirmed by the determination of CFU. Extracellular bacteria were removed 2 h post-infection by incubating cells in cDMEM with 20 μg/ml lysostaphin and 100 μg/ml gentamicin for 2 h, which eliminates extracellular bacteria without altering intracellular bacteria [START_REF] Raupach | Caspase-1-mediated activation of interleukin-1beta (IL-1beta) and IL-18 contributes to innate immune defenses against >Salmonella enterica</i> serovar Typhimurium infection[END_REF][START_REF] Cohen | S. aureus evades macrophage killing through NLRP3-dependent effects on mitochondrial trafficking[END_REF], followed by incubation in cDMEM containing 25 μg/ml of gentamicin. After the indicated periods adherent and floating, cells were collected, centrifuged, and prepared either for Western blot analysis or for analysis by cytofluorometry. For the determination of the number of internalized bacteria, we used the method we have previously described [START_REF] Cohen | S. aureus evades macrophage killing through NLRP3-dependent effects on mitochondrial trafficking[END_REF][START_REF] Ip | Phagocytosis and phagosome acidification are required for pathogen processing and MyD88-dependent responses to Staphylococcus aureus[END_REF]. Briefly, to determine the number of internalized bacteria after 2 h of infection, infected cells were lysed with 0.05% Triton X-100 in PBS, cell lysates were plated on BHI agar at different dilutions, and CFU was determined after overnight incubation as described [START_REF] Cohen | S. aureus evades macrophage killing through NLRP3-dependent effects on mitochondrial trafficking[END_REF][START_REF] Ip | Phagocytosis and phagosome acidification are required for pathogen processing and MyD88-dependent responses to Staphylococcus aureus[END_REF].

Western blot analysis

Pro-caspase-1 and cleaved caspase-1 were detected by Western blot analysis as described (66). Briefly, 2.5x10 5 of cells were grown in 12-well plates for 12 h. Pro-caspase-1 was detected in non-stimulated and stimulated cells, while active caspase-1 was detected solely in stimulated cells. Wild type (WT) MG-63 or CASP1 -/-MG-63 cells were primed for 3 h with LPS (1μg/ml) (Sigma) and selected samples were stimulated with ATP (5 mM) for 15 min or after the exposure to S. aureus strains. Six hours after the beginning of the treatment cells together with cell-culture supernatants were collected and diluted in RIPA lysis buffer supplemented with complete protease inhibitor cocktail, P8340 (Sigma Aldrich ® ). Protein samples were prepared in 5x polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer.

Proteins were separated on 12% SDS-PAGE gels and transferred onto 0.2 µM PVDF membranes. The membranes were washed three times in PBS-T (Tris-buffered saline containing 0.5% Tween 20) and blocked in 10% skim milk in PSB-T for 1 h at room temperature before being incubated with primary antibody overnight at 4°C. Caspase-1 was detected with anti-human caspase-1 antibody (1:1,000 dilution, mAb p/20, AdipoGen). Membranes were washed four times in PBS-T and incubated with HRP-conjugated secondary anti-mouse IgG-HRP antibody 1:10,000 dilution, Cell Signaling Ozyme) for 1 h at room temperature, washed another four times, and incubated with Western chemiluminescent HRP substrate (ECL kit) (GE Healthcare). The image was processed using a GBOX imaging system (Syngene, Ozyme, Poitiers, France). To assess the quantity of loaded protein, membranes were re-probed with rabbit anti-tubuline (Cell Signaling Ozyme France) (1:1000) and bands were visualized using the ECL kit and a GBOX imaging system.

Flow cytometry analysis

For an estimation of NLRP3 protein expression in WT MG-63 vs CASP1 -/-MG-63 cells, cells were exposed to S. aureus LAC for 2 h and were fixed in 4% paraformaldehyde/PBS followed by permeabilization in 0.1% Triton/0.5% BSA/PBS. After centrifugation, cells were resuspended in 200 μl of 0.5% BSA/PBS, 5μl of Alexa Fluor 488 rat anti-human NLRP3 antibody (RDsystems, France, IC7578G) per 106 cells was added, and the samples were incubated for 45 min. NLRP3 expression was analyzed with an Accuri C6 flow cytometer. Data were collected from 20,000 cells, and analyzed with CFlow software (Becton Dickinson) as described [67].

Confocal microscopy

WT MG-63 or CASP1 -/-MG-63 cells were grown on coverslips and exposed to S. aureus SA113 (MOI 50:1) for 2 h. Six and 24 h post-infection, cells were fixed with 4% paraformaldehyde/PBS for 20 min, followed by permeabilization in 0.1% triton/PBS and incubation with 20% goat serum (Sigma) as described [START_REF] Grahames | Pharmacological characterization of ATP-and LPS-induced IL-1beta release in human monocytes[END_REF]. Rabbit anti-PYCARD antibody (Coger France) diluted 1:50 in 1% BSA/PBS was applied overnight at 4°C, followed by incubation with Alexa Fluor 488 labeled goat anti-rabbit antibody (Cell Signaling Ozyme)

IL-1β quantification by ELISA

Undiluted cell culture supernatants were subjected to human IL-1β detection by sandwich-ELISA (Thermofischer Life Technologies) according to the manufacturer's instructions. Briefly, wells of 96-wells plates were coated with capture anti-human IL-1β antibody and were incubated overnight at 4°C. After washing the wells were incubated in the blocking solution.

Then tested samples were added to the appropriate wells. A standard curve was measured with 2-fold serial dilutions of the maximal standard concentration of 150 pg/ml. Recombinant human IL-1β was used for generating the standard curve and calibrating samples. After 2 h of incubation, biotin-conjugated anti-human IL-1β antibody was added to the wells followed by incubation with avidin-HRP for 30 min. After the addition of the tetramethylbenzidine substrate solution for 15 min, the reaction was stopped with stop solution and absorbance was read at 450 nm. The sensitivity of IL-1β detection was 2 pg/ml.

Statistical analysis

At least three independent assays were performed per experiment. The differences among the groups were assessed by analysis of variance (ANOVA). P-values < 0.05 were considered to be significant. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as mean ± standard deviation (±SD). 63 cells by Western blot analysis was performed using anti-caspase-1 antibody (AdipoGen) as described in Material and Methods. The protein load was verified with anti-tubulin antibody.

Three independent assays were performed.

B, C. MG-63 or ThP1 cells were incubated in 12-well plates. Afterwards, cells were primed for 2 h with LPS (1 μg/ml) and stimulated with ATP (5 mM) for 15 min. ThP1 were treated with PMA before LPS+ATP treatment as described above. Two or six hours after the beginning of the treatment cell supernatants were collected, centrifuged, and the level of IL-1β was determined by a commercial sandwich-ELISA (Thermofischer Life Technologies) as described in Material and Methods. Supernatants of ThP-1+LPS+ATP were diluted 1:5. The values are presented as concentrations in pg/ml. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). Comparisons with P-values < 0.05 (*) were considered to be significant. *, P < 0.05; **, P < 0.01. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups.

The values are expressed as means ± standard deviation (±SD). Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). P-values < 0.05 were considered to be significant. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups.

The values are expressed as means ± standard deviation (±SD).

four PSMα peptides (LAC∆psmαβhld-pTX∆α1-4), the two PSMβ peptides (LAC∆psmαβhld-pTX∆β1-2), or the δ-toxin (LAC∆psmαβhld-pTX∆hld) at MOI 1:50 for 2 h followed by antibiotic treatment as described in Material and Methods. After various times post-infection (2 days, 5 days, 7 days and 9 days) cell supernatants were collected, centrifuged and the level of IL-1β was determined by a sandwich-ELISA (Thermofischer Life Technologies) as described in Material and Methods. The values are presented as concentrations in pg/ml. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). P-values< 0.05 (*) were considered to be significant. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups.

The values are expressed as means ± standard deviation (±SD). The evaluation of the activation of the inflammasome was made based on the activation of caspase-1 subunit p/10 and measured by western blotting. Cells were exposed to S. aureus strains at MOI 50:1. A maximal MOI value of 50:1 was selected to prevent the induction of a host cell death. Bacterial concentrations were estimated spectrophotometrically and were confirmed by determination of colony-forming unit (CFU). CFU was determined after overnight incubation as described previously (ALEKSEEVA et al., 2013). Our results demonstrated that MG-63 cells in culture medium with Fetal Calf Serum (FCS) infected by S. aureus strains USA 300 and MW2 (USA 400), as well as with the combination of LPS and nigericin, which are known activators of inflammasome (LATZ; XIAO; STUTZ, 2013a; MAN; [START_REF] Man | Regulation of inflammasome activation[END_REF] were able to activate caspase-1.

Interestingly, as observed in fig. 11, the band of 10-kDa was also identified in the negative control . Therefore, in the first instance, we consider that the band present in the negative control was some protein from the FCS. In fact, the use of the complete medium (FCS) may lead to the binding of the caspase-1 to albumin and/or other serum components causing the effective concentration of caspase-1 in western blotting analysis to be reduced. Besides, cells infected with S. aureus strains were not well labeled with Anti- In addition, human monocytic cell line (ThP1) (BOSSHART; HEINZELMANN, 2016), extensively described as a cellular model able to activate caspase-1 and produce large quantities of IL-1β notably, after differentiation by phorbol 12-myristate 13-acetate (PMA) otherwise known as 12-O-Tetradecanoylphorbol-13-acetate (TPA) was investigated in our newest protocol for identification of mature caspase-1 by Western blot [START_REF] Akita | Involvement of Caspase-1 and Caspase-3 in the Production and Processing of Mature Human Interleukin 18 in Monocytic THP.1 Cells[END_REF][START_REF] Franchi | The Inflammasome: A Caspase-1 Activation Platform Regulating Immune Responses and Disease Pathogenesis[END_REF][START_REF] Gaidt | Human Monocytes Engage an Alternative Inflammasome Pathway[END_REF][START_REF] Kim | NLRP3 Inflammasome Activation in THP1 Target Cells Triggered by Pathogenic Naegleria fowleri[END_REF]LI;[START_REF] Li | Differential activation of the inflammasome in THP1 cells exposed to chrysotile asbestos and Libby "six-mix" amphiboles and subsequent activation of BEAS-2B cells[END_REF][START_REF] Li | Differential activation of the inflammasome in THP1 cells exposed to chrysotile asbestos and Libby "six-mix" amphiboles and subsequent activation of BEAS-2B cells[END_REF][START_REF] Schumann | Lipopolysaccharide Activates Caspase-1 (Interleukin-1-Converting Enzyme) in Cultured Monocytic and Endothelial Cells[END_REF][START_REF] Shamaa | Monocyte Caspase-1 Is Released in a Stable, Active High Molecular Weight Complex Distinct from the Unstable Cell Lysate-Activated Caspase-1[END_REF][START_REF] Starr | The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP1 macrophages with Salmonella Typhimurium[END_REF]. This differentiation has been described for several years in the literature and is accompanied by a change in the morphology and cell adhesion [START_REF] Tsuchiya | Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester[END_REF].

Initially, different protocols were used for sample preparation and active caspase-1 detection (Table 3) tested with different incubation times and using inducers of caspase-1 activation such as used anteriorly (LPS, Nigericin) and ATP (LOPEZ-CASTEJON;BROUGH, 2011;[START_REF] Piccini | ATP is released by monocytes stimulated with pathogen-sensing receptor ligands and induces IL-1β and IL-18 secretion in an autocrine way[END_REF] as positive control and the activation of caspase-1 was measured by western blotting. Surprisingly, we observed that in contrast to (KAHLENBERG; [START_REF] Kahlenberg | Differing caspase-1 activation states in monocyte versus macrophage models of IL-1beta processing and release[END_REF][START_REF] Laliberte | ATP Treatment of Human Monocytes Promotes Caspase-1 Maturation and Externalization[END_REF][START_REF] Laliberte | ATP Treatment of Human Monocytes Promotes Caspase-1 Maturation and Externalization[END_REF][START_REF] Laliberte | ATP Treatment of Human Monocytes Promotes Caspase-1 Maturation and Externalization[END_REF][START_REF] Niu | Caspase-1 promotes monocyte-macrophage differentiation by repressing PPARγ[END_REF][START_REF] Shamaa | Monocyte Caspase-1 Is Released in a Stable, Active High Molecular Weight Complex Distinct from the Unstable Cell Lysate-Activated Caspase-1[END_REF] literature findings, caspase-1 activation did not occur in response to the stimuli used. Indeed, studies reported discrepancies in the expression of caspase-1 in human monocytes (ThP1) ranging from different activators to different activation times, as well as conflicting evidence regarding the expression of active caspase-1 in this cell line [START_REF] Baker | NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5[END_REF]. These results suggest a degree of variability among these ThP1 cells differentiated with (PMA) grown in separate facilities.

It is well characterized in the literature that inactive pro-caspase-1 is converted into an active enzyme via dimerization, followed by an autocatalytic reaction that generates an active molecule composed of two large and two small subunits. As well as, that the autocatalysis of pro-caspase-1 to active caspase-1 is tightly controlled by the caspase-1 inflammasome [START_REF] Broz | Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1[END_REF][START_REF] Lamkanfi | Emerging inflammasome effector mechanisms[END_REF]RATHINAM;VANAJA;FITZGERALD, 2012;SCHRODER;TSCHOPP, 2010). To evaluate the activation of the inflammasome by activation of caspase- , 2002). Caspase-1 is present in the cytoplasm of phagocytic cells as a 45-kDa inactive zymogen, which needs to be cleaved to acquire its biological function [START_REF] Broz | Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1[END_REF]RATHINAM;VANAJA;FITZGERALD, 2012;VANAJA;RATHINAM;[START_REF] Vanaja | Mechanisms of inflammasome activation: recent advances and novel insights[END_REF]. It is known that activation of some inflammasomes, such as NLRP3, is characterized by two signals: the first signal is an exogenous derived microbial (PAMPs), which triggers the production of cytokine precursors e.g., pro-IL-1β; and a second signal, represented by the interaction of DAMPs with their PRRs. There is accumulated evidence indicating the relevance of this second sign of activation, particularly extracellular ATP in the regulation of the NLRP3 inflammasome through purinergic receptors (P2X), which are important mediators of inflammation and apoptosis (GOMBAULT; BARON; COUILLIN, 2013;LOPEZ-CASTEJON;BROUGH, 2011;[START_REF] Mccall | Osteoblasts express NLRP3, a nucleotide-binding domain and leucine-rich repeat region containing receptor implicated in bacterially induced cell death[END_REF][START_REF] Piccini | ATP is released by monocytes stimulated with pathogen-sensing receptor ligands and induces IL-1β and IL-18 secretion in an autocrine way[END_REF][START_REF] Savio | The P2X7 Receptor in Inflammatory Diseases: Angel or Demon[END_REF].

Therefore, we investigate whether activation caspase-1 and secretion of IL-1β both occur in MG-63 cells, non-professional phagocytes and professional phagocytes, human monocytes.

For this purpose, human osteoblastic cells (MG-63) and ThP1 cells were used. Differentiation of ThP1 monocytes into macrophages was obtained by the treatment with 200 nM phorbol 12myristate 13-acetate (PMA). This differentiation is following by a change in morphology and cell adhesion, described in the literature for several years [START_REF] Tsuchiya | Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester[END_REF]. The cells were stimulated as follows: i) primates with 1 μg/ml LPS per 3 h for MG-63 cells and 30 min for ThP1 cells, as the first sign of inflammasome activation, and stimulated with 5 mM of ATP for 15 min as the second signal of activation of inflammasome and ii) stimulated with only 1 μg/ml of LPS. After different times post-infection (2h, 6h, 2 days, 5 days, 7 days and 9 days) cell supernatants were collected and the level of IL-1β was determined by commercial sandwich-ELISA (Invitrogen, France) and the activation of caspase-1 in MG-63 cells was measured by Western blot.

As shown in fig. 13, differentiation of ThP1 cells by PMA, induced IL-1β secretion significantly 24 h post-treatment, corroborating with the findings of [START_REF] Park | Optimized THP1 differentiation is required for the detection of responses to weak stimuli[END_REF], which demonstrate that PMA-differentiated cells are able to induce IL-1β production, showing that PMA plays an important role in the production of detectable levels of IL-1β (PARK et al., 2007). Besides, there was an elevation of this secretion when we primed the monocytes with LPS when we compared the times of 4h and 24h after the treatment (Fig. 13). It is well established in the literature that stimulation with LPS activates the TLR4 pathway and induces inflammation [START_REF] Guijarro-Muñoz | Lipopolysaccharide Activates Toll-like Receptor 4 (TLR4)-mediated NF-κB Signaling Pathway and Proinflammatory Response in Human Pericytes[END_REF][START_REF] Souza | Suppressor of cytokine signaling 1 expression during LPS-induced inflammation and bone loss in rats[END_REF]WU;CHEN;[START_REF] Wu | Lipopolysaccharide triggers macrophage activation of inflammatory cytokine expression, chemotaxis, phagocytosis, and oxidative ability via a toll-like receptor 4-dependent pathway: validated by RNA interference[END_REF][START_REF] Zhang | Columbianadin Suppresses Lipopolysaccharide (LPS)-Induced Inflammation and Apoptosis through the NOD1 Pathway[END_REF]. However, there is controversy over the use of TLR ligands to activate the secretion of caspase-1 and IL-1β. It was suggested by [START_REF] Martinon | Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases[END_REF] that stimulation by LPS alone would be ineffective as an activator of the inflammasome and successful secretion of IL-1β (MARTINON;[START_REF] Martinon | Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases[END_REF]. Our results showed that In PMA-differentiated ThP1 cells, LPS-treatment provides the "first signal" to induce a pro-inflammatory response that culminates with the secretion of IL-1β in the supernatants. Further, according to the literature, PMA treatment induces the characteristic expression of CD14, a TLR4 co-receptor, which suggests that, without PMA treatment, ThP1 cells are not able to respond to the LPS stimulus, and can not then induce the production of IL-1β, demonstrating that induction can occur through the LPS-dependent signaling pathway and thus TLR4/CD14 [START_REF] Kohro | A comparison of differences in the gene expression profiles of phorbol 12-myristate 13-acetate differentiated THP1 cells and human monocyte-derived macrophage[END_REF][START_REF] Schwende | Differences in the state of differentiation of THP1 cells induced by phorbol ester and 1,25dihydroxyvitamin D3[END_REF]. The results showed that there was a potentiation of IL-1β secretion in ThP1 cells that received ATP as the second signal (Fig. 13). These data corroborate with findings from the literature that subsequent stimulation with inflammatory inducers such as ATP as a second signal for activation of the inflammasome induces caspase-1 activation and IL-1β maturation and secretion [START_REF] He | Mechanism and Regulation of NLRP3 Inflammasome Activation[END_REF][START_REF] He | Mechanism and Regulation of NLRP3 Inflammasome Activation[END_REF][START_REF] He | Mechanism and Regulation of NLRP3 Inflammasome Activation[END_REF][START_REF] Jo | Molecular mechanisms regulating NLRP3 inflammasome activation[END_REF]. It is well known that ATP acts via the purinergic pathway to activate inflammasomes such as NLRP3 [START_REF] Mariathasan | Cryopyrin activates the inflammasome in response to toxins and ATP[END_REF].

ATP also involves another intermediate signal, which is the generation of reactive oxygen species (ROS) [START_REF] Abais | Redox Regulation of NLRP3 Inflammasomes: ROS as Trigger or Effector?[END_REF][START_REF] Martínez-Reyes | The H+-ATP synthase: A gate to ROS-mediated cell death or cell survival[END_REF][START_REF] Martínez-Reyes | The H+-ATP synthase: A gate to ROS-mediated cell death or cell survival[END_REF][START_REF] Zheng | Reactive oxygen species activated NLRP3 inflammasomes prime environment-induced murine dry eye[END_REF].

It has been shown that both ATP and ROS have different activation pathways, however, both lead to caspase-1 activation and IL-1β production through the purinergic pathway (COUILLIN; GOMBAULT; BARON, 2013;[START_REF] Cruz | ATP Activates a Reactive Oxygen Species-dependent Oxidative Stress Response and Secretion of Proinflammatory Cytokines in Macrophages[END_REF]LATZ;XIAO;STUTZ, 2013a). Indeed, ATP causes the activation of the NLRP3 inflammasome by the production of ROS, which will then act on another inflammasome and give rise to the production of IL-1β. ROS would also cause the production of endogenous ATP, which would be excreted via through pannexin hemichannels (mainly Panx1) or P2X7R and which, in turn, would act in the purinergic channels to activate the NLRP3 inflammasome [START_REF] Chen | Inhibition of the P2X7-PANX1 complex suppresses spreading depolarization and neuroinflammation[END_REF][START_REF] Dahl | ATP release through pannexon channels[END_REF][START_REF] Dahl | ATP release through pannexon channels[END_REF][START_REF] Dahl | The bizarre pharmacology of the ATP release channel pannexin1[END_REF][START_REF] Dahl | The bizarre pharmacology of the ATP release channel pannexin1[END_REF][START_REF] Muñoz-Planillo | K+ efflux is the Common Trigger of NLRP3 inflammasome Activation by Bacterial Toxins and Particulate Matter[END_REF][START_REF] Shoji | Pannexin1 channels act downstream of P2X 7 receptors in ATP-induced murine T-cell death[END_REF].

In order to monitor inflammasome activation in the human osteoblast-like MG-63 cells the detection of active caspase-1 by Western blot analysis was set up in samples containing cells and cell supernatants. As shown in Fig. 14-A, exposing MG-63 cells to LPS+ATP led to the appearance of 20 kDa band corresponding to the active caspase-1 p20 fragment. The p20 subunit of mature caspase-1 was investigated in culture supernatants plus cell lysates (Sup+CL) by Western blot. In fact, in the absence of the activation of the inflammasome, caspase-1 is detected in its precursor form, pro-caspase-1, in cell lysates. Upon activation, pro-caspase-1 is cleaved proteolytically in its p20 and p10 active forms which are detectable in cell lysates early in the activation of the inflammasome, and then most of these active forms are secreted and detected in the supernatant [START_REF] Boucher | Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity[END_REF]BROZ;[START_REF] Broz | Molecular mechanisms of inflammasome activation during microbial infections: Mechanisms of inflammasome activation[END_REF][START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Tsuchiya | Caspase-1 initiates apoptosis in the absence of gasdermin D[END_REF][START_REF] Wang | Inflammasome Activation Triggers Caspase-1-Mediated Cleavage of cGAS to Regulate Responses to DNA Virus Infection[END_REF]. The activation of caspase-1 was associated with the production of IL-1β, as the exposure of cells to LPS+ATP resulted in the production of mature IL-1β (Fig. 14-B).

These results suggest that MG-63 cells form functional inflammasomes and that their activation can be estimated by IL-1β release.

To compare the ability of inflammasome activation by professional versus non-professional phagocytes, PMA-stimulated ThP1 cells differentiated into macrophagesor MG-63 osteoblast-like cells were exposed to inflammasome activators LPS+ATP and the level of IL-1β production was estimated 2 h and 6 h after the beginning of the treatment. PMA-treated control ThP1 cells produce ~ 7 pg/ml of IL-1β 2 h at the beginning of the treatment. Exposure of PMAdifferentiated ThP1 cells to LPS+ATP resulted in a significant increase to ~ 30 pg/ml 2 h post treatment. After 6 h PMA-treated control ThP1 cells produce ~ 10 pg/ml of Il-1β, while the level of IL-1β produced by PMA-treated ThP1 cells after an exposure to LPS+ATP was ~ 650 pg/ml. Supernatants of ThP-1+LPS+ATP (6 h) were diluted 1:5, the result presented in Fig. 14-C is multiplied by the dilution factor. In contrast, IL-1β was not detected 2 h post-treatment, while only 4 pg/ml of IL-1β was detected in MG-63 cells 6 h after the exposure to LPS+ATP.

Thus, we demonstrated that human osteoblast-like MG-63 cells release a soluble IL-1β starting from 6 h post-treatment with inflammasomes activator LPS+ATP, matching the appearance of active caspase-1 20-kDa subunit (Fig. 14) that suggests inflammasomes activation in MG-63 cells. The production of IL-1β by MG-63 cells started later and IL-1β level was much lower than IL-1β production by ThP1 cells whose level of IL-1β production corresponds to the findings of other researchers [START_REF] Grahames | Pharmacological characterization of ATP-and LPS-induced IL-1beta release in human monocytes[END_REF]. The discrepancy between levels of the soluble A. MG-63 cells were incubated in 12-well plates. Afterwards, cells were primed with LPS (1 μg/ml) and stimulated with ATP (5 mM). Six hours after the beginning of the treatment cells together with cell-culture supernatants were diluted in RIPA lysis buffer. Protein samples were prepared in 5 x SDS-PAGE sample loading buffer. Detection of 45-kDa pro-caspase-1 in MG-63 cells by Western blot analysis was performed using anti-caspase-1 antibody (AdipoGen) as described in Material and Methods. The protein load was verified with anti-tubulin antibody. Three independent assays were performed. B, C. MG-63 or ThP1 cells were incubated in 12-well plates. Afterwards, cells were primed for 2 h with LPS (1 μg/ml) and stimulated with ATP (5 mM) for 15 min. ThP1 were treated with PMA before LPS+ATP treatment as described above. Two or six hours after the beginning of the treatment cell supernatants were collected, centrifuged, and the level of IL-1β was determined by a commercial sandwich-ELISA (Thermofischer Life Technologies) as described in Material and Methods. Supernatants of ThP-1+LPS+ATP were diluted 1:5. The values are presented as concentrations in pg/ml. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). Comparisons with P-values < 0.05 (*) were considered to be significant. *, P < 0.05; **, P < 0.01. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD).

5.2. Deletion of caspase-1 gene in human osteoblasts MG-63 using the CRISPR/Cas9 gene editing system NLRP3 is one of the most studied and best-characterized inflammasomes and has been associated with several diseases and conditions characterized by chronic inflammation, including gout, cancer, type 2 diabetes, and rheumatoid arthritis, as well as periodontal diseases, among others (SCHRODER; TSCHOPP, 2010). Thus, in the first part of this study, we were interested to set up an experimental model for the investigation of the involvement of the NLRP3 inflammasome in the response of S. aureus-infected MG-63 cells.

It has been shown that stimulation is required for IL-1β transcription and protein expression by MG-63 cells (KOH et al., 1997). Since MG-63 cells are usually used as a model to study bone and joint infections (BJI) [START_REF] Deplanche | Staphylococcus aureus induces DNA damage in host cell[END_REF], we used MG-63 cells for CRISPR/Cas9mediated caspase-1 knockout to study the involvement of inflammasomes in S. aureus infection of osteoblasts cells. Indeed, the study of the response of WT MG-63 vs CAS1 -/-MG-63 human cell line, non-professional phagocytes, allowed us to estimate the role of inflammasomes and its component caspase-1 in staphylococcal infection of osteoblasts.

To develop this model, we aimed to achieve the generation of a cell line deficient for the caspase-1 gene that will help to investigate the involvement of the inflammasome activation 122 during infection of MG-63 cells. The selection of caspase-1 as a potential candidate for the gene editing is linked to the pivotal role of this protease in the processing and maturation of the pro-inflammatory cytokine IL-1β by canonical inflammasome pathway.

The caspase-1 gene in MG-63 cells was deleted using CRISPR/Cas9 technology [START_REF] Mali | RNAguided human genome engineering via Cas9[END_REF]. The knockout was achieved via lentiviral infection of MG-63 cells. Lentiviral productions have been performed as recommended (http://tronolab.epfl.ch) and previously described [START_REF] Gilot | A non-coding function of TYRP1 mRNA promotes melanoma growth[END_REF], more detail in the main article materials and methods session. There are 6 isoforms of caspase-1 encoded by the CASP1 gene, located in chromosome 11. Caspase-1 alpha and beta are the main transcripts in human cells. To generate a caspase-1 knockout in MG-63 cells we used a previously validated sgRNA-ATTGACTCCGTTATTCCGAA [START_REF] Schmid-Burgk | A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation[END_REF]. Based on sequences analysis, the sgRNA is predicted to target four caspase-1 isoforms including alpha and beta.

Four major groups of DNA bind ing proteins used in gene editing are known: meganucleases from microbial mobile genetic elements, nucleases based on eukaryotic transcription factors such as zinc fingers (ZFN), transcription activator-like effectors (TALENs), from the Xanthomonas bacterium and the Cas9 endonuclease of the adaptive immune system of some bacteria such as S. pyogenes [START_REF] Adli | The CRISPR tool kit for genome editing and beyond[END_REF][START_REF] Boch | TALEs of genome targeting[END_REF]HSU;[START_REF] Hsu | Development and Applications of CRISPR-Cas9 for Genome Engineering[END_REF]ZHANG, 2014;[START_REF] Jinek | A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[END_REF][START_REF] Kim | NLRP3 Inflammasome Activation in THP1 Target Cells Triggered by Pathogenic Naegleria fowleri[END_REF][START_REF] Kim | Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain[END_REF][START_REF] Kim | Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain[END_REF][START_REF] Ledford | CRISPR, the disruptor[END_REF][START_REF] Ramirez | Unexpected failure rates for modular assembly of engineered zinc fingers[END_REF].

The great difference between Cas9 and the rest is the fact that recognition between RNA and DNA is based on the complementarity of bases. The Cas9 nuclease is directed by a small RNA sequence that recognizes the target DNA by complementarity making it accessible to the action of Cas9, which has innumerable advantages compared to the previous nucleases, e.g. in CRISPR/Cas9 technology, several target genetic sites can be targeted simultaneously using multiple RNA sequences guide (gRNA) and all redirecting of the Cas9 nuclease depends on two RNA sequences, which is much easier and simpler [START_REF] Adli | The CRISPR tool kit for genome editing and beyond[END_REF][START_REF] Cong | Multiplex genome engineering using CRISPR/Cas systems[END_REF][START_REF] Liu | Efficient CRISPR-based genome editing using tandem guide RNAs and editable surrogate reporters[END_REF][START_REF] Maggio | Genome editing at the crossroads of delivery, specificity, and fidelity[END_REF][START_REF] Maggio | Genome editing at the crossroads of delivery, specificity, and fidelity[END_REF][START_REF] Mali | RNAguided human genome engineering via Cas9[END_REF]. Thus, after obtaining the genetically modified lineage, using the CRISPR/Cas9 technique, we used the single cell clonal expansion method to select the monoclones, which did not express the caspase-1 gene. For this, first, we needed to select several unique clones to obtain a true knockout cell lineage. This process is important after engineering mutations, especially with something like CRISPR/Cas9 that can generate heterogeneous mutants. Thus, based on the literature showing the use of multiple approaches that were developed for the isolation of 123 monoclonal cell lines as Fluorescence-activated cell sorting (FACS), Magnetic-activated cell sorting (MACS), Laser capture microdissection (LCM), Manual cell picking and Microfluidic [START_REF] Citri | Comprehensive qPCR profiling of gene expression in single neuronal cells[END_REF][START_REF] Datta | Laser capture microdissection: Big data from small samples[END_REF][START_REF] Gross | Technologies for Single-Cell Isolation[END_REF][START_REF] Lecault | Microfluidic single cell analysis: from promise to practice[END_REF][START_REF] Welzel | Magnetic-activated cell sorting (MACS) can be used as a large-scale method for establishing zebrafish neuronal cell cultures[END_REF][START_REF] Welzel | Magnetic-activated cell sorting (MACS) can be used as a large-scale method for establishing zebrafish neuronal cell cultures[END_REF][START_REF] Welzel | Magnetic-activated cell sorting (MACS) can be used as a large-scale method for establishing zebrafish neuronal cell cultures[END_REF]. According to Hu and colleagues (2016), in the single cell clonal expansion method, the probability of obtaining a single cell in an aliquot is statistical in nature, and although limiting dilution provides the most versatile approach, the experimental procedure requires optimization to increase the chance of successful isolation of the single clone [START_REF] Gross | Technologies for Single-Cell Isolation[END_REF][START_REF] Hu | Single Cell Isolation and Analysis[END_REF].

Therefore, after establishing the method for the isolation of the monoclones MG-63 cells were transduced and selected with 0.2 μg/ml of puromycin for 3 days. Individual cells from a stable pool of cells were then expanded for another 3 weeks under a selection of puromycine.

Western blotting with anti-caspase-1 antibody was performed with cell lysates to detect the 45-kDa precursor form of caspase-1 (pro-caspase-1). And WT MG-63 cells expressing procaspase-1 were included as a control. Thus, clones exhibiting an absence of 45-kDa bands were selected as potential knockout cells for the caspase-1 gene. Our results showed that of the 142 monoclones selected by the single cell clones method (data not are shown), only three were selected as possible candidates for the Casp1 knockout gene (Fig. 15). Interestingly, as shown in fig. 15, there was the expression of a weak band that could still correspond to pro-caspase-1 of 45-kDa in the cellular extract. We assume that in our experimental model the development of wild type cells occurred which could seriously confuse the data obtained. In fact, the presence of wild cells can overcome the growth of a mutant clone over time. We decided to perform an additional selection of monoclones using a higher concentration of puromycin, so the selection was made with 1μg/mL of puromycin for 3 days.

Individual cells from a stable pool of cells were then expanded for another 3 weeks under a selection of puromycine. The detection of the 45-kDa band corresponding to pro-caspase-1 was performed by Western blotting of cell extracts with anti-caspase 1 antibody. Wild type MG-63 (WT MG-63) cells expressing pro-caspase-1 were included as a control. Our data demonstrated that of the total of 35 monoclones tested only 3 did not present the band of 45-kDa in cellular extracts corresponding to pro-caspase-1 (Fig. 16 and Fig. 17). Thus, in order to ensure the generation of Cas1 -/-MG-63 cells we performed a second cloning by limited dilution of these 3 monoclones. Monoclonal cell colonies were tested by western blotting (Fig. 18). As shown in fig. 18, our results demonstrated the obtaining of monoclones with the deletion of the 45-kDa band from the reference to pro-caspase-1 and, therefore, we selected to be subjected to DNA sequencing the monoclones: N2E11-G2 (G2), N2E11-B9 (B9) (Fig. 18) and N5G2-C3 (C3) (data not shown). D, E. WT MG-63 or CASP1 -/-MG-63 cells were exposed to LPS+ATP (D) or S. aureus strain SA113 at MOI 1:50 for 2 h (E) followed by antibiotic treatment as described in Material and Methods. Six hours post-infection or 6 h after the beginning of LPS+ATP treatment cell supernatants were collected, centrifuged and the level of IL-1β was determined by a commercial sandwich-ELISA (by sandwich-ELISA (Thermofischer Life Technologies) as described in Materials and Methods. The values are presented as concentrations in pg/ml. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). P-values < 0.05 were considered to be significant. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD).

Taking into account that both types of cells contain NLRP3 (Fig. 20-B), while pro-caspase-1 is present only in WT MG-63 cells (Fig. 3-A) we next determined whether an exposure of both cell lines to S. aureus SA113 leads to the formation of ASC specks, since their formation does not require the presence of pro-caspase-1. Consequently, we analyzed immunofluorescence staining for the adaptor ASC in WT and CASP1 -/-MG-63 cells. As shown in Fig. 20-C, S. aureus infection triggers assembly of the ASC specks in the cytosols of WT as well as CASP1-/-MG-63 cells (green). These results corroborate with the findings of [START_REF] Guey | Caspase-1 autoproteolysis is differentially required for NLRP1b and NLRP3 inflammasome function[END_REF], that shows that in WT cells (macrophages and dendritic cells) treated with lethal factor (LT), are able to formed ASC specks and also that in the absence of caspase-1, ASC specks are still formed [START_REF] Guey | Caspase-1 autoproteolysis is differentially required for NLRP1b and NLRP3 inflammasome function[END_REF]. Moreover, it has been shown that ASC specks can accumulate in the microenvironment of cells dying by pyroptosis. Interestingly, these aggregates of ASC specks can then be phagocytosed by surrounding immune cells and, by their "prion" activity, spread the inflammation by activating caspase-1 in these cells [START_REF] Fernandes-Alnemri | The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation[END_REF][START_REF] Franklin | The intra-and extracellular functions of ASC specks[END_REF]LATZ;[START_REF] Schmidt | Thus, it is necessary that the expression of all these upstream components occurs so that the subsequent activation of the inflammasome[END_REF].

Previously it was reported that the transcripts of IL-1β and IL-18 were observed after an exposure of mice osteoblasts to S. aureus, in contrast, secretion of IL-1β and IL-18 was not detected despite the presence of active caspase-1 (MARRIOTT; HUGHES; BOST, 2002).

To understand whether S. aureus activates inflammasomes in human osteoblast-like MG-63 cells, we measured IL-1β production in WT and CASP1 -/-MG-63 cells. The presence of NLRP3 and the lack of pro-caspase-1 in CASP1 -/-MG-63 cells allows these cells to be used as a control for the estimation of NLRP3 inflammasome activity in MG-63 cells by the measurement of IL-1β, which is processed due to caspase-1. The assessment of IL-1β in WT and CASP1 -/- MG-63 cells exposed to inflammasomes activators LPS+ATP (Fig. 20-D) as well as to S.

aureus LAC (Fig. 20-E) revealed the lack of IL-1β production in CASP1 -/-MG-63 in contrast to WT MG-63 cells.

Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD).

B. WT MG-63 cells were exposed to S. aureus strains LAC (USA300, red color), MW2 (USA 400, green color), or SA113 (yellow color) at MOI 1:50 for 2 h followed by antibiotic treatment as described in Material and Methods. After various times post-infection (6 h, 2 days, 5 days, 7, days and 11 days) cell supernatants were collected, centrifuged and the level of IL-1β was determined by a commercial sandwich-ELISA (Invitrogen, France) as described in Material and Methods. The values are presented as concentrations in pg/ml. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). P-values < 0.05 (*) were considered to be significant. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD).

Besides, the lack of production of IL-1β in the other clones proposed also was evaluated. For this, WT MG-63 cells, (CASP1 -/-MG-63 clone B9) and (CASP1 -/-MG-63 clone C3) were infected with S. aureus SA113 at MOI 50:1. Cultured cells were monitored during (9 days postinfection), and the supernatant was used to quantify IL-1β levels by ELISA. Our results show that there was no secretion of IL-1β in the (CASP1 -/-MG-63 clone B9) and (CASP1 -/-MG-63 clone C3), after the stimulation with S. aureus SA113, when compared to WT MG-63 infected with the strain SA113, that presented high levels of IL-1β (Fig. 22).

Thus, the equal NLRP3 protein expression together with the similar formation of ASC specks in both WT MG-63 and CASP1 -/-MG-63 cells, as well as the absence of active caspase-1 and IL-1β release following the stimulation with inflammasomes activators in CASP1 -/-MG-63 cells, validate caspase-1 knockout in osteoblastic cells. Therefore, we generated CASP1 -/-MG-63 cell line that is a powerful tool that allows the function of inflammasomes to be investigated.

We result show that S. aureus-induced IL-1β release is strongly strain-dependent. Rising IL-1β production in S. aureus-infected MG-63 cells in contrast to its lack in infected CASP1 -/-MG-63 cells demonstrated caspase-1 dependent mechanisms of IL-1β production by human osteoblasts. Our data corroborate observations of others that demonstrated caspase-1 depending mechanism of IL-1β processing and its release by mice macrophages during infection [START_REF] Raupach | Caspase-1-mediated activation of interleukin-1beta (IL-1beta) and IL-18 contributes to innate immune defenses against >Salmonella enterica</i> serovar Typhimurium infection[END_REF]. Besides, the absence of IL-1β production by cells exposed to heat-killed bacteria indicates that factors associated with viable bacteria are involved in the activation of the NLRP3 inflammasome. FITZGERALD, 2012). Certain bacteria such as Yersinia pseudotuberculosis, Yersinia enterocolitica, Salmonellae species, Pseudomonas aeruginosa, among others, use strategies that manipulate inflammasome activation, in most cases, interfere in the production or recognition of bacterial ligands that trigger inflammasomes [START_REF] Brodsky | A Yersinia effector protein promotes virulence by preventing inflammasome recognition of the type III secretion system[END_REF].

The quorum-sensing system in S. aureus known as the accessory gene regulator (Agr) regulates the expression of many virulence factors. Agr regulates main S. aureus toxins, among which regulation of Phenol-soluble modulins (PSMs) reviewed (PESCHEL; OTTO, 2013) contain five α-peptides (δ-toxin and PSMα1-4) and two β-peptides (PSMβ1-2), which all share an amphipathic α-helical structure, thereby acting as biological detergents. Thus, PSMs are regarded as a new class of Staphylococcal leukocidins [START_REF] Kretschmer | Human formyl peptide receptor 2 senses highly pathogenic Staphylococcus aureus[END_REF][START_REF] Nakamura | Staphylococcus δ-toxin induces allergic skin disease by activating mast cells[END_REF]PESCHEL;[START_REF] Peschel | Phenol-soluble modulins and staphylococcal infection[END_REF][START_REF] Richardson | PSM Peptides From Community-Associated Methicillin-Resistant Staphylococcus aureus Impair the Adaptive Immune Response via Modulation of Dendritic Cell Subsets in vivo[END_REF]. PSMs first attract innate immune cells like neutrophils, macrophages, and dendritic cells (DCs) by binding to the formyl peptide receptor 2 (FPR2) [START_REF] Richardson | PSM Peptides From Community-Associated Methicillin-Resistant Staphylococcus aureus Impair the Adaptive Immune Response via Modulation of Dendritic Cell Subsets in vivo[END_REF][START_REF] Schreiner | Staphylococcus aureus Phenol-Soluble Modulin Peptides Modulate Dendritic Cell Functions and Increase In Vitro Priming of Regulatory T Cells[END_REF][START_REF] Wang | Inflammasome Activation Triggers Caspase-1-Mediated Cleavage of cGAS to Regulate Responses to DNA Virus Infection[END_REF]. Was been reported that PSMs can activate inflammasome-like signaling and trigger IL-1β and IL-18 secretion (MELEHANI; DUNCAN, 2016). In addition, was demonstrated that PSMs stimulate the production of inflammatory cytokines by infected keratinocytes [START_REF] Syed | Staphlyococcus aureus phenolsoluble modulins stimulate the release of proinflammatory cytokines from keratinocytes and are required for induction of skin inflammation[END_REF], thus it is essential that this role is unraveled in our experimental model. Furthermore, our results on strain dependence of IL-1β stimulation in osteoblasts suggested an involvement of Agr, as the low levels of IL-1β in cells infected with strain SA113 as opposed to those infected with strains MW2 and LAC correlate with levels of Agr functionality in those strains. SA113 is functionally Agr-defective, while MW2 and LAC show high Agr activity. These considerations prompted us to investigate the role of PSMs in IL-1β production by infected osteoblasts.

First, we analyzed LAC (USA300) wild-type and its isogenic mutant LACΔpsmαβhld (S. aureus strain lacking PSMα, PSMβ and δ-toxin) for their ability to stimulate the release of IL-1β. As shown in Fig. 23-A, the level of IL-1β was significantly decreased in the supernatants of WT MG-63 cells exposed to LAC Δpsmαβhld compared to WT MG-63 cells exposed to LAC wildtype on days 5 and 9 post-infection. To further investigate which PSM were involved in the simulation of IL-1β release, we used the PSM deletion strain LACΔpsmαβhld and complemented strains, expressing either the four PSMα peptides (LACΔpsmαβhld-pTXΔα1-4), the two PSMβ peptides (LACΔpsmαβhld-pTXΔβ1-2), or the δ-toxin (LACΔpsmαβhld-pTXΔhld) and monitored IL-1β levels in cell exposed to those strains up to 9 days postinfection. As shown in Fig. 23-B, there was a significant decrease in the IL-1β released by cells exposed to the LACΔpsmαβhld strain compared to LAC. Exposure to complemented applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD).

B. MG-63 cells were exposed to USA300 LAC (pTX∆16), which carries the control plasmid, the deletion mutant LAC∆psmαβhld (pTX∆16) and the complemented strains expressing the four PSMα peptides (LAC∆psmαβhld-pTX∆α1-4), the two PSMβ peptides (LAC∆psmαβhld-pTX∆β1-2), or the δ-toxin (LAC∆psmαβhld-pTX∆hld) at MOI 1:50 for 2 h followed by antibiotic treatment as described in Material and Methods. After various times post-infection (2 days, 5 days, 7 days and 9 days) cell supernatants were collected, centrifuged and the level of IL-1β was determined by a sandwich-ELISA (Thermofischer Life Technologies) as described in Material and Methods. The values are presented as concentrations in pg/ml. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). P-values< 0.05 (*) were considered to be significant. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD).

In addition, we evaluated the secretion of IL-1β in osteoblast cells exposed to S. aureus strains obtained from patients with osteomyelitis. We analyzed MG-63 cells infected by six S. aureus human clinical isolates. The level of IL-1β was measured by ELISA. Three pairs of S. aureus strains used were obtained from patients with staphylococcal BJI provided by CNF Staphylococcus (National Reference Center for Staphylococci) of Lyon. Three couples of isolates were selected from patients P1, P2, P3 who were diagnosed with initial acute (i) and recurrent (r) staphylococcal BJI: isolates from the same patient were named 45i and 46r (P1), 47i and 48r (P2), 51i and 53r (P3) for initial and recurrent BJI correspondently. (Supplementary Material). Previous studies conducted by our group have demonstrated that the eukaryotic cell cycle delay induced by S. aureus human clinical isolates is associated with the production of PSMα1 that reveals a new mechanism to promote infection (DEPLANCHE et al., 2015).

The analysis of the kinetics of IL-1β release by WT MG-63 cells exposed to either six clinical isolates or S. aureus USA400 strain (MW2) demonstrated an increase of the level of IL-1β in the supernatants from the 2nd day post-infection up to 9 days post-infection. Furthermore, we found different levels of IL-1β production induced by clinical strains collected from three patients. When comparing its acute (i) and recurrent (r) forms, we find that IL-1β production induced by the strains (45i and 46r) was similar (Fig. 24-A). The IL-1β production induced by the acute strain (47i) was significantly higher then induced by the recurrent strain (48r) (Fig.

24-B).

A comparison of strains (51i) and (52r) showed that strain (52r) induced higher IL-1β levels compared to strain (51i) (Fig. 24-C). These results demonstrated that clinical strains of S. aureus obtained from patients with staphylococcal BJI induce IL-1β secretion in infected demonstrating that bacterial invasion of these cells can occur in vivo (REILLY et al., 2000). [START_REF] Bosse | Internalization of bacteria by osteoblasts in a patient with recurrent, long-term osteomyelitis. A case report[END_REF] revealed in the interior of the osteoblasts and osteocytes removed from a patient suffering from a chronic infection of the fibula, the presence of gram-positive cocci intracellular bacteria presented a suggestive morphology by transmission electron microscopy [START_REF] Bosse | Internalization of bacteria by osteoblasts in a patient with recurrent, long-term osteomyelitis. A case report[END_REF][START_REF] Bosse | Internalization of bacteria by osteoblasts in a patient with recurrent, long-term osteomyelitis. A case report[END_REF][START_REF] Bosse | Internalization of bacteria by osteoblasts in a patient with recurrent, long-term osteomyelitis. A case report[END_REF]. In this context, the hypothesis that the facultative intracellular character of S. aureus provides the bacterium with a protective niche, protected from the immune system and most antibiotics, was gradually accepted by most authors; is also considered a plausible explanatory phenomenon of the chronic character of certain bone infections [START_REF] Ellington | Intracellular Staphylococcus aureus. A mechanism for the indolence of osteomyelitis[END_REF][START_REF] Ellington | Intracellular Staphylococcus aureus and antibiotic resistance: implications for treatment of staphylococcal osteomyelitis[END_REF].

Previous studies reported that the professional phagocytes lacking caspase-1 are permissive to intracellular pathogen replication (KHWEEK; AMER, 2010; SIMON; HILBI, 2015). [START_REF] Sokolovska | Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function[END_REF] demonstrated that activation of caspase-1 is required for the processing and killing of intracellular S. aureus by professional phagocytes [START_REF] Sokolovska | Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function[END_REF]. According to our knowledge, the role of caspase-1 in bacterial clearance in nonprofessional phagocytes, which include osteoblasts, has not been investigated.

Consequently, we analyzed whether the lack of caspase-1 in CASP1 -/-MG-63 cells is correlated with a failure to control the intracellular replication of S. aureus bacteria. The quantity of intracellular bacteria during S. aureus infection of WT and CASP1 -/-MG-63 cells was determined after gentamicin treatment of infected cells as described in Material and Methods.

According to CFU determination bacterial internalization was not impaired in CASP1 -/-MG-63 cells, as we observed equivalent levels of internalized bacteria in WT MG-63 cells at 2 h postinfection at ~ 10 2 CFU/10 5 host cells. Six and 24 hours post-infection significantly higher numbers of viable bacteria were recovered from CASP1 -/-MG-63 cells compared to WT MG-63 cells (Fig. 25-A). These results suggest that the lack of caspase-1 impairs the ability of osteoblasts to restrict S. aureus growth. These findings were further confirmed using confocal microscopy. As shown in Fig. 25-B, 6 h post-infection a considerably higher number of intracellular S. aureus bacteria (red) was observed in CASP1 -/-MG-63 cells than WT MG-63 cells, while assembly of the ASC specks was observed in both cell types (green). These results corroborate with the findings of [START_REF] Amer | Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf[END_REF], which demonstrate that caspase-1 activation is critical for the clearance of the bacteria independently of IL-1β activity [START_REF] Amer | Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf[END_REF]. B. WT MG-63 or CASP1 -/-MG-63 cells were grown on the slides of 12-well plates overnight, then cells were exposed to a fluorescent derivative of strain S. aureus SA113 fluorescent, which carries plasmidencoded mCherry (red fluorescence), at MOI 1:50 for 2 h followed by antibiotic treatment as described.

Six hours post-infection cells were immune-stained with rabbit anti PYCARD antibody (Coger France), followed by incubation with Alexa Fluor 488 labeled goat anti-rabbit antibody (Cell Signaling Ozyme) at a dilution of 1:50 for 2 h at room temperature (green staining). Nuclear DNA was labeled with DAPI (blue staining). Samples were viewed with a Zeiss laser-scanning microscope equipped with a 63× plan Apo-NA 1.4 immersion objective driven by Zen software. Scale bar: 3 µm.

General conclusion and perspective

Staphylococcus aureus is a bacterium that is widely studied due to its great impact on human and animal health as well as its role in the food industry. The purpose of this thesis was to investigate the contributions of internalized S. aureus on the alteration of host defense events such as inflammasomes activation and bacterial clearance in infected osteoblasts-like MG-63 cells that are widely used for the study of bone and joint infections (BJI).

We demonstrated that human osteoblast-like MG-63 cells treated with inflammasomes activators, form inflammasomes resulting in the activation of IL-1β-converting enzyme (ICE; caspase-1) and secretion of Pro-inflammatory cytokine Interleukin-1β (IL-1β).

The production of IL-1β by osteoblastic MG-63 cells started later and its level was much lower then IL-1β production by differentiated ThP1 cells that are professional phagocytes, macrophages. This discrepancy is likely related to different origins in both types of cells.

Due to the ability of the inflammasomes compound in the activation of caspase-1 to process IL-1β and IL-18 we used CRISPR/Cas9 gene editing to generate osteoblast-like MG-63 deficient for caspase-1 gene that are a powerful tool, which allows the study of the function of inflammasomes to be investigated. We observed a progressive increase of IL-1β levels in infected MG-63 cells from several hours to several days in contrast to the lack of IL-1β production in infected CASP1 -/-MG-63 cells demonstrated caspase-1-dependent mechanisms and IL-1β production. Besides, the effect was strongly S. aureus strains dependent. The similar results were obtained with freshly isolated clinical S. aureus isolates: this emphasizes the biological significance of our findings. The absence of IL-1β production by cells exposed to heat-killed bacteria indicates that factors associated with viable bacteria are involved in the activation of the NLRP3 inflammasome. Employment of deletion and complemented PSMs mutants demonstrated a pivotal role of S. aureus toxins PSMs (especially PSM-β and PSM-γ) in inflammasomes related to IL-1β production by infected osteoblasts cells. This is noteworthy, as we don't know any specific phenotypes attributable to those PSMs so far. However, the presence of other factors can not be excluded since complemented mutants did not restore entirely the level of secreted IL-1β.

The investigation of another host cell defense event, such as the control of intracellular bacterial proliferation, reveals a drastic increase in the proliferation of internalized bacteria in osteoblast-like CASP1 -/-MG-63 cells. This finding, in addition to the previous reports demonstrating the correlation of the lack of caspase-1 activation with a failure to restrict the replication of S. aureus inside of phagocytic cells points out that the active caspase-1 prevent exacerbated intracellular replication of S. aureus in professional as well as in non-professional phagocytes.

Collectively, our results demonstrated that human osteoblast-like MG-63 cells play an important role in the immune response against S. aureus infection. Infected osteoblasts are not passive bystanders but active players in defense against S. aureus infection. The outcome of the infection depends on the balance of the immune response of the host and the action of multiple S. aureus virulence factors among which PSMs play a pivotal role and whose production may differ among the S. aureus. Besides our observations of inflammasome activation (through measurement of IL-1β production), we demonstrated that caspase-1 deficient osteoblast-like cells were unable to restrict the intracellular replication of S. aureus suggesting the universal pivotal role of caspase-1 in pathogens clearance independently from the type of cells. These findings deserve further investigation to improve our understanding of S. aureus-host interactions and to uncover the underlying mechanisms.

And the next steps

The developed model is a powerful tool for tracking inflammation in vitro. Although this model is based on a single cell line, it opens new perspectives for the generation of similar models of non-phagocytic cells that allow the infectious process associated with S. aureus to be explored. Furthermore, we consider other action mechanisms for the formation of the NLRP3 inflammasome in S. aureus-infected MG-63 cells. It is known that process like lysosomal membrane disturbance, ionic imbalance (K + efflux) and induction of oxidative stress, signals critical for the activation of the NLRP3 inflammasomes, are processes depend on an active participation of the bacterium [START_REF] Halle | The NALP3 inflammasome is involved in the innate immune response to amyloid-beta[END_REF][START_REF] Jin | Novel Insights Into the NLRP3 Inflammasome in Atherosclerosis[END_REF][START_REF] Jin | Novel Insights Into the NLRP3 Inflammasome in Atherosclerosis[END_REF][START_REF] Latz | The inflammasomes: mechanisms of activation and function[END_REF][START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Zhou | A role for mitochondria in NLRP3 inflammasome activation[END_REF]. Reactive oxygen species (ROS) are oxygen metabolites that, because of their ability to gain and lose electrons, are prone to participate in oxidation-reduction reactions. Mammalian cells have developed various mechanisms to limit the production of ROS, including deactivating them and repairing cell damage. However, when the rate of ROS production increases dramatically and/or the antioxidant defenses are insufficient or fail, oxidative stress occurs. This oxidative stress is a secondary signal for the activation of the inflammasome (DOSTERT et al., 2008b;[START_REF] Zhou | Thioredoxin-interacting protein links oxidative stress to inflammasome activation[END_REF][START_REF] Zhou | A role for mitochondria in NLRP3 inflammasome activation[END_REF]. In the context of the implemented cellular model, it would be interesting to verify the role of ROS in IL-1β production through the activation of the NLRP3 inflammasome in order to elucidate the underlying mechanisms involved in the activation of the NLRP3 inflammasome induced during considered a key factor in refractory infections. Also, the investigation of the underlying mechanisms of the activation of the canonical and non-canonical pathways of the NLRP3 inflammasome in the modulation of the immune response during S. aureus infection will be interesting and new knowledge and open up new perspectives for the development of more selective treatments for the intracellular persistence of S.aureus in Bone Joint Infection. L'étude d'un autre événement de défense de la cellule hôte, tel que le contrôle de la prolifération bactérienne intracellulaire, révèle une augmentation considérable de la prolifération des bactéries internalisées dans les cellules CASP1 -/-MG-63 de type ostéoblastique. Cette découverte, en plus des rapports précédents démontrant la corrélation de l'absence d'activation de la caspase-1 avec un manque de restriction de la réplication de S. aureus à l'intérieur de cellules phagocytaires, indique que la caspase-1 active empêche la réplication intracellulaire exacerbée de S. aureus (SA113) chez les phagocytes professionnels et non professionnels.

Conclusion générale et perspectives

Ensemble, nos résultats ont démontré que les cellules MG-63 de type ostéoblaste humain jouent un rôle important dans la réponse immunitaire contre l'infection à S. aureus. Les ostéoblastes infectés ne sont pas des passants passifs, mais des acteurs actifs de la défense contre l'infection à S. aureus. L'issue de l'infection dépend de l'équilibre entre la réponse immunitaire de l'hôte et l'action de multiples facteurs de virulence de S. aureus parmi lesquels les PSM jouent un rôle pivot et dont la production peut différer chez les S. aureus. Outre nos observations d'activation de l'inflammasome (par mesure de la production d'IL-1β), nous avons démontré que les cellules de type ostéoblastes déficientes en caspase-1 étaient incapables de restreindre la réplication intracellulaire de S. aureus, ce qui suggère le rôle central de la caspase-1 dans la clairance des agents pathogènes indépendamment du type de cellules.

Ces découvertes méritent d'être approfondies afin d'améliorer notre compréhension des interactions entre S. aureus et l'hôte et de découvrir les mécanismes sous-jacents.

Et les prochaines étapes

Le modèle que nous avons développé est un outil puissant pour suivre l'inflammation in vitro.

Bien que ce modèle soit basé sur une seule lignée cellulaire, il ouvre de nouvelles perspectives pour la génération de modèles similaires de cellules non phagocytaires permettant d'explorer le processus infectieux associé à S. aureus.

De plus, nous spéculons sur l'action des autres mécanismes dans la formation de l'inflammasome NLRP3 dans les cellules MG-63 infectées par S. aureus. Une fois que ces processus dépendent de la participation active de la bactérie, tels que perturbation de la membrane lysosomale, déséquilibre ionique (efflux de K +) et induction du stress oxydatif, signaux essentiels à l'activation des inflammasomes de NLRP3 [START_REF] Halle | The NALP3 inflammasome is involved in the innate immune response to amyloid-beta[END_REF][START_REF] Jin | Novel Insights Into the NLRP3 Inflammasome in Atherosclerosis[END_REF][START_REF] Jin | Novel Insights Into the NLRP3 Inflammasome in Atherosclerosis[END_REF][START_REF] Latz | The inflammasomes: mechanisms of activation and function[END_REF][START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Zhou | A role for mitochondria in NLRP3 inflammasome activation[END_REF]. Les Reactive oxygen species (ROS) sont des métabolites de l'oxygène qui, en raison de leur capacité à gagner et à perdre des électrons, ont tendance à participer aux réactions d'oxydoréduction.

Les cellules de mammifères ont mis au point divers mécanismes pour limiter la production de ROS, y compris leur désactivation et la réparation des dommages cellulaires. Cependant, Comparing bacteriocins with conventional antibiotics as treatment options for

Staphylococcus aureus infections

Elma Lima Leite 1-2 , Alberto F. de Oliveira Jr 1 , Eric Guédon 1 , Nadia Berkova 1 , Fillipe L. R. do Carmo 2 , Debmalya Barh 3 , Preetam Ghosh 4 , Yves Le Loir 1 , Vasco Azevedo 2 * enzyme, a sensor of the presence of penicillin and a transcriptional repressor of blaZ expression, respectively. β-lactamase acts on penicillin outside the cell by changing its molecular structure to an inactive form, penicilloic acid, by the hydrolysis of its β-lactam ring.

The expression of blaZ is controlled by external penicillin availability. Penicillin interacts with the transmembrane protein BlaR1. This interaction leads to the autocatalytic activation of BlaR1 to BlaR2 (or BlaR1 in active form) that can promote the inactivation of the BlaI repressor and therefore blaZ to synthesize enzyme. The three genes are located on a transposable element of a large S. aureus plasmid called b-lactamase-encoding transposon Tn552 that shows persistence over time and geographic spread. Furthermore, it was also described to carry cadmium resistance genes, which can strengthen the role of resistance by attributing greater persistence among strains that carry this genetic content [START_REF] Shearer | Major Families of Multiresistant Plasmids from Geographically and Epidemiologically Diverse Staphylococci[END_REF]. Like the others β-lactams, methicillin impends the synthesis of bacterial cell walls. It inhibits crosslinkage between the linear peptidoglycan polymer chains, which compose a major component of the cell wall of gram-positive bacteria, by binding to and competitively inhibiting penicillinbinding proteins (PBPs). These PBP molecules can also behave like transpeptidases (Dalanyl-alanine). The mechanism that describes the methicillin-resistance of S. aureus (MRSA) proceeds in a similar way to Penicillin. However, several works have discussed that the mecA gene, that is responsible for methicillin resistance, may be a mobile genetic element (MGE), which confers the ability to respond to environmental stresses.

Conversely, mecA codes for the enzyme PBP2a, which belong to the class of glycopeptide antibiotics, and acts in exactly the same way as the β-lactamases by hydrolyzing and inactivating Penicillin. Its activation leads to the cleavage of mecR1 that inactivates mecI releasing the operator region to the expression of mecA.

Another glycopeptide antibiotic described as susceptible to resistance in S. aureus is Vancomycin [START_REF] Walters | Vancomycin-Resistant Staphylococcus aureus -Delaware, 2015[END_REF]. Currently, two groups of strains set up the scenario of vancomycin resistance: Vancomycin-sensitive S. aureus (VSSA) and Vancomycin-resistant S. aureus (VRSA) (McGuinness et al., 2017). Vancomycin alters the peptidoglycan density in the cell wall, being capable of interacting with it forming non-covalent hydrogen bonds, in the exposed D-D-Ala-D-Ala peptides [START_REF] Hanaki | Activated cell-wall synthesis is associated with vancomycin resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50[END_REF] which inhibits cell wall synthesis in the VSSA strains. The mechanism of resistance is associated with either the presence of an enterococcal plasmid vanA or by the transposition of elements related to Tn1546 [START_REF] Zhu | Dissemination of an Enterococcus Inc18-Like vanA Plasmid Associated with Vancomycin-Resistant Staphylococcus aureus[END_REF]. The affinity of vancomycin to the polypeptide is heavily reduced due to the vanA operon provided by the conjugation of the plasmid in the VRSA strains, which is able to produce the different polypeptide D-Ala-D-Lac.

S. aureus may also become resistant to the class of quinolones antibiotics (Lowy, 2003;[START_REF] Oizumi | Relationship between mutations in the DNA gyrase and topoisomerase IV genes and nadifloxacin resistance in clinically isolated quinolone-resistant Staphylococcus aureus[END_REF][START_REF] Tanaka | Mechanism of quinolone resistance in Staphylococcus aureus[END_REF]. This resistance is achieved due to specific mutations in two (~1.0%). Interest has grown around this approach in the academic world, and recent studies have shown better results when the PEG / salt-based ATPS type is used, reaching yield values around 93% [START_REF] Sabo | Bacteriocin partitioning from a clarified fermentation broth of Lactobacillus plantarum ST16Pa in aqueous two-phase systems with sodium sulfate and choline-based salts as additives[END_REF].

In addition, the AMTPS results trying to improve the nisin extraction in the presence of electrolytes, show the advances in using the approach compared to conventional methods [START_REF] Jozala | Aqueous Two-Phase Micellar System for Nisin Extraction in the Presence of Electrolytes[END_REF].

Hence, the potential of ATPS and AMTPS as primary recovery methods for bacteriocins from a complex fermentation broth can be explored at an industrial scale, considering the easy handling and speed in obtaining these biomolecules, compared to the production stages already developed in the research laboratory.

A future applicability to S. aureus: bacteriocins becoming green?

Recent research on fermented vegetable extract has been shown to be a good strategy and a new application in the use of bacteriocins [START_REF] Feng | Recent research process of fermented plant extract: A review[END_REF]. Receiving the name of Fermented Plant Extract (FPE), this approach -most commonly performed as a plain liquid manure or plant extract can be used as a tool that assists in obtaining active substances providing a diversity of health benefits [START_REF] Altay | A review on traditional Turkish fermented non-alcoholic beverages: Microbiota, fermentation process and quality characteristics[END_REF][START_REF] Marsh | Fermented beverages with health-promoting potential: Past and future perspectives[END_REF]. Some plants of the family Labiatae which contain a diversity of herbs were shown to have antimicrobial activity [START_REF] Mahboubi | Total Phenolic Content and Antibacterial Activity of Five Plants of Labiatae against Four Foodborne and Some Other Bacteria[END_REF]. FEP are also described as being fermented microorganisms, which includes yeast and bacteria [START_REF] Blandino | Cereal-based fermented foods and beverages[END_REF][START_REF] Manzanilla | Effects of butyrate, avilamycin, and a plant extract combination on the intestinal equilibrium of early-weaned pigs[END_REF]. This motivates the specific use of bacteria and plants, which may constitute a synergy in the antimicrobial treatment and a better understanding of the control by bacteriostatic phenotypes. The approach describing a composition comprising a bacteriocin and an extract from a plant is reported in a patent of [START_REF] Coyne | Composition comprising a bacteriocin and an extract from a plant of the labiatae family[END_REF]. This kind of strategy has been gaining strength and are evidenced in numerous cases where the interruption of bacterial growth appears to be ineffective due to the use of these vegetable broths in conjunction with specific microbiotas [START_REF] Marbun | Analysis of Antibacterial, Antioxidant, and In Vitro Methane Mitigation Activities of Fermented Scutellaria baicalensis Georgi Extract*[END_REF]. In addition, extracts of tea and soybean showed dosedependent growth inhibition of pathogens (Zhao and Shah, 2015). This study used phenolicenriched milk (PEM), fermented with lactic acid bacteria (LAB) and ultrafiltered, concluding that multiple agents, such as bacteriocins secreted by LAB, may exhibit synergistic antibacterial activity. This evidence on the role of FPE reveal their importance and applicability not only considering their use for health, thus presenting a new approach in treatments for S. aureus infections, but also the production at an industrial scale, and amongst other considerations, the environment.

Figure 1 : 8 Figure 2 :

 182 Figure 1: Overview of the immune system. .................................................................. 8 Figure 2: Accessory gene regulatory system (Agr). .................................................... 22

Figure 3 :

 3 Figure 3: Overview of phenol-soluble modulin activities in S. aureus. ........................ 23

Figure 4 : 24 Figure 5 : 35 Figure 6 :

 4245356 Figure 4: Phenol-soluble modulins peptides (PSMs) define the virulence potential of S. aureus. ....................................................................................................................... 24 Figure 5: Scheme of NLR receptor subfamilies and their structure in humans. .......... 35 Figure 6: Scheme of the domain structure of the NLRP3 inflammasome components. ................................................................................................................................... 36

Figure 7 :

 7 Figure 7: Human NLRP1 responds to muramyl dipeptide, while Anthrax lethal toxin triggers murine Nlrp1b.. .............................................................................................. 39

Figure 9 :

 9 Figure 9: AIM2 and NLRC4 inflammasomes are activated by specific PAMPs. .......... 44

Figure 8 : 46 Figure 10 :

 84610 Figure 8: NLRP3 inflammasome activation mechanism. ............................................ 46 Figure 10: Schematic of the proposed objectives for the first and second part of the thesis. ......................................................................................................................... 52

Figure 11 :Figure 12 :Figure 13 :Figure 14 :Figure 15 :Figure 16 :Figure 17 :Figure 18 :Figura 19 :Figure 20 :Figure 21 :VIIFigure 22 :

 111213141516171819202122 Figure 11: Detection of active caspase-1 in MG-63 cell supernatants by Western blot. ................................................................................................................................. 114 Figure 12: Detection of pro-caspase-1 in lysates of MG-63 and THP1 cells by western blotting. ..................................................................................................................... 116 Figure 13: Influence of PMA on IL-1β production in culture supernatants of THP1 cells. ................................................................................................................................. 118 Figure 14: Caspase-1 activation and IL-1β release triggered in human osteoblasts. 120 Figure 15: Detection of pro-caspase-1 in lysates of MG-63-CRISPR/cas9 cells by Western blot. ............................................................................................................ 123 Figure 16: Detection of pro-caspase-1 in lysates of CASP1 -/-MG-63 cells by Western blotting. ..................................................................................................................... 124 Figure 17: Detection of pro-caspase-1 in lysates of CASP1 -/-MG-63 cells by Western blotting. ..................................................................................................................... 125 Figure 18: Detection of pro-caspase-1 in lysates of CASP1 -/-MG-63 cells by Western blot. ........................................................................................................................... 125 Figura 19: Deletion of caspase-1 gene in human osteoblasts MG-63 using the CRISPR/Cas9 gene editing system .......................................................................... 126 Figure 20: NLRP3 protein expression, speck-like aggregate formation and IL-1β secretion by WT MG-63 vs CASP1 -/-MG-63 cells exposed to S. aureus. ................. 128 Figure 21: S. aureus strain-dependent release of IL-1β by infected MG-63 cells. ..... 130

Figure 23 :

 23 Figure 23: S. aureus phenol-soluble modulins stimulate IL-1β release from infected osteoblasts. ..............................................................................................................

Figure 24 :

 24 Figure 24: kinetics of IL-1β production between 6 h and 9 days post-infection in MG-63 cells by S. aureus clinical strains...............................................................................

Figure 25 :

 25 Figure 25: Involvement of caspase-1 in bacterial clearance. ....................................

Table 1 :Table 2 :Table 3 :

 123 Virulence factors of S. aureus and their characteristics................................. List of activators known to activate of the NLRP3 inflammasome. ................ Different protocols used to detect active caspase-1 in THP1 cells differentiated with (PMA) by western blotting. ................................................................................. IX List of Abbreviations ANOVA: Analysis of variance ASC: Apoptosis-associated speck-like protein containing a caspase recruitment domain ATP: Adenosine triphosphate BHI: Brain Heart Infusion CA-MRSA: Community-Acquired Methicillin-Resistant S. aureus CARD: Caspase recruitment domain DC: Dendritic cells DMEM: Dulbecco's Modified Eagle Medium DNA: Deoxyribonucleic acid ELISA: enzyme-linked immunosorbent assay FACS: Fluorescence-activated cell sorting Fn: Fibronectins FnBPA and FnBPB: Fibronectins Binding Proteins (A and B) Gent100: Gentamicin 100 μg/mL Gent25: Gentamicin 25 μg/mL HA-MRSA: Hospital-Associated Methicillin-Resistant S. aureus ICB: Institudo de Ciências Biológicas ICE: IL-1-converting enzyme ou enzyme transformant IL-1 IFN-γ: interferon gamma IL-1β: interleukin-1 beta INRA: French National Institute for Agricultural Research LDH: Lactate dehydrogenase LPS: lipopolysaccharide MOI: Multiplicity of infection MRSA: Methicillin-resistant Staphylococcus aureus MS: Mass spectrometry MSCRAMMs: Microbial Surface Components Recognizing Adhesive Matrix Molecules NK: Natural killers NLR: NOD-like receptor NOD: nucleotide binding oligomerization domain PAMPs: Pathogen associated molecular pattern PBMC: Peripheral blood mononuclear cells PBS: Phosphate Buffered Saline PMA: phorbol 12-myristate 13-acetate X PSM: Phenol-soluble modulins PVDF: Polyvinylidene fluoride membrane ROS: reactive oxygen species/espèces réactives de l'oxygène RPMI-1640: Roswell Park Memorial Institute medium S. aureus: Staphylococcus aureus FCS: Fetal Calf Serum SCCmec: staphylococcal cassette chromosome mec SCV: Small colony variant SD: Standard deviation SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis SE: Staphylococcal enterotoxins TBS: Tris-buffered saline Th: T helper cells TLR: Toll-like receptor TNF-α: Tumor Necrosis Factor-α TPA: 12-O-tetradecanoylphorbol 13-acetate UFMG: Universidade Federal de Minas Gerais V: Volts WB: Western Blotting WT: Wild type

  Mots clés: Ostéoblastes, Staphylococcus aureus, inflammasomes, caspase-1, IL-1β Collaborations This PhD thesis was prepared in collaboration between the Federal University of Minas Gerais (UFMG) (Belo Horizonte, Brazil) and the Institute of Agricultural Sciences, Agro-Food, Horticulture and Landscape (Rennes, France). The work presented in this manuscript was performed at the Laboratory Science & Technologie du Lait & de l'Oeuf (STLO) at the Institut National de la Recherche Agronomique (INRA), located in Rennes, France under the supervision of Dr. Nadia Berkova and co-supervision of Dr. Yves Le Loir and at the Laboratory of Cellular and Molecular Genetics in the Institute of Biological Sciences, Federal University of Minas Gerais under the supervision of Prof. Vasco Azevedo. In this context, the objective of my project was to investigate whether Staphylococcus aureus invasion can induce the activation of inflammasomes in non-professional phagocytes, using osteoblast-like cells as a model of cells infections and which S. aureus virulence factors are involved in inflammasomes activation during infection. To address these questions, we generated knock-out osteoblasticlike cells for the caspase-1 gene using CRISPR/Cas9-based genome editing in collaboration with Dr. Arthur Gautron and Dr. David Gilot, IGDR, Rennes, France and Pr. Petr Broz, University of Lausanne, Switzerland. Inflammasomes activation was investigated in infected WT MG-63 vs CASP1 -/ -MG-63 cells through immunological detection of active caspase-1 and active IL-1β. Moreover, bacterial clearance in WT MG-63 vs CASP1 -/ -MG-63 cells was investigated using gentamicin protection assay and immunofluorescence methodology. To decifer the role of principal S. aureus virulence factors in inflammasomes activation of infected osteoblasts, we used wild type and mutant strains of S. aureus kindly provided by Pr. Michael Otto, NIH, USA and Pr. Friedrich Goetz, Tubiengen University, Germany. Our work brings new knowledge on the pathogenic mechanisms of S. aureus, a major cause of chronic infection in humans and animals. This work is supported by different funding agencies in Brazil by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, CAPES/COFECUB) -Finance Code 001 and in France by Institut National de la Recherche Agronomique (INRA).

( 6 .

 6 I) microbiological aspects of Staphylococcus aureus, as well as diseases caused by this pathogen and associated pathogenic processes; phenol-soluble modulins (PSMs) and their role in pathogenesis; (II) the inflammasome, a primordial complex of innate immunity; (III) innate immunity receptors; (IV) molecular platform: the inflammasome. II. Chapter 2. Problem statement of the project thesis. III. Chapter 3. Literature review. Strain and cell type-specificity of host cell response to Staphylococcus aureus invasion. IV. Chapter 4. Original article -Involvement of caspase-1 in inflammasomes activation and bacterial clearance in S. aureus-infected osteoblasts. In this part it will be described (the methodology, the results and the discussion) of the work. General conclusion and perspectives of the work performed during PhD project.

Figure 1 :

 1 Figure 1: Overview of the immune system.

Figure 1 :

 1 Figure 1: Vue d'ensemble du système immunitaire. La réponse innée est caractérisée par le fait qu'elle constitue la première ligne de défense contre l'infection dans laquelle interviennent des leucocytes tels que des neutrophiles, des monocytes, ou des macrophages. Ces cellules de défense détectent et attaquent d'autres cellules portant des structures pathogen-associated molecular patterns (PAMP) et de petites protéines qui signalent une invasion d'agents pathogènes (cytokines et chimiokines) ou de courts peptides antimicrobiens se liant directement aux agents pathogènes microbiens. Les cellules du système imunitaire adaptatif s'appuient sur des lymphocytes T et B et des protéines (les anticorps) qui détectent et éliminent spécifiquement des agents pathogènes et prodiosent également une cytokine/chimiokine afin de signaler et de recruter d'autres cellules immunitaires. Les lymphocytes T et B jouent un rôle essentiel dans l'immunité contre l'invasion pathogène. Le système du complément, ainsi que les cellules tueuses naturelles (NK) et les cellules dendritiques, interviennent à la fois dans l'immunité innée et adaptative. Reproduit de (SPIERING, 2015).

Figure 2 :

 2 Figure 2: Accessory gene regulatory system (Agr).

Figure 3 :

 3 Figure 3: Overview of phenol-soluble modulin activities in S. aureus.

  the genome. The PSMα1-PSMα4 genes are encoded in the PSMα operon. The PSMβ1 and PSMβ2 peptides encoded in the PSMβ operon and the δ-toxin is encoded within the RNAIII sequence (Fig. 4) (CHATTERJEE et al., 2011; OLIVEIRA; BORGES; SIMÕES, 2018).

Figure 4 :

 4 Figure 4: Phenol-soluble modulins peptides (PSMs) define the virulence potential of S. aureus.

  another proinflammatory cytokine belonging to the IL-1 family(DINARELLO, 2018a;[START_REF] Girard-Guyonvarc'h | Unopposed IL-18 signaling leads to severe TLR9-induced macrophage activation syndrome in mice[END_REF]. The maturation stage of IL-18 is mediated by caspase-1 and the inflammasome, and has a similar structure to IL-1β (DINARELLO, 2018b; FENINI; CONTASSOT; FRENCH, 2017). IL-18 is constitutively expressed in cells of the immune system but also in keratinocytes and epithelial cells[START_REF] Nakanishi | Unique Action of Interleukin-18 on T Cells and Other Immune Cells[END_REF]. This cytokine plays a key role in T-cell stimulation and is an important inducer of IFN-γ. Th1 and T cells are involved in the Th17 cytokine production by T cells, NK cells, basophils and mast cells[START_REF] Nakanishi | Unique Action of Interleukin-18 on T Cells and Other Immune Cells[END_REF][START_REF] Silvestre | Innate immunity and effector and regulatory mechanisms involved in allergic contact dermatitis[END_REF] SATO;[START_REF] Silvestre | Innate immunity and effector and regulatory mechanisms involved in allergic contact dermatitis[END_REF] YASUDA;[START_REF] Nakanishi | Unique Action of Interleukin-18 on T Cells and Other Immune Cells[END_REF][START_REF] Yasuda | Interleukin-18 in Health and Disease[END_REF].

  inflammatory activity (BOUTET; NERVIANI; PITZALIS, 2019). In 2001, cloning of the coding genes located on chromosome 2 was described byKumar and colleagues 2000 (KUMAR; KAWAI;[START_REF] Kumar | Pathogen Recognition by the Innate Immune System[END_REF]. There are five isoforms of the IL-37 gene encoding five isoforms (IL-37a, IL-37b, IL-37c, IL-37d and IL-37e), however, to date the IL-37b isoform is the most characterized (BOUTET; NERVIANI; PITZALIS, 2019). IL-37 has a similar receptor and structure to IL-18. However, to date it is not known whether its activity occurs via caspase-1 cleavage or whether it is active as a precursor (BOUTET; NERVIANI; PITZALIS, 2019; DINARELLO, 2018a). In a study developed by Ellidson and colleagues in 2017, additional sites of IL-37 cleavage predicted in silica have been shown to include cathepsin K, elastase-2 and matrix metalloproteinase (MMP) -9 cleavage sites (BOUTET; NERVIANI; PITZALIS, 2019;[START_REF] Ellisdon | Homodimerization attenuates the anti-inflammatory activity of interleukin-37[END_REF].

  Currently, four families of PRRs are known to operate cooperatively to recognize endogenous microbial pathogens or endogenous danger signals: toll-like receptor system (TLRs) and Ctype lectin receptors (CLRs), which are transmembrane receptors that monitor the extracellular environment, as well as the interior of the endosomes, and the intracellular receptors like [RIG-I-like receptors (RLRs) and nucleotide-binding domain leucine-rich repeats (NLRs)] that act on the cytosol (BAUERNFEIND; HORNUNG, 2013; TEMPLETON; MOEHLE, 2014). Among the transmembrane receptors, the TLRs were first discovered and therefore are the most studied class of receptors (AGIER; PASTWIŃSKA;[START_REF] Agier | An overview of mast cell pattern recognition receptors[END_REF] JANG et al., 2015; KAWAI;[START_REF] Kawai | The roles of TLRs, RLRs and NLRs in pathogen recognition[END_REF]. TLRs can be expressed in various cell types, such as monocytes, macrophages, B and T lymphocytes, dendritic cells, neutrophils, NK cells, epithelial cells, endothelial cells, and Mast cells. Currently, 10 TLR genes have been identified in humans (TLR1-TLR10) and 12 (TLR1-TLR9, TLR11-TLR13) in mice[START_REF] Goulopoulou | Toll-like Receptors in the Vascular System: Sensing the Dangers Within[END_REF] MCCARTHY;[START_REF] Goulopoulou | Toll-like Receptors in the Vascular System: Sensing the Dangers Within[END_REF][START_REF] Kielian | Overview of Toll-Like Receptors in the CNS[END_REF].

  central nucleotide-binding domain/NOD (NACHT); and a caspase activation and recruitment domains (CARD), pyrin domain (PYD), or baculovirus inhibitor of apoptosis protein repeat (BIR) (BAUERNFEIND; HORNUNG, 2013). In humans, there are 22 known NLRs characterized by N-terminals composed of five receptor subfamilies, which resulted in the splitting of NLR family members into two main subfamilies: the NLRC -NLR family CARD domain-containing family (NOD1, NOD2, NLRC3, NLRC4 and NLRC5) and NLRP -NACHT, LRR, and the PYD domain-containing family (NLRP1-14), which accompanies three other small subfamilies NLRA (CIITA), NLRB (NAIP -neuronal apoptosis inhibitory protein) and NLRX (NLRX1) (BAUERNFEIND; HORNUNG, 2013) (Fig. 5).The best characterized NLRs are NOD1 (NLRC1), NOD2 (NLRC2), NLR family containing a CARD domain 4 (NLRC4 -previously known as IPAF), and NLR family pyrin domain containing 3 (NLRP3)[START_REF] Di | The therapeutic potential of modifying inflammasomes and NOD-like receptors[END_REF]. Almost two decades ago, research began on the role of NLR receptors as inflammation mediators surrounding the discovery of a multiprotein complex called the inflammasome. This inflammasome is the basis of this work and will be described in the next topic.

Figure 5 :

 5 Figure 5: Scheme of NLR receptor subfamilies and their structure in humans. NLRA (CIITA), NLRB (NAIP), NLRC (NOD1, NOD2, NLRC3, NLRC4 and NLRC5), NLRP3 (NLRP1, NLRP2-9, NLRP11-14 and NLRP10) and NLRX (NLRX1). According to (BAUERNFEIND et al., 2011).

4 . 6 )

 46 Molecular platform: The inflammasomeInflammasomes are multi-protein signaling complexes that assemble after recognition of danger signals and/or pathogens by a family of cytosolic receptors called NLRs (nucleotide binding domain and leucine-rich repeats containing receptors) or PYHIN protein family members. Once assembled, inflammasomes initiate signaling by activation of downstream proteases, most notably caspase-1 and caspase-11, which then proteolytically mature pro-IL-1β, pro-IL-18, and pro-IL-33, and promote their secretion from the cell. Most inflammasomes also use an adaptor molecule known as ASC (Apoptosis-associated speck-like protein) (Fig.[START_REF] Gross | Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1[END_REF][START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Martinon | Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases[END_REF].After the activation of caspase-1, the cleavage of pro-IL-1β and pro-IL-18 occurs, producing their active forms IL-1β and IL-18, which stimulate inflammatory response[START_REF] Gross | Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1[END_REF]. Furthermore, activation of Caspase-1 via the inflammasome may induce pyrophosis, a type of inflammatory cell death (MIAO; RAJAN; ADEREM, 2011). The inflammasomes play an important role in the regulation of host immune response in response to different stimuli, whether endogenous or exogenous[START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF].Currently, five members of the NLRP family capable of forming the inflammasome have been described: NLRP1, NLRP2, NLRP3, NLRP6 and NLRP12. However, NLRB and NLRC4, are also able to form the inflammasome together with AIM2 (AIM2-Like receptor, ALR) (LAMKANFI;DIXIT, 2014;[START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Sá | Inflammasomes and dermatology[END_REF][START_REF] Sá | Inflammasomes and dermatology[END_REF] SCHRODER; TSCHOPP, 2010).

Figure 6 :

 6 Figure 6: Scheme of the domain structure of the NLRP3 inflammasome components.NLR (the nod-like receptor) is composed of a C-terminal LRR domain, a NACHT domain and an Nterminal PYD. ASC is composed of a PYD and a CARD, and Pro-caspase-1 has a CARD and caspase-1 (subunits p20 and p10) which is activated within the inflammasome multiprotein complex. Adapted by[START_REF] Inouye | The Emerging Role of Inflammasomes as Central Mediators in Inflammatory Bladder Pathology[END_REF].

  activation was only well elucidated from the year 2002 when Tschopp and Martinon discovered the inflammasome[START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF]. Caspase-1 is present in the cellular cytoplasm in an inactive form of 45 kDa (pro-caspase-1), that consist, of an N-terminal CARD domain and a domain containing subunits (p20) and (p10) in C-terminal contains the small catalytic subunit of the enzyme (NAIK;[START_REF] Naik | Modulation of Inflammasome Activity for the Treatment of Auto-inflammatory Disorders[END_REF] SCHRODER; TSCHOPP, 2010;[START_REF] Shamaa | Monocyte Caspase-1 Is Released in a Stable, Active High Molecular Weight Complex Distinct from the Unstable Cell Lysate-Activated Caspase-1[END_REF].However, the functions of caspase-1 are not exclusively cytokine maturation. It also participates in the control of a type of programmed cell death called pyroptosis[START_REF] Di | The therapeutic potential of modifying inflammasomes and NOD-like receptors[END_REF][START_REF] Gross | Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1[END_REF]. According to PetrBroz and colleagues (2011), the long form of caspase-1 induces pyroptosis after the assembly of the NLRC4 inflammasome in Salmonella typhimurium and Legionella pneumophila (BROZ; MONACK

  polymorphic among different mouse strains(BROZ;[START_REF] Broz | Molecular mechanisms of inflammasome activation during microbial infections: Mechanisms of inflammasome activation[END_REF][START_REF] Sá | Inflammasomes and dermatology[END_REF][START_REF] Sá | Inflammasomes and dermatology[END_REF]. The NLRP1 inflammasome is composed of the NLRP1 protein from C-terminal to Nterminal, CARD, a domain with function to find (FIIND), LRR, NACHT, and PYD (Fig4).However, the PYD domain is only found in human NLRP1 (CHAVARRÍA-SMITH; VANCE, 2015; LOPEZ-CASTEJON; BROUGH, 2011). The presence of the CARD domain suggests that NLRP1 can interact directly with caspase-1 and caspase-5, leading to maturation ofIL-1β and IL-18 (FRANCHI et al., 2009;[START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF].

  7) (LATZ; XIAO;STUTZ, 2013b;[START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Martinon | Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases[END_REF]. However, the mechanism of activation is indirect and depends on ionic changes between the intracellular and extracellular medium suggesting a link between the activation of NLRP1 inflammasome and the NLRP3 inflammasome (DI VIRGILIO, 2013).

Figure 7 :

 7 Figure 7: Human NLRP1 responds to muramyl dipeptide, while Anthrax lethal toxin triggers murine Nlrp1b. Adapted by (BROZ; MONACK, 2011).

  regarding the NLRP3 inflammasome activation mechanism, supported by several studies, include potassium efflux out of the cell, reactive oxygen species (ROS), cathepsin release into the cytosol after lysosomal destabilization, and cardiolipin or release of mitochondrial DNA (GRAZIOLI;[START_REF] Grazioli | Mitochondrial Damage-Associated Molecular Patterns: From Inflammatory Signaling to Human Diseases[END_REF] VANAJA; RATHINAM;[START_REF] Vanaja | Mechanisms of inflammasome activation: recent advances and novel insights[END_REF][START_REF] Wei | The Potential Role of the NLRP3 Inflammasome Activation as a Link Between Mitochondria ROS Generation and Neuroinflammation in Postoperative Cognitive Dysfunction[END_REF].The activation of the NLRP3 inflammasome can occur by three different. i) Canonical pathway (Canonical NLRP3 inflammasome activation requires two parallel and independent steps: transcription and oligomerization), described in detail below. ii) Non-canonical (caspase-11dependent NLRP3 activation. In particular, Gram-negative bacteria (i.e., Escherichia coli, Salmonella typhimurium), activate the TLR4-MyD88 and toll/IL-1 receptor homology-domaincontaining adapter-inducing interferon-β (TRIF) pathways, with a consequent nuclear translocation of NF-κB, which in turn promotes the transcription ofIL-1β, IL-18 (PELLEGRINI et al., 2017). iii) Alternative pathways (This alternative pathway is independent of K efflux and leads to a low and gradual IL-1β response without pyroptosome and depends on TRIF, RIPK1, FADD, and caspase-8 signaling pathway).

Figure 8 :

 8 Figure 8: AIM2 and NLRC4 inflammasomes are activated by specific PAMPs. AIM2 functions as a cytosolic DNA sensor, detecting DNA introduced by transfection, infection with the cytosolic bacterial pathogens F. tularensis or L. monocytogenes or DNA viruses. NLRC4 detects flagellin or the T3SS rod subunit in the cytosol. Adapted by (BROZ; MONACK, 2011).

Figure 9 :

 9 Figure 9: NLRP3 inflammasome activation mechanism.

Figure 8 :

 8 Figure 8: NLRP3 inflammasome activation mechanism. The activation of a TLR allows the release of the transcription factor NF-kB, responsible for the synthesis of IL-1β and NLRP3 expressed in its basal state. The first signal called "priming" also allows for the activation of BRCC3, leading to the unubiquitination of NLRP3, a step necessary for its activation. The assembly of the NLRP3 inflammasome can occur through three known signals: 1) release of cathepsin B through lysosomal rupture; 2) ionic fluid via pore opening of the plasma membrane; 3) ROS synthesis. Activation of NLRP3 leads to its oligomerization and to the recruitment of ASC and pro-caspase-1, causing autoproteolysis and Caspase-1 activation. After activation, Caspase-1 cleaves and induces the release of IL-1β and IL-18 and may also induce pyroptosis.

Figure 10 :

 10 Figure 10: Schematic of the proposed objectives for the first and second part of the thesis.

  entre les cellules ostéoblastiques et S. aureus restent relativement peu étudiées. Dans ce contexte, nous avons émis l'hypothèse que l'infection persistante par S. aureus implique la reprogrammation transcriptionnelle de cellules ostéoblastiques afin de tolérer le portage asymptomatique des bactéries intracellulaires, conduisant à une altération de la réponse immunitaire et plus spécifiquement à une modification de l'activation des inflammations et du traitement ultérieur de l'IL-1β.Afin de mettre en évidence l'activation de l'inflammasome, l'activation de la pro-caspase-1 qui subit un traitement protéolytique pour produire 2 sous-unités qui se dimérisent pour former la caspase-1 active doit être surveillée. En outre, il est essentiel de surveiller la caspase-1 active (comprenant les sous-unités p20 et p10) et leur effet sur l'induction de l'IL-1β. Pour démontrer que l'IL-1β est effectivement activée par l'inflammasome, il a été nécessaire de générer un mutant de délétion du gène caspase-1, Cas1 -/-MG-63 sur des cellules MG-63 de type ostéoblaste en utilisant le système d'édition de gène CRISPR/Cas9. Ainsi, ce travail a été divisé en deux parties:• Dans la première partie de l'étude, nous avons développé un modèle qui nous permettrait d'étudier l'implication de l'inflammasome NLRP3 dans l'infection à S. aureus. Tout d'abord, nous avons effectué l'inactivation du gène de la Caspase-1 sur des cellules MG-63, en utilisant l'approche CRISPR/cas9. Ensuite, l'activation de la Caspase-1 dans des cellules de type sauvage (WT) par rapport à des cellules (CASP1 - /-MG-63) sera surveillée (Fig.10-A).• Dans la deuxième partie de l'étude, la production d'une forme active soluble d'IL-1β sera surveillée dans les cellules WT MG-63 vs CASP1 -/-MG-63 dans un modèle cellulaire in vitro d'infection chronique utilisant des infections à long terme (de 6 h à 11 jours) d'ostéoblastes avec différentes souches de S. aureus (Fig.10 -B).

Figure 10 :

 10 Figure 10: Schéma des objectifs proposés pour la première et la deuxième partie de la thèse.

Figure 1 -

 1 Figure 1-B shows a schematic presentation of the different receptors involved in S. aureus internalization in non-professional phagocytes.

4 .

 4 Cell response to S. aureus infection is strain-dependent S. aureus is a highly variable and versatile pathogen. Its variability relies in the set virulence factors borne by S. aureus strains and in the strains capability to adapt, grow and express their virulence in a given context. Such variability affects each step of the pathogenesis, from adhesion onto host cells to the host cell response. A very first step of bacteria-host cell

Figure 1 :

 1 Figure 1: Staphylococcal mechanisms of adherence to and internalization into host cells. (A). The current model of some signaling events involved in the invasion of S. aureus into non-professional phagocytic cells. Fibronectin-binding proteins (FnBPs) expressed on the bacterial surface bind via host fibronectin in the extracellular matrix, which forms a bridge with receptors integrin α5β1 on the host cell membrane. The integrin-linked kinase (ILK) provides a link via α5β1 and the cytoskeleton and interacts through the cytoplasmic domain β1. The subsequent recruitment of paxillin (PAX) and focal adhesion kinase (FAK) lead to remodeling of the actin cytoskeleton. Other focal adhesion proteins such as vinculin (VLC) and tensin are also recruited to the site of bacterial uptake. (B). Different receptors involved in S. aureusinternalization in non-professional phagocytes. The main mechanism of internalization involves the α5β1 integrin receptor, which is mediated by Fibronectin (Fn) and fibronectin-binding protein (FnBP). FnBP can interact with heat shock protein 60 (Hsp60) thus contributing to efficient S. aureus internalization by host cells. Extracellular adherence protein (Eap) also contributes to staphylococcal adherence and internalization if sufficiently expressed, but a host cell receptor has not been described yet. S. aureus autolysin (Atl) can bind to heat shock protein, Hsc70 or integrin α5β1 integrin via Fn. Wall teichoic acid (WTA) is required for nasal colonization by S. aureus and there is evidence that scavenger receptors are involved in WTA binding. Adapted by[START_REF] Hirschhausen | A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor: Atl-mediated staphylococcal internalization[END_REF].

Figure 2 :

 2 Figure 2: Staphylococcus aureus can also cause apoptosis of osteoblasts.The induction of apoptosis of osteoblasts by the intrinsic and extrinsic caspase pathways. Both can lead to the release of intracellular S. aureus after the death of the osteoblast. Adapted by[START_REF] Josse | Staphylococcus aureus vs. Osteoblast: Relationship and Consequences in Osteomyelitis[END_REF]

Figure 1 :

 1 Figure 1:

Figure 2 :

 2 Figure 2:

1 .

 1 Caspase-1 activation and IL-1β release triggered by inflammasomes activators LPS and ATP in human osteoblasts and PMA-treated ThP1 cells In order to monitor inflammasome activation in the human osteoblast-like MG-63 cells the detection of active caspase-1 by Western blot analysis was set up in samples containing cells and cell supernatants. As shown in Fig. 1A, exposing MG-63 cells to LPS+ATP led to the appearance of 20 kDa band corresponding to the active caspase-1 p20 fragment. The activation of caspase-1 was associated with the production of IL-1β, as the exposure of cells to LPS+ATP resulted in the production of mature IL-1β (Fig.1B). These results suggest that MG-63 cells form functional inflammasomes and that their activation can be estimated by IL-1β release. To compare the ability of inflammasome activation by professional versus non-professional phagocytes, PMA-stimulated ThP1 cells differentiated into macrophagesor MG-63 osteoblastlike cells were exposed to inflammasome activators LPS+ATP and the level of IL-1β production was estimated 2 h and 6 h after the beginning of the treatment. PMA-treated control ThP1 cells produce ~ 7 pg/ml of IL-1β 2 h at the beginning of the treatment. Exposure of PMAdifferentiated ThP1 cells to LPS+ATP resulted in a significant increase to ~ 30 pg/ml 2 h post treatment. After 6 h PMA-treated control ThP1 cells produce ~ 10 pg/ml of Il-1β, while the level of IL-1β produced by PMA-treated ThP1 cells after an exposure to LPS+ATP was ~ 650 pg/ml.

  analyzed in WT MG-63 and CASP1 -/-MG-63 cells. NLRP3 protein expression was estimated by FACS. As shown in Fig. 3B, equal amounts of NLRP3 was observed in WT and CASP1 -/- MG-63 cells. The exposure of both types of cells to S. aureus SA113 resulted in similarly increased levels of fluorescence. Mean fluorescence intensity (MFI) increased from 8000 to 13500 in WT MG-63 cells after S. aureus infection, similarly to CASP1 -/-MG-63 cells, which

6 h

 6 post treatment with inflammasomes activator LPS+ATP, matching the of active caspase-1 20 kDa subunit (Fig.1) that suggests inflammasomes activation in MG-63 cells. The production of IL-1β by MG-63 cells started later and IL-1β level was much lower than IL-1β production by ThP1 cells whose level of IL-1β production corresponds to the findings of other researchers[START_REF] Syed | Staphlyococcus aureus phenolsoluble modulins stimulate the release of proinflammatory cytokines from keratinocytes and are required for induction of skin inflammation[END_REF]. The discrepancy between levels of the soluble IL-1β released by MG-63 and ThP1 is likely related to the different origin of both cells' types: osteoblast-like MG-63 cells and PMAtreated ThP1 differentiated into macrophages[START_REF] Trouillet-Assant | Adaptive processes of Staphylococcus aureus isolates during the progression from acute to chronic bone and joint infections in patients[END_REF].Taking into account the pivotal role of caspase-1 in the maturation of IL-1β we generated the CASP1 -/-MG-63 cell line using CRISPR/CAS9 gene editing for the investigation of inflammasomes involvement in S. aureus infected osteoblasts. The equal NLRP3 protein expression together with the similar formation of ASC specks in both WT MG-63 and CASP1 - /-MG-63 cells, as well as the absence of active caspase-1 and IL-1β release following the stimulation with inflammasomes activators in CASP1 -/-MG-63 cells, validate caspase-1 knock out in osteoblastic cells. Therefore, we generated CASP1 -/-MG-63 cell line that is a powerful tool that allows the function of inflammasomes to be investigated. We observed that an exposure of MG-63 cells to S. aureus results in the progressive increase of IL-1β from several hours to several days in contrast to the findings ofMarriott et al. describing the increase of IL-1β RNA expression along with the absence of IL-1β protein synthesis in 24 h infected mice osteoblasts. The discrepancy between our and Marriott et al. results are likely related to the different origin of S. aureus strains used in both laboratories, because S. aureus-induced IL-1β release is strongly strain-dependent as we have demonstrated in the present work.

( 1 :

 1 1000) for 2 h. Coverslips were mounted with ProLong antifade containing DAPI (Vectashield, Biovalley). Confocal laser scanning microscopy (CLSM) observation of specimens was performed using a ZEISS LSM 880 inverted confocal microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with immersion objective 63× (Plan Apochromat objective, oil immersion, NA 1.4) driven by Zen software.

Figure legend Figure 1 .

 legend1 Figure legend

Figure 2 .

 2 Figure 2. Deletion of caspase-1 gene in human osteoblasts MG-63 using the CRISPR/Cas9 gene editing system A. Scheme showing the caspase-1 deletion targeting exon 2 of the CASP1 gene (Sequence ID NG 029124.2). B. Trace file from Sanger sequencing of the targeted deletion in the exon 2 of the CASP1 gene (WT MG-63 and CASP1 -/-MG-63 clone).

Figure 3 .

 3 Figure 3. NLRP3 protein expression, speck-like aggregates formation and IL-1β secretion by WT MG-63 vs CASP1 -/-MG-63 cells exposed to S. aureus A. WT MG-63 or CASP1 -/-MG-63 cells were incubated in 12-well plates. Cells were then detached by trypsin treatment and cell pellets were obtained by centrifugation. Protein samples were prepared in 5 x SDS-PAGE sample loading buffer. Detection of 45-kDa pro-caspase-1 in WT MG-63 and CASP1 -/-MG-63 cells by Western blot analysis using anti-caspase-1 antibody (AdipoGen) was performed as described in Material and Methods. Three independent assays were performed. B. WT MG-63 or CASP1 -/-MG-63 cells were exposed to S. aureus LAC (USA300) for 2 h. Following fixation and permeabilization of cells, NLRP3 expression at the protein level was determined by FACS using Alexa Fluor 488 anti-NLRP3 antibody. NLRP3 expression was analyzed with an Accuri C6 flow cytometer. Data were collected from 20,000 cells, and analyzed with CFlow software (Becton Dickinson). Cells are analyzed using FSC-A x SSC-A plot. The major density of events is captured by the gate. The events that represent debris, cell fragments and pyknotic cells are eliminated. Values shown on the right side of the graph refer to the respective mean fluorescence intensities (MFIs). Three independent assays were performed. C. WT MG-63 or CASP1 -/-MG-63 cells were exposed to a fluorescent derivative of S. aureus SA113, which carries plasmid-encoded mCherry (red fluorescence), at MOI 1:50 for 2 h followed by antibiotic treatment as described in Materials and Methods. Six hours postinfection cells were immunostained with rabbit anti PYCARD antibody (Coger France), followed by incubation with Alexa Fluor 488 labeled goat anti-rabbit antibody (Cell Signaling Ozyme) at a dilution of 1:50 for 2 h at room temperature (green staining, red arrow). Nuclear DNA was labeled with DAPI (blue staining). Samples were viewed with a Zeiss laser-scanning microscope equipped with a 63 × plan Apo-NA 1.4 immersion objective driven by Zen software. Scale bar: 1 µm. D, E. WT MG-63 or CASP1 -/-MG-63 cells were exposed to LPS+ATP (D) or S. aureus strain SA113 at MOI 1:50 for 2 h (E) followed by antibiotic treatment as described in Material and Methods. Six hours post-infection or 6 h after the beginning of LPS+ATP treatment cell supernatants were collected, centrifuged and the level of IL-1β was determined by a commercial sandwich-ELISA (by sandwich-ELISA (Thermofischer Life Technologies) as described in Materials and Methods. The values are presented as concentrations in pg/ml.

Figure 6 .

 6 Figure 6. Involvement of caspase-1 in bacterial clearance

  Figure 1:

Figure 3 :

 3 Figure 3:

Figure 4 :

 4 Figure 4:

Figure 5 :

 5 Figure 5:

Figure 6 :

 6 Figure 6:

caspase- 1 (

 1 Polyclonal Anti-human caspase-1 antibody, Acris-Origene, dilution of 1:250) used to recognize the p10 subunit of active caspase-1, showing a non-specificity interaction. Based on this observation, detection criteria were modified, such as: use of serum-free medium to avoid the cross-reaction of our antibodies with FCS proteins and the use of another primary monoclonal antibody to the caspase-1 subunit p/20 (Anti-human caspase-1 antibody, mAb p/20, dilution 1:1000), which has high affinity for active 20-kDa caspase-1.

Figure 11 :

 11 Figure 11: Detection of active caspase-1 in MG-63 cell supernatants by Western blot.The supernatant proteins were precipitated with trichloroacetic acid (TCA) 100%. Polyclonal Anti-human caspase-1 antibody, Acris-Origene, dilution of 1:250.

1 ,

 1 we performed assays to detect pro-caspase-1 in the cytosol of the ThP1 differentiated and MG-63 cells. In fig. 12, the MG-63 cells and PMA-differentiated ThP1 cells with the combination of LPS and nigericin demonstrated the presence of 45-kDa pro-caspase-1 in the cytosol by Western blot.

Table 3 :

 3 Different protocols used to detect active caspase-1 in ThP1 cells differentiated with (PMA) by western blotting.

Figure 12 :

 12 Figure 12: Detection of pro-caspase-1 in lysates of MG-63 and ThP1 cells by western blotting. Negative controls: WT MG-63 and PMA-differentiated ThP1 cells and positive controls: 1µg/ml of LPS and 5 mg/ml of nigericin were used. Pro-caspase-1 (45-kDa) is present in the cytosol as an inactive zymogen.

  Caspase-1 activation and IL-1β release triggered by inflammasome activators LPS and ATP in human osteoblasts and PMA-treated ThP1 cells Several mechanisms are associated with the activation of inflammasome and secretion of IL-1β. Previous studies have extensively demonstrated the importance of caspase-1 cleavage for its activation (MARTINON; BURNS; TSCHOPP

Figure 13 :

 13 Figure 13: Influence of PMA on IL-1β production in culture supernatants of THP1 cells. IL-1β production in ThP1 supernatants was measured between 4 and 24 hours after treatment with 200 nM PMA and stimulated with LPS (1 μg/ml) for 30 min and 5 mM ATP for 15 min. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as mean ± standard deviation (±SD). Experiments are performed in triplicate and are representative of data obtained in three independent experiments. The asterisk represents a statistically significant difference: **** p <0.0001 by two-way ANOVA.

  IL-1β released by MG-63 and ThP1 is likely related to the different origin of both cells' types: osteoblast-like MG-63 cells and PMA-treated ThP1 differentiated into macrophages (SPANO; BARNI; SCIOLA, 2013).

Figure 14 :

 14 Figure 14: Caspase-1 activation and IL-1β release triggered in human osteoblasts.

Figure 15 :

 15 Figure 15: Detection of pro-caspase-1 in lysates of MG-63-CRISPR/cas9 cells by Western blot.

Figure 16 :

 16 Figure 16: Detection of pro-caspase-1 in lysates of CASP1 -/-MG-63 cells by Western blotting. Negative control: WT MG-63 cells. Absence of the 45-kDa subunit band of the caspase-1 gene for the monoclone N2E11, in bold. Anti-human caspase-1 antibody, mAb p / 20 1: 1000, Adipogene.

  125

Figure 17 :

 17 Figure 17: Detection of pro-caspase-1 in lysates of CASP1 -/-MG-63 cells by Western blotting. Negative control: WT MG-63 cells. Absence of the 45-kDa subunit band of the caspase-1 gene for the monoclone: N5G2 and N5G3, in bold. Anti-human caspase-1 antibody, mAb p / 20 1: 1000, Adipogene.

Figure 18 :

 18 Figure 18: Detection of pro-caspase-1 in lysates of CASP1 -/-MG-63 cells by Western blot. Negative control: WT MG-63 cells. Selection of monoclones: N2E11-G2 (monoclone G2) and N2E11-B9 (monoclone B9), in bold, for sequencing. Anti-caspase-1 human antibody, mAb p/20 1:1000, Adipogene.

Figure 20 :

 20 Figure 20: NLRP3 protein expression, speck-like aggregate formation and IL-1β secretion by WT MG-63 vs CASP1 -/-MG-63 cells exposed to S. aureus.

Figure 22 :

 22 Figure 22: Caspase-1 is required for secretion of IL-1β in S. aureus-infected MG-63 cells. WT MG-63 cells were infected or not with S. aureus SA113 at MOI 50:1 and compared with (CASP1 -/- MG-63 clone B9) and (CASP1 -/-MG-63 clone C3) infected or not with S. aureus SA113 at MOI 50: 1. IL-1β production was measured in the cell-free supernatant by ELISA. Data show monitoring for a period of 6 h to 9 days post-infection. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as mean ± standard deviation (±SD). Experiments are performed in triplicate and are representative of data obtained in three independent experiments. Asterisk represent statistically significant difference: *p < 0.05 by two-way ANOVA.

5. 4 .

 4 Pivotal role of S. aureus PSM toxins in stimulation of IL-1β release by infected osteoblasts Pathogens have developed strategies to avoid and subvert the defense functions of infected cells for extended periods (RATHINAM; FITZGERALD, 2016; RATHINAM; VANAJA;

Figure 25 :

 25 Figure 25: Involvement of caspase-1 in bacterial clearance. A. WT MG-63 or CASP1 -/-MG-63 cells were grown in 12-well plates overnight, then were exposed to S. aureus MW2 strain at MOI 1:50 for 2 h followed by antibiotic treatment as described. Two hours, 6 h and 24 h post-infection cells were lysed with 0.05% Triton X-100 in PBS, cell lysates were plated on BHI agar, and CFU was determined after overnight incubation. CFU values were normalized to 10 5 host cells. Experiments were performed in triplicate. The data are presented as means ± SD. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. **, P ˂ 0.01, for the comparison of the number of internalized bacteria in CASP1 -/-MG-63 cells with those in WT MG-63 cells.

  Staphylococcus aureus est une bactérie largement étudiée en raison de son impact important sur la santé humaine et animale ainsi que de son rôle dans l'industrie alimentaire.Le but de cette thèse était d'étudier les contributions de S. aureus intériorisé sur l'altération d'événements de défense de l'hôte tels que l'activation de l'inflammasome et la clairance bactérienne dans des cellules MG-63 ressemblant à des ostéoblastes infectés qui sont largement utilisées pour l'étude des infections des os et des articulations (BJI). Nous avons démontré que les cellules humaines MG-63 ressemblant à des ostéoblastes, traitées avec des activateurs de l'inflammasome, forment des inflammations entraînant l'activation de IL-1β-converting enzyme (ICE; caspase-1) et la sécrétion de la cytokine proinflammatoire Interleukine-1β (IL-1β). La production d'IL-1β par des cellules ostéoblastiques MG-63 a débuté plus tard et son niveau était beaucoup plus bas que la production d'IL-1β par des cellules ThP1 différenciées qui sont des phagocytes professionnels, des macrophages. Cette divergence est probablement liée à des origines différentes dans les deux types de cellules. En raison de la capacité du composé inflammasomes dans l'activation de la caspase-1 à traiter l'IL-1β et l'IL-18, nous avons utilisé l'édition de gènes CRISPR/Cas9 pour générer des cellules CASP1 -/-MG-63, un outil puissant étude de la fonction des inflammations à étudier. L'augmentation progressive des taux d'IL-1β dans les cellules MG-63 infectées de plusieurs heures à plusieurs jours, contrairement à l'absence de production d'IL-1β dans les cellules CASP1 -/-MG-63 infectées, a démontré des mécanismes dépendant de la caspase-1 et d'IL-1β production. En outre, l'effet était fortement dépendant des souches de S. aureus. Des résultats similaires ont été obtenus avec des isolats cliniques de S. aureus fraîchement isolés: cela souligne la signification biologique de nos résultats. L'absence de production d'IL-1β par les cellules exposées à des bactéries tuées par la chaleur indique que des facteurs associés aux bactéries viables sont impliqués dans l'activation de l'inflammasome NLRP3. L'emploi de mutants PSM de délétion et de complément a démontré le rôle pivot des toxines de S. aureus PSM (en particulier PSM-β et PSM-γ) dans les inflammations liées à la production d'IL-1β par les cellules infectées. Cependant, la présence d'autres facteurs ne peut pas être exclue car les mutants complémentés ne restituent pas entièrement le niveau d'IL-1β sécrétée.

Annexe 1 .

 1 Review: Comparing bacteriocins with conventional antibiotics as treatment options for Staphylococcus aureus infections (Submitted to the peerreviewed journal PeerJ on 20th May 2019).

  . S. aureus est notamment une cause fréquente d'infections des os et des articulations (BJI, pour l'anglais Bone and Joint Infection), maladie associée à une morbidité élevée et à d'importants coûts de santé dans le monde (DEL POZO; PATEL, 2009; TUCHSCHERR;

GERACI; LÖFFLER, 2017; WRIGHT; NAIR, 2010;[START_REF] Zimmerli | Prosthetic-joint infections[END_REF][START_REF] Zimmerli | Prosthetic-joint infections[END_REF][START_REF] Zimmerli | Prosthetic-joint infections[END_REF]

.

Les infections osseuses induites par S. aureus peuvent évoluer en infections chroniques, devenant très réfractaires aux traitements antibiotiques

(LOWY, 1998)

. Cela est probablement dû aux différentes stratégies que cette bactérie à développer pour s'adapter et survivre à l'intérieur des cellules hôtes (EIFF; PETERS; PROCTOR, 2001; MONTGOMERY; DAVID; DAUM, 2015; TUCHSCHERR; GERACI; LÖFFLER, 2017).

  Cependant, en dépit de nombreux travaux sur le mécanisme utilisé par S. aureus à internaliser dans et survivre dans les phagocytes non professionnels, comme les cellules épithéliales(KREMSEROVA; NAUSEEF, 2019;[START_REF] Ma | LncRNA XIST mediates bovine mammary epithelial cell inflammatory response via NF-κB/NLRP3 inflammasome pathway[END_REF] l'implication des inflammasomes et de l'IL-1β traitée, qui joue un rôle crucial dans l'homéostasie osseuse dans le BJI associée à S. aureus, n'a pas encore été démontrée. Ceci est particulièrement intéressant dans le cas de BJI que S. aureus est représenté à internaliser et à survivre dans les ostéoblastes, générant des infections chroniques réfractaires aux traitements antimicrobiens. Il est donc essentiel de développer de nouvelles approches pour mieux comprendre les mécanismes par lesquels les staphylocoques interagissent avec ostéoblastes et, in fine, génère des dommages aux os.

	Bibliographic synthesis
	1. General characteristics of Staphylococcus aureus
	majeure connue sous le nom d'inflammasomes (MARTINON; BURNS; TSCHOPP, 2002;
	SCHRODER; TSCHOPP, 2010a). 1.1. Definition and morphology
	Les inflammasomes sont des complexes de signalisation multi-protéines qui s'assemblent Staphylococcus aureus (S. aureus) was described by Alexander Ogston and Louis Pasteur as
	après la reconnaissance des signaux de danger et/ou des agents pathogènes par une famille cocci with a mean diameter of 0.5 and 1.5 μm, isolated from furuncle and abscesses in 1880
	de récepteurs cytosoliques appelés NLR (nucleotide-binding domain leucine-rich repeat (OGSTON, 1881). The genus Staphylococcus belongs to the family Micrococcaceae, which is
	containing receptors) ou de membres de la famille de protéines PYHIN (MARTINON; BURNS; composed of Gram-positive bacteria (ROSYPAL; ROSYPALOVÁ; HOREJS, 1966). The name
	TSCHOPP, 2002). Une fois assemblés, les inflammasomes initient l'activation de protéases "Staphylococc" derives from the Greek words Staphyle (Grape-like clusters) and aureus (gold)
	Chapter 1: Bibliographic Synthesis
	L'inflammasome NLRP3 est le NLR le mieux caractérisé. NLRP3, ainsi que la protéine ASC et
	la Caspase-1, forment un complexe oligomérique cytoplasmique appelé inflammasome
	NLRP3, qui joue un rôle essentiel dans l'initiation de la réponse immunitaire innée. Cependant,
	les mécanismes moléculaires de son activation ne sont pas entièrement compris (DAVIS;
	WEN; TING, 2011; DEIGENDESCH; ZYCHLINSKY; MEISSNER, 2018; MELEHANI et al.,
	2015). L'assemblage de l'inflammasome NLRP3 a été associé à plusieurs pathologies telles

Ainsi, la réponse immunitaire innée joue un rôle central dans la défense contre les agents pathogènes et est initiée par les pattern recognition receptors (PRR). Les PRR reconnaissent les pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) et les endogenous danger-associated molecular pattern (DAMPs), ce qui conduit à l'activation des voies de défense de l'hôte qui entraînent l'élimination de l'infection

[START_REF] Carrillo | Physiology and Pathology of Innate Immune Response Against Pathogens[END_REF] JANG et al., 2015;[START_REF] Kumar | Pathogen Recognition by the Innate Immune System[END_REF] KAWAI;[START_REF] Kumar | Pathogen Recognition by the Innate Immune System[END_REF][START_REF] Mogensen | Pathogen Recognition and Inflammatory Signaling in Innate Immune Defenses[END_REF][START_REF] Roh | Damage-Associated Molecular Patterns in Inflammatory Diseases[END_REF][START_REF] Roh | Damage-Associated Molecular Patterns in Inflammatory Diseases[END_REF]

. Les récepteurs Toll-like (TLR), un groupe de récepteurs extracellulaires et endosomaux liés à la membrane, détectent la présence d'une infection par la reconnaissance des PAMP. La réponse immunitaire innée contre les microbes implique également une voie inflammatoire en aval, notamment la caspase-1 et la caspase-11, qui ensuite maturent protéolytiquement la pro-IL-1β et la pro-IL-18 et favorisent leur sécrétion cellulaire (BROZ;

[START_REF] Broz | Newly described pattern recognition receptors team up against intracellular pathogens[END_REF][START_REF] Latz | The inflammasomes: mechanisms of activation and function[END_REF] LATZ; XIAO; STUTZ, 2013a;[START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Wilson | Structure and mechanism of interleukin-1 beta converting enzyme[END_REF]

.

De plus, l'activation de l'inflammmasome déclenche la pyroptose, une forme inflammatoire de mort cellulaire

[START_REF] Bergsbaken | Pyroptosis: host cell death and inflammation[END_REF] FINK;[START_REF] Bergsbaken | Pyroptosis: host cell death and inflammation[END_REF][START_REF] Yang | Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors[END_REF][START_REF] Yuan | Inflammatory caspase-related pyroptosis: mechanism, regulation and therapeutic potential for inflammatory bowel disease[END_REF]

. Le rôle de NLRP1, NLRP3, NLRC4, NLRP6, NLRP7, NLRP12 et des protéines AIM2 et IFI16 de PYHIN dans l'assemblage de l'inflammasome et le contrôle de la réponse immunitaire innée contre divers microbes, y compris différentes bactéries, est maintenant bien documenté. De même, l'implication de divers facteurs bactériens dans le déclenchement de l'inflammasome a été intensément explorés au cours des dernières années

[START_REF] Caneparo | The Absent in Melanoma 2-Like Receptor IFN-Inducible Protein 16 as an Inflammasome Regulator in Systemic Lupus Erythematosus: The Dark Side of Sensing Microbes[END_REF] LIN; ZHANG, 2017;[START_REF] Sá | Inflammasomes and dermatology[END_REF][START_REF] Sá | Inflammasomes and dermatology[END_REF] VLADIMER et al., 2013a)

. Ces inflammasomes se forment en réponse à divers stimuli et sont également soumis à des mécanismes de régulation négative qui modulent la production de cytokines inflammatoires après le déclenchement de la réponse inflammatoire (NAIK; DIXIT, 2010). que des syndromes auto-inflammatoires, l'inflammation chronique et certaines maladies métaboliques (DEIGENDESCH; ZYCHLINSKY; MEISSNER, 2018).

Le rôle des inflammasomes formés dans les phagocytes professionnels infectés par S. aureus qui ingèrent et/ou tuent les bactéries invasives a été étudié de manière approfondie.

(BROWN; GRILLI, 1998). S. aureus is immobile, non-spore-forming, produces coagulase and is often unencapsulated or has a limited capsule (OGSTON, 1881).

1.2. General cultural, biochemical characteristics and genome of S. aureus S. aureus is an aerobic and facultative anaerobic bacterium. It readily grows on basal media and forms golden-yellow colonies on nutrient agar. It is positive for catalase, negative for oxidase and capable of fermenting mannitol

(LOWY, 1998)

. S. aureus can grow in a wide range of temperatures. Its optimal growth temperature is 30º-37ºC, but it can grow between 7º and 48.5ºC. Its optimum pH is 7.2 (but it can grow in a range of

4.2 -9.3)

. It also grows in a wide range of sodium chloride concentration (7.5-10% NaCl) (LE LOIR; BARON; GAUTIER, 2003; SCHMITT; SCHULER-SCHMID; SCHMIDT-LORENZ

Table

Table 1 :

 1 Virulence factors of S. aureus and their characteristics. Adapted by (GNANAMANI;

	Factors	Characteristics	References
	Helping attachment to host tissues
	Microbial Surface Components Recognizing	Cell surface proteins which interact with host molecules
	adhesive matrix molecules (MSCRAMM)	such as collagen, fibronectin and fibrinogen, thus, facilitate
		the tissue attachment. Staphylococcal protein A, fibronectin-binding proteins A and B, collagen-binding	Vazquez et al., 2011
		protein and clumping factor A and B belong to this family.
		They are also involved in host immune evasion
	Breaking/evading the host immunity
	Polysaccharide microcapsule	Resist the phagocytosis and killing by polymorphonuclear phagocyte	Nilsson et al., 1997
	Protein A (SpA)	Binding of the Fcγ domain of immunoglobulin (Igs)
			Hong et al., 2016
	Panton-Valentine leukocidin (PVL)	PVL is found in most of community-associated MRSA	Genestier et al., 2005
		(CA-MRSA). PVL consists of two subunits, LukS and LukF.	Voyich et al., 2006
		LukS binds to human complement receptors C5aR and
		C5L2 and allows docking of LukF. The Luk subunits
		oligomerizes and form a pore lysing the cell.
	α-Hemolysin	Disruption of epithelial or endothelial surfaces
			Bhakdi, and Tranum-Jensen, 1991
	Chemotaxis-inhibitory protein of S. aureus (CHIPS): Impediment of neutrophil chemotaxis
			Postma et al., 2004
	Tissue invasion
	Extracellular adherence protein (Eap)	Blockade of neutrophil adherence to endothelia
			Edwards et al., 2012
	Proteases, lipases, nucleases, hyaluronatelyase,	These extracellular enzymes cause tissue destruction
	phospholipase C, metalloproteases (elastase),	and,
	and
	Staphylokinase
	Induces toxinosis
	Staphylococcal Enterotoxins (SE)	There are more than 25 known SEs. SEs bind MHC class
		II receptors on T-cells and hyperstimulate the T-cells. S.
		aureus produces battery of enterotoxins which are
		potent gastrointestinal exotoxins. The Staphylococcal food poisoning is an intoxication which results from	Argudin et al., 2010
		consumption of foods containing sufficient amount of
		preformed enterotoxins
	Toxic shock syndrome toxin -1 (TSST-1)	TSST-1 and some of enterotoxins are called as pyrogenic
		toxin super antigens. TSST-1 causes toxic shock syndrome especially in menstrual women	Dinges et al., 2000
	Exfoliative toxins A and B	Serine proteases which selectively recognize and
		hydrolyze desmosomal proteins in the skin. ETs cause staphylococca-scalded skin syndrome, a disease	Bukowski et al., 2010
		predominantly affecting infants

HARIHARAN; PAUL-SATYASEELA, 2017).

  [START_REF] Bauernfeind | Inflammasomes: current understanding and open questions[END_REF][START_REF] Bauernfeind | Of inflammasomes and pathogens -sensing of microbes by the inflammasome: Of inflammasomes and pathogens[END_REF][START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Martinon | Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases[END_REF].

	The NLRP3 inflammasome can be activated by PAMPs and damage-associated molecular

patterns (DAMPs) such as Sendai virus, influenza A virus, adenovirus, Staphylococcus aureus, L. monocytogenes, E. coli, Mycobacterium marinum, S. flexneri, Neisseria gonorrhoe, Candida albicans, bacterial RNA, ATP, pore forming toxins, and structural aggregates such as monosodium urate and calcium pyrophosphate (SeeTable 2)

[START_REF] Allen | The NLRP3 inflammasome mediates in vivo innate immunity to influenza A virus through recognition of viral RNA[END_REF]

Table 2 :

 2 List of activators known to activate of the NLRP3 inflammasome.

  Les avancées sur plusieurs fronts de recherche ont permis de considérablement améliorer notre connaissance des déclencheurs et des modulateurs de l'inflammation. La découverte récente des inflammasomes a été d'une grande importance dans les domaines de l'immunologie et de la médecine. Cette importance est due à l'identification et à la caractérisation des mécanismes moléculaires qui lient la stimulation cellulaire (par des agents pathogènes, des lésions physiques ou chimiques) à l'activation des réponses cellulaires immunitaires précoces chez l'hôte.Concernant le processus d'inflammation par des bactéries pathogènes, Staphylococcus aureus est un agent pathogène humain majeur qui colonise les narines et la peau et envahit fréquemment les tissus mous et le sang. S. aureus dispose de nombreux mécanismes pour contourner et neutraliser le système immunitaire, provoquant diverses infections invasives et En outre, plusieurs maladies inflammatoires associées à une résorption osseuse accrue ont été bien rapportées[START_REF] De | What Are the Peripheral Blood Determinants for Increased Osteoclast Formation in the Various Inflammatory Diseases Associated With Bone Loss[END_REF][START_REF] Hardy | Bone loss in inflammatory disorders[END_REF][START_REF] Hardy | Bone loss in inflammatory disorders[END_REF][START_REF] Iseme | Is osteoporosis an autoimmune mediated disorder?[END_REF][START_REF] Lacativa | Osteoporosis and inflammation[END_REF][START_REF] Lacativa | Osteoporosis and inflammation[END_REF]. L'IL-1β, ainsi que d'autres cytokines inflammatoires (par exemple, IL-6 et TNFα), jouent un rôle important dans le remodelage osseux. L'infection à S. aureus peut déclencher la production locale et systémique de ces trois cytokines par le biais de PRR hôtes (

	Plusieurs types de cellules, notamment les monocytes, les macrophages, les neutrophiles et
	les cellules dendritiques (CD) expriment les différents PRR reconnaissant les PAMP et les
	DAMP, ce qui conduit à l'activation rapide des cytokines pro-inflammatoires IL-1β et IL-18
	(CARRILLO et al., 2017; FULLARD; O'REILLY, 2015; SCHRODER; TSCHOPP, 2010).

superficielles

[START_REF] Tong | Staphylococcus aureus Infections: epidemiology, pathophysiology, clinical manifestations, and management[END_REF]

. Cela se voit, par exemple, dans des maladies telles que les infections des os et des articulations, en particulier en présence d'appareils orthopédiques

[START_REF] Saavedra-Lozano | Bone and Joint Infections[END_REF]

. Le traitement de cette infection est particulièrement difficile et prend du temps, avec des coûts énormes en soins de santé

(DEL POZO; PATEL, 2009;[START_REF] Zimmerli | Prosthetic-joint infections[END_REF][START_REF] Zimmerli | Prosthetic-joint infections[END_REF][START_REF] Zimmerli | Prosthetic-joint infections[END_REF]

. En dépit d'un traitement chirurgical et d'un traitement antimicrobien adéquat, ces infections donnent lieu à un pourcentage élevé de rechutes à court terme, conduisant souvent au développement d'une maladie chronique

[START_REF] Ferry | DAIR' (debridement, antibiotics and implant retention) with local injection of a selected cocktail of bacteriophages: is it an option for an elderly patient with relapsing S. aureus prosthetic-joint infection[END_REF][START_REF] Peeters | Teicoplanin-based antimicrobial therapy in Staphylococcus aureus bone and joint infection: tolerance, efficacy and experience with subcutaneous administration[END_REF]

. De nombreuses études sont axées sur les stratégies antibactériennes. On en sait moins sur le rôle du système immunitaire dans la lutte contre ce type d'infection.

Le rôle des inflammasomes formés dans les phagocytes professionnels tels que les macrophages et les phagocytes non professionnels, tels que les cellules épithéliales infectées par S. aureus et d'autres micro-organismes, a fait l'objet de nombreuses études

[START_REF] Flannagan | Antimicrobial Mechanisms of Macrophages and the Immune Evasion Strategies of Staphylococcus aureus[END_REF][START_REF] Flannagan | Antimicrobial Mechanisms of Macrophages and the Immune Evasion Strategies of Staphylococcus aureus[END_REF][START_REF] Flannagan | Antimicrobial Mechanisms of Macrophages and the Immune Evasion Strategies of Staphylococcus aureus[END_REF] KREMSEROVA; NAUSEEF, 2019;[START_REF] Ma | LncRNA XIST mediates bovine mammary epithelial cell inflammatory response via NF-κB/NLRP3 inflammasome pathway[END_REF]

.

De nombreuses études ont clarifié les mécanismes responsables de l'activation de la procaspase-1 et de la maturation de l'IL-1β. Une fois activée, l'IL-1β joue un rôle crucial dans les maladies inflammatoires chroniques, les troubles métaboliques et le cancer.

Table 1 :

 1 Strain-specific EVs cytotoxicity

	continue…

Chapter 4. Original article -Involvement of caspase-1 in inflammasomes activation and bacterial clearance in S. aureus-infected osteoblasts.

Table 2 .

 2 Biological functions affected by the action of bacteriocins and antibiotics.

	Targeted biological functions	Bacteriocins	Antibiotics
	Cell Wall Biosynthesis	Nisin A, nukasin ISK-1, NAI-107	β-lactams, glycopeptides
	DNA Replication and Transcription	Microcin B17, colicins, carocin S2	Quinolenes
		Geobacillin I, bac-GM17,
		plantaricins, dysgalacticin,
	Membrane Disruption	lactococcin, pediocin-like bacteriocins, mesentericin	Lipopetides
		Y105, lacticin Q, nisin A,
		Uberolysin, AS-48 Bacteriocin
	Protein Synthesis	Colicins, cloacin DF13	Aminoglycosides, tetracyclines, chloramphenicol, macrolides
			Benzamide derivade, N-
	Septum Formation	Garvicin A, lactococcin 972	heterocycles, phenols/polyphenols,
			carboxylic acids
	Vitamins Biosynthesis	-	Sulfonamides

Table 3 .

 3 Role of bacteriocins on preventing and control of the S. aureus growth.

	Bacteriocins	Class	Organism source	Reference
	Aureocins A70, A53 and 215FN (aureus) Pep5, Epicidin 280 (epidermidis) Epidermin K7 and	II and I	Staphylococcus aureus epidermidis and Staphylococcus	(Coelho et al., 2007)
	Morricin 269, Kurstacin
	287, Kenyacin 404, Entomocin 420 and	II	Bacillus thuringiensis	(Corona et al., 2009)
	Tolworthcin 524
			Staphylococcus	(Coelho et al., 2017;
	Lysostaphin	III	simulans biovar	SCHINDLER and
			staphylolyticus	SCHUHARDT, 1964)
	Epidermicin NI01		Staphylococcus epidermidis	(Sandiford and Upton, 2012)
	Pediocina PA-1	II	Lactococcus lactis	(Rodrıguez et al., 2005)
	Nisin A, lacticin Q, and Nukacin ISK-1	I	Lactococcus lactis QU 5 warneri ISK-1 and Staphylococcus	(Okuda et al., 2013)
	Lacticin 3147	I	Lactococcus lactis subsp. lactis	(Twomey,Wheelock, Flynn, Meaney, 2000)
	Enterocin CCM 4231	II	Enterococcus faecium CCM 4231	(Laukova and Czikkova, 1999)
			Enterococcus faecium
	E 50-52 and OR-70	II	NRRL B-30746 and Lactobacillus salivarius	(Svetoch et al., 2008)
			NRRL B-30514
	Duracin 61A and Reuterin		Enterococcus durans 61A and Lactobacillus	(Hanchi et al., 2017)
			reuteri
	Pentocin JL-1	I	Lactobacillus pentosus	(Jiang et al., 2017)
	TA6	II	Pseudomonas aeruginosa TA6	(Arumugam et al., 2018)

lorsque le taux de production de ROS augmente considérablement et/ou que les défenses anti-oxydantes sont insuffisantes ou défaillantes, un stress oxydatif se produit. Ce stress oxydatif est un signal secondaire pour l'activation de l'inflammasome(DOSTERT et al., 2008b;[START_REF] Zhou | Thioredoxin-interacting protein links oxidative stress to inflammasome activation[END_REF][START_REF] Zhou | A role for mitochondria in NLRP3 inflammasome activation[END_REF]. Dans le contexte du modèle cellulaire mis en oeuvre, il serait intéressant de vérifier le rôle des ROS dans la production d'IL-1β via l'activation de l'inflammasome NLRP3 afin d'élucider les mécanismes sous-jacents impliqués dans l'activation de l'inflammasome NLRP3 induit pendant S. aureus-infection dans les cellules MG-63. L'analyse de la génération de ROS extracellulaires dans les cellules MG-63 sera mesurée en utilisant l'inhibition de la réduction du cytochrome C par la superoxyde dismutase (SOD), comme décrit par[START_REF] Johnston | Elaboration of toxic oxygen by-products by neutrophils in a model of immune complex disease[END_REF][START_REF] Johnston | Elaboration of toxic oxygen by-products by neutrophils in a model of immune complex disease[END_REF]. Pour la détection des ROS intracellulaires, les cellules MG-63 seront infectées ou non avec les souches de S. aureus. Après une période d'incubation de 24 h, on utilise la sonde 2',7'dichlorodihydrofluorescein diacetate (H2DCFDA) perméable à la membrane cellulaire et non fluorescente. En présence de ROS, ce composé s'oxyde à l'intérieur de la cellule et produit un composé fluorescent, le diacétate de 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), qui reste à l'intérieur de la cellule. La lecture se fera par cytométrie en flux (cytomètre en flux Accuri C6). Ainsi, il pourra éventuellement identifier la formation de ROS dans l'analyse proposée dans les cellules MG-63 après ces stimuli. En outre, il a récemment été découvert qu'outre la caspase-1, d'autres caspases, telles que la caspase-11 et la caspase-8, pourraient également participer au traitement et à la maturation de l'IL-1β par l'inflammation (RATHINAM; FITZGERALD, 2016; VANAJA; RATHINAM; FITZGERALD, 2015). Ainsi, il serait intéressant d'élucider le rôle des voies d'inflammasome non canoniques dans la modulation de la réponse immunitaire lors d'une infection à S. aureus dans une cellule MG-63. La caspase-11, ainsi que la caspase-1, sont présentes dans le cytoplasme des cellules en tant que zymogène inactif de 43 kDa, qui doit être clivé (gros 23 kDa et petits 10 kDa) (RATHINAM; VANAJA; FITZGERALD, 2012). Par la technique du Western Blot, nous avons pu mesurer les formes de précurseur et de clivage exprimées de la caspase-11 après stimulation avec S. aureus dans des cellules MG-63. Ces résultats pourraient élucider si, en plus de la voie canonique de la caspase-1, la voie non canonique de la caspase-11 fonctionne également pendant l'infection à S. aureus.Aussi, nous pourrions inclure l'évaluation de la caspase-8 pour élucider davantage l'existence d'une voie supplémentaire non canonique, car la caspase-8 peut directement cliver la pro-IL-1β et initier la caspase-1 en réponse à différents agents pathogènes[START_REF] Maelfait | Stimulation of Toll-like receptor 3 and 4 induces interleukin-1beta maturation by caspase-8[END_REF]

(Promega, Madison, WI, USA). En résumé, cet essai l'intensité du signal généré par la réaction de luciférase est proportionnelle au clivage du substrat de la caspase-8. Ainsi, nous étudierons l'implication de voies non canoniques de la caspase-11 et de la caspase-8 dans la libération de IL-1β dans des cellules MG-63 après ces stimuli.Les interactions directes de S. aureus avec les ostéoblastes sont cruciales dans la pathogenèse de l'ostéomyélite. La capacité de S. aureus à envahir et à accéder au cytoplasme de phagocytes non professionnels, tels que les ostéoblastes, attire de plus en plus l'attention et est désormais considérée comme un facteur clé des infections réfractaires. En outre, l'étude des mécanismes sous-jacents de l'activation des voies canoniques et non canoniques de l'inflammasome NLRP3 dans la modulation de la réponse immunitaire pendant l'infection à S. aureus sera une nouvelle connaissance intéressante et ouvrira de nouvelles perspectives pour traitements plus sélectifs pour la persistance intracellulaire de S. aureus dans l'infection des articulations osseuses.

Acknowledgments

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brazil (CAPES) -Finance Code 001 and by grants from INRA -Institut National de la Recherche Agronomique, France.

Acknowledgment

The authors thank Pr. Petr Broz, Department of Biochemistry, University of Lausanne, Epalinges, Switzerland for invaluable suggestions, discussion and corrections of the manuscript. We thank Mary Bret for English corrections. The program Geraine de Stael, bilateral France-Switzerland collaboration (to N.B. and M.D.), French National Institute for Agricultural Research (INRA), LONGhealth -MPP10573 (to N.B. and M.D), the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), U.S. National Institutes of Health (NIH), project number 1 ZIA AI000904 (to M.O.), the Deutsche Forschungsgemeinschaft (DFG) SFB766 (to F.G. and M.T). E. L. L. is a recipient of a Ph.D. fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Brazil). The ZEISS LSM880 confocal microscope was funded by the European Union (FEDER), the French Ministry of Education, Research and Innovation, the Regional Council of Brittany and INRA.

Acknowledgements

The authors would like to acknowledge the INRA -Institut national de la recherche agronomique for the support of this work and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) -Brazil. The authors would like to thank Mary Bret for the English revision.

Funding

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Funding

This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and, Finance Code 001 and by grants from INRA -Institut national de la recherche agronomique, France.

Aim of the PhD project

This research aims to investigate whether S. aureus infection activates inflammasomes in nonprofessional phagocytes, such as human osteoblast-like cells, and which S. aureus virulence factors are involved in inflammasomes activation during infection.

Objectif du projet de thèse

Le but de cette recherche est de déterminer si l'infection à S. aureus active les inflammasomes dans les phagocytes non professionnels tels que les cellules ressemblant à des ostéoblastes humains et quels facteurs de virulence de S. aureus sont impliqués dans l'activation de l'inflammasomes lors de l'infection.

Author Contributions

NB, YLL supervised the work and corrected the manuscript. ELL, BSRL, EG, FC, VA did the main part of the bibliographical survey. All the authors took part in the writing of the manuscript.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Conflict of Interest

The authors declare no competing financial and/or non-financial interests, all authors concur with the submission. A. WT MG-63 or CASP1 -/-MG-63 cells were grown in 6-well plates, then were exposed to S.

aureus strain SA113 at MOI 1:50 for 2 h followed by antibiotic treatment as described in Materials and Methods. After various times post-infection (6 h, 2 days, 5 days, 7, days and 11 days) cell supernatants were collected, centrifuged and the level of IL-1β was determined by a sandwich-ELISA (Thermofischer Life Technologies) as described in Material and Methods.

The values are presented as concentrations in pg/ml. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). P-values < 0.05 ( ⃰ ) were considered to be significant. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD).

B. WT MG-63 cells were exposed to S. aureus strains LAC (USA300, red color), MW2 (USA 400, green color), or SA113 (yellow color) at MOI 1:50 for 2 h followed by antibiotic treatment as described in Material and Methods. After various times post-infection (6 h, 2 days, 5 days, 7, days and 11 days) cell supernatants were collected, centrifuged and the level of IL-1β was determined by a commercial sandwich-ELISA (Invitrogen, France) as described in Material and Methods. The values are presented as concentrations in pg/ml. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). P-values < 0.05 (*) were considered to be significant. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD). Caspases consist of a family of proteases classically divided into two groups according to their function: inflammatory or apoptotic (CHANG;[START_REF] Chang | Proteases for Cell Suicide: Functions and Regulation of Caspases[END_REF]CHEN;KANG;[START_REF] Chen | The independence of and associations among apoptosis, autophagy, and necrosis[END_REF][START_REF] Denes | Caspase-1: is IL-1 just the tip of the ICEberg?[END_REF]LOPEZ-CASTEJON;[START_REF] Denes | Caspase-1: is IL-1 just the tip of the ICEberg?[END_REF]. Caspases are produced in their inactive form and activated through inflammasome (DENES; LOPEZ-CASTEJON;[START_REF] Denes | Caspase-1: is IL-1 just the tip of the ICEberg?[END_REF][START_REF] Latz | The inflammasomes: mechanisms of activation and function[END_REF]. Activation of caspase-1 has been associated with the response of cells of the phagocytic system such as (monocytes, dendritic cells, and macrophages) against diff erent bacterial pathogens (BROZ;[START_REF] Broz | Molecular mechanisms of inflammasome activation during microbial infections: Mechanisms of inflammasome activation[END_REF][START_REF] Kubica | A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages[END_REF][START_REF] Lamkanfi | Emerging inflammasome effector mechanisms[END_REF]. This process occurs through the release of several antigens (PAMPs) into the intracellular medium that binds to cytosolic standard recognition receptors culminating in the activation of caspase-1 that leads to cleavage and activation of pro-inflammatory cytokines IL-1β and IL-18 (SCHRODER;TSCHOPP, 2010).

We initially evaluated whether S. aureus was able to activate signaling pathways that culminated in the endogenous activation of caspase-1 in human osteoblastic (MG-63) cells (non-professional phagocytes). Classically, the detection of mature subunits of caspase-1 (p20 and/or p10) extracellularly serve as markers for the activation of inflammasome in vitro [START_REF] Shamaa | Monocyte Caspase-1 Is Released in a Stable, Active High Molecular Weight Complex Distinct from the Unstable Cell Lysate-Activated Caspase-1[END_REF]. Thus, based on observations provide by Shamaa and collaborators (2015) and in others studies [START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF][START_REF] Burns; Tschopp | Upon inflammasome activation, ASC is rapidly depleted from its steady-state homogeneous cellular distribution and self associates to form a single punctum inside the cell of about 1µm in diameter, called a "speck" detectable by immunofluorescence as a single perinuclear focus[END_REF]SCHRODER;TSCHOPP, 2010), reporting that mature caspase-1 is released into the supernatant after stimulation through signals such as PAMPs or DAMPs, we used the solution of trichloroacetic acid (TCA) 100 % to precipitate proteins from the supernatant since it is well reported that TCA is a very effective protein precipitating agent [START_REF] Novák | Protein Extraction and Precipitation[END_REF][START_REF] Novák | Protein Extraction and Precipitation[END_REF] we investigated the activation of caspase-1.

To clarify the possible role of human osteoblasts in the production of IL-1β, we analyzed the kinetics (from 2 to 11 days) of IL-1 β production by WT and CASP1 -/-MG-63 cells exposed to S. aureus SA113. As demonstrated in Fig. 21-A, the level of IL-1β increased from day 2 to 11.

In contrast, IL-1β was not detected in supernatants from infected CASP1 -/-MG-63 cells during the tested period.

To verify whether IL-1β release by infected osteoblasts was strain-dependent, we analyzed the kinetics of IL-1 β release by MG-63 exposed to different S. aureus strains: LAC (USA300), MW2 (USA 400) and SA113. As shown in Fig. 21-B, all strains induce IL-1β release; however, MW2 and LAC induced significantly higher levels of IL-1β than SA113. IL-1β was not detected in the supernatants of MG-63 cells exposed to killed bacteria of any of the three strains, suggesting that factors associated with viable bacteria are involved in inflammasome activation. A. WT MG-63 or CASP1 -/-MG-63 cells were grown in 6-well plates, then were exposed to S. aureus strain SA113 at MOI 1:50 for 2 h followed by antibiotic treatment as described in Materials and Methods.

After various times post-infection (6 h, 2 days, 5 days, 7, days and 11 days) cell supernatants were collected, centrifuged and the level of IL-1β was determined by a sandwich-ELISA (Thermofischer Life Technologies) as described in Material and Methods. The values are presented as concentrations in pg/ml. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). P-values < 0.05 ( ⃰ ) were considered to be significant. Tukey's mutants demonstrated that the release was partially restored when the strains were complemented with PSMα (LACΔpsmαβhld-pTXΔα1-4), PSMβ (LACΔpsmαβhld pTXΔβ1-2) or δ-toxin (LACΔpsmαβhld-pTXΔhld). However, the difference was statistically significant only when strains were complemented with PSMβ or, at earlier time points, δ-toxin (LACΔpsmαβhld-pTXΔhld) (Fig. 23).

Employment of deletion and complemented PSMs mutants demonstrated a pivotal role of S. aureus toxins PSMs in inflammasomes related IL-1β production by infected osteoblastic cells.

We would like to highlight that IL-1β production by infected osteoblasts appears to be specifically dependent on PSM betas among PSMs. This is noteworthy, as we don't know any specific phenotypes attributable to those PSMs so far. In addition to PSMs the action of other factors cannot be excluded since complemented mutants did not restore entirely the level of secreted IL-1β. A. WT MG-63 cells were exposed to wild type LAC (USA300) and its isogenic mutant LAC ∆psmαβhld at MOI 1:50 for 2 h followed by antibiotic treatment as described in Material and Methods. After various time post-infection (2 days and 5 days) cell supernatants were collected, centrifuged and the level of IL-1β was determined by a sandwich-ELISA (Thermofischer Life Technologies) as described in Material and Methods. The IL-1β values are presented as concentrations in pg/ml. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). P-values < 0.05 ( ⃰ ) were considered to be significant. Tukey's Honestly Significant Difference test was Chapter 6. General conclusion and perspectives of the work performed during PhD project S. aureus-infection in MG-63 cells. Analysis of extracellular ROS generation in MG-63 cells will be measured using the inhibition of cytochrome C reduction by superoxide dismutase (SOD), as described by [START_REF] Johnston | Elaboration of toxic oxygen by-products by neutrophils in a model of immune complex disease[END_REF][START_REF] Johnston | Elaboration of toxic oxygen by-products by neutrophils in a model of immune complex disease[END_REF]. For the detection of intracellular ROS, MG-63 cells will be infected or not with S. aureus strains. After a 24 h incubation period, the 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is used which is cell membrane permeable and non-fluorescent. In the presence of ROS, this compound is oxidized within the cell and produces a fluorescent compound, the 2', 7'dichlorodihydrofluorescein diacetate (H2DCFDA), which remains inside the cell. The reading will be performed by flow cytometry (Accuri C6 flow cytometer). Thus, it possibly will identify the formation of ROS in the analysis proposed in MG-63 cells after these stimuli.

Besides, it has recently been discovered that in addition to caspase-1, other caspases, such as caspase-11 and caspase-8, may also be involved in the processing and maturation of IL- These results could elucidate whether, in addition to the canonical pathway of caspase-1, the non-canonical pathway of caspase-11 is also operating during S. aureus infection.

Besides, we could include the evaluation of caspase-8 to further elucidate the existence of an additional non-canonical pathway, since caspase-8 can directly cleave pro-IL-1β as well as initiate caspase-1 in response to different pathogens [START_REF] Maelfait | Stimulation of Toll-like receptor 3 and 4 induces interleukin-1beta maturation by caspase-8[END_REF][START_REF] Shenderov | Cutting edge: Endoplasmic reticulum stress licenses macrophages to produce mature IL-1β in response to TLR4 stimulation through a caspase-8-and TRIF-dependent pathway[END_REF]. For this, the activity of caspase-8 in the osteoblastic cell lysates will be measured according to the recommendations of the Caspase-Glo 8 Assay kit (Promega, Madison, WI, USA). Summarizing, this assay the signal intensity generated by the luciferase reaction is proportional to the cleavage of the caspase-8 substrate. Thus, we will possibly identify the presence of caspase-8 in MG-63 cells in these analyzes.

The direct interactions of S. aureus with osteoblasts are crucial in the pathogenesis of osteomyelitis. The ability of S. aureus to invade and gain access to the cytoplasm of nonprofessional phagocytes, such as osteoblasts, is gaining increasing attention and is now we considered the first cases of resistance and evaluated the progress until the present day.

Additionally, in order to answer possible ways to deal with this protective advance in bacteria, the activity of bacteriocins was explored in the research studies considered here, as a good substitute capable of replacing in some cases the antibiotics currently used. The criteria for inclusion of possible bactericidal agents were evaluated considering experimental cases published in scientific journals. Our study highlighted some of the cases analyzed experimentally and we also show methods to explore the production of these potential molecules at an industrial scale using protocols from other review works.

Results: Our work reviews the current informative content and brings the applicability of the use of bacteriocins overlapping with the use of conventional antibiotics in the context of S.

aureus infections. Here we ascertained the main antibiotic-resistance mechanisms and analyzed the current bacteriocins that stand out as strong competitors in the struggle against S. aureus infections. At the same time, our work highlights perspectives for the production of these biomolecules with implications at an industrial scale, using aqueous two-phase system (ATPS) and Aqueous micellar two-phase system (AMTPS).

Conclusions: The use of Fermented Plant Extract (FPE) is explored showing itself to be an innovative tool, serving as a carrier for numerous health benefits, which includes the control of microbial activity.

Introduction

Mammal's skin is the main environment for the colonization of members of genus

Staphylococcus which can facilitate infection in immunologically suppressed individuals, found in cases of S. epidermis as reported on a leukemic patient, but not exclusively [START_REF] Otto | Staphylococcus colonization of the skin and antimicrobial peptides[END_REF]Senthilkumar et al., 2001;[START_REF] Weisser | Hypervariability of Biofilm Formation and Oxacillin Resistance in a Staphylococcus epidermidis Strain Causing Persistent Severe Infection in an Immunocompromised Patient[END_REF]. Specifically, S. aureus is the main trigger of several diseases such as osteomyelitis (Olson and Horswill, 2013), septic arthritis [START_REF] Corrado | Staphylococcus aureus-dependent septic arthritis in murine knee joints: local immune response and beneficial effects of vaccination[END_REF], bacteremia (Holland et al., 2014), endocarditis [START_REF] Fowler | Staphylococcus aureus endocarditis: a consequence of medical progress[END_REF], pneumonia (Chickering, 1919) and mastitis [START_REF] Kiku | Effect of intramammary infusion of recombinant bovine GM-CSF and IL-8 on CMT score, somatic cell count, and milk mononuclear cell populations in Holstein cows with Staphylococcus aureus subclinical mastitis[END_REF]. This bacterium is also the major causal agent of numerous implant infections (Arciola et al., 2018). Bacterial infections have been treated by the use of antibiotics since the beginning of the 40's, but their efficiency tends to decrease due to the high rate of antibiotic resistance developed by several microorganisms, such as S. aureus (Ventola, 2015b). The inappropriate use of antibiotics, particularly their overuse, has been considered one of the most important factors contributing to the development of microbial resistance. This constitutes a serious global public health problem since the frequency of established and emerging infectious diseases has increased because of the ineffectiveness of antibiotics. This augurs a warning about the inefficacy of our current drug arsenal and pushes us to look for new strategies in the struggle against bacterial infection [START_REF] Ventola | The antibiotic resistance crisis: part 2: management strategies and new agents[END_REF].

The World Health Organization (WHO) has been monitoring this danger with worldwide alert, since the mechanisms of resistance are emerging and spreading around the world faster than usual. In order to prevent and control the antibiotic resistance, the WHO advised the healthcare industry to invest in the development of new antibiotics and novel strategies to treat bacterial infections. At the same time, it is estimated that close to 80% of all antibiotics sold just in the US are intended for animal use (Martin et al., 2015). One study some years ago estimated that the global average annual consumption of antibiotics in livestock considering cattle, chicken and pigs will increase by 67% from 63,151 ± 1,560 tons to 105,596 ± 3,605 tons (Van Boeckel et al., 2015).

In the context of human and animal health, bacteriocins appear to have a different strategy and may act as strong candidates to overlap with or even replace the role of conventional antibiotics. The bacteriocins have been described with potential application as biopreservatives, but they are mainly defined as proteins and peptides produced by bacteria that inhibit the growth or kill other related and unrelated microorganisms [START_REF] Balciunas | Novel biotechnological applications of bacteriocins: A review[END_REF].

This review gathers recent information about the possible use of bacteriocins replacing antibiotic treatment during S. aureus infection considering the advantages offered by these natural compounds, as well as the differences and comparisons between them.

Survey methodology

A comprehensive review of the relevant literature was made through a collection of original articles and review articles that were selected by an academic search engine and academic databases, such as (e.g. https://scholar.google.com/; https://www.ncbi.nlm.nih.gov/pubmed/; http://www.scielo.br/ and http://www.periodicos.capes.gov.br/). We searched the following keywords: "Staphylococcus aureus, antibiotic, resistance, bacteriocins, biotechnology and bacteriocins". A total of 65 articles were accumulated and categorized into four main categories. The research criteria adopted in this review took into account important studies that addressed a high degree of observation of resistance to antibiotics by strains of S. aureus.

As a method criteria of inclusion, we considered the first cases of resistance and evaluated the progress until the present day. Additionally, in order to answer possible ways to deal with this protective advance in bacteria, the activity of bacteriocins was explored in the research studies considered here, as a good substitute capable of replacing in some cases the antibiotics currently used. The criteria for inclusion of possible bactericidal agents were evaluated considering experimental cases published in scientific journals. Our study highlighted some of the cases analyzed experimentally and we also show methods to explore the production of these potential molecules at an industrial scale using protocols from other review works.

Mechanisms of Antibiotic Resistance in S. aureus

Since the discovery of the first case of resistance to penicillin in mid of 20's, the number of key events of antibiotic resistance to S. aureus only increases. At the same time it is unstable, since the speed of response to antibiotics may be a few years or several (Figure 1) (Andriole, 2005;Fleming, 1929b;Humphreys and Mulvihill, 1985;Kaiser, 2000). Considering the broad spectrum of pathogenic bacteria currently observed in nature, S. aureus is pointed out as the most worrisome due to its capacity to manifest several diseases, a set of virulence factors [START_REF] Rozemeijer | Evaluation of approaches to monitor Staphylococcus aureus virulence factor expression during human disease[END_REF], the ability to survive in different environmental conditions [START_REF] Mäder | Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions[END_REF] and due to the plasticity of response to antibiotics. Presently, S. aureus is described as being resistant to a broad class of antibiotics including β-lactams, glycopeptides, quinolones, oxazolidinone etc.

In the class of β-lactam, S. aureus can acquire resistance to penicillin and methicillin (Fuda et al., 2005). Penicillin inhibits the formation of peptidoglycan (PG) cross-links in the bacterial cell wall. PG cross-links generate a three-dimensional structure around the cell, which ensures bacterial integrity. Penicillin binds to DD-transpeptidase, the enzyme responsible for the formation of PG cross-links, and prevents its catalytic activity. While a part of PG synthesis is inhibited by penicillin, bacterial enzymes involved in PG turnover remain active such as hydrolases and autolysins. The activity of penicillin that causes an imbalance between PG synthesis and degradation weakens PG and leads to cell death. Penicillin, which was discovered by Alexander Fleming in 1929, was the first antibiotic used to fight S. aureusmediated infections (Fleming, 1929b). Only two years after its introduction, S. aureus penicillinresistant strains appeared. Theses strains contained an enzyme, β-lactamase, able to destroy penicillin (Dietz and Bondi, 1948). The gene blaZ together with blaR1 and blaI, are the genetic determinants of the resistance of S. aureus to penicillin. They code for the β-lactamase chromosomal genes: grlA coding for a subunit of DNA topoisomerase IV, the primary quinolone target reported [START_REF] Ferrero | Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones[END_REF], and gyrA coding for DNA gyrase A subunit. The two proteins are intimately associated with the overlapping and opening of the double DNA strand during DNA replication. Specific mutations in grlB and gyrB also cause resistance to quinolones (Andriole, 2005). Both proteins have also been described as having the B subunit (grlB and gyrB) intrinsically associated with resistance in S. aureus. These mutations stand out in a region known as quinolone-resistance determining region (QRDR), which trigger several codon alteration in synonymous and non-synonymous amino acid mutations [START_REF] Tanaka | Mechanism of quinolone resistance in Staphylococcus aureus[END_REF]. Mutations reduce the affinity of enzyme-DNA complex for quinolones. Figure 4 displays the mechanism of resistance to fluoroquinolones.

Considering the oxazolidinone class, resistance of S. aureus to linezolid has also been observed in the last decade [START_REF] Besier | Linezolid Resistance in Staphylococcus aureus: Gene Dosage Effect, Stability, Fitness Costs, and Cross-Resistances[END_REF]. The main feature of the mechanism of action describes the linezolid interaction with the 50S subunit of prokaryotic ribosomes, which prevents initiation factors such as formylmethionyl-tRNA from acting to form the complex with the 30S subunit. This disruption precludes the formation of the 70S complex, which sequentially disrupts protein synthesis (Figure 5) [START_REF] Swaney | The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria[END_REF].

However, some cases of bacteria resistant to linezolid bearing point mutations at the specific targets in 50S, more precisely in the 23S ribosomal portion have been reported [START_REF] Afşar | Linezolid-resistant Enterococcus faecium: the first G2576T mutation in Turkey][END_REF][START_REF] Meka | Linezolid resistance in sequential Staphylococcus aureus isolates associated with a T2500A mutation in the 23S rRNA gene and loss of a single copy of rRNA[END_REF]. Furthermore, a new mechanism of linezolid resistance, which was first explored in a plasmid that included the gene cfr, was detected (Schwarz et al., 2000).

Several years ago a novel variant of the phenicol resistance transposon Tn558 was detected on the plasmid pSCFS6 suggesting the ability of horizontal transfer between staphylococci (Kehrenberg et al., 2007). The transcription of cfr gene produces a methyltransferase protein, which catalyzes the methylation of 23S rRNA at position A2503 offering resistance to some antibiotics such as chloramphenicol, florfenicol and clindamycin [START_REF] Kehrenberg | A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503[END_REF] and later to linezolid [START_REF] Toh | Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid: Linezolid resistance through ribosome modification[END_REF].

Considering all the information explored here, the broad spectrum of antibiotics capable of inhibiting the bacterial growth has been gradually decreasing over the last 70 years. All mechanisms of bacterial resistance to the action of antibiotics are embedded in a complex interactions among genes and its mobile elements and also with their interactions with the bacterial hosts. Moreover, the misuse of antibiotics by humans intensifies the selection pressure, making the resistance even more pronounced.

Bacteriocins: as alternative way in the treatment of S. aureus infections Bacteriocins are molecules usually produced by bacteria, that can be used with biopreservative applications (Bali et al., 2016) but mainly in an antibiotic role (Egan et al., 2017). These biomolecules can be differently classified over the gram-positive and gram-negative bacteria (Table 1).

It is suggested that the primary function of bacteriocins with regard to their killing ability is directly associated to maintaining the population around, in order to reduce the number of nutritional competitors in the environment. The targets for bacteriocins may have a broad spectrum of action similar to those of antibiotics (Table 2), preventing several biologically important phases in the cell. However, so far there has been no report on preventing vitamin biosynthesis [START_REF] Cavera | Bacteriocins and their position in the next wave of conventional antibiotics[END_REF].

In relation to the increased rate of S. aureus resistance, bacteriocins show great potential as candidates that are able to overlap the function of a large number of antibiotics (Table 2) [START_REF] Arumugam | Inhibition of Methicillin Resistant Staphylococcus aureus by Bacteriocin Producing Pseudomonas aeruginosa[END_REF][START_REF] Cavera | Bacteriocins and their position in the next wave of conventional antibiotics[END_REF][START_REF] Ceotto-Vigoder | Nisin and lysostaphin activity against preformed biofilm of Staphylococcus aureus involved in bovine mastitis[END_REF]. Several studies have described the bactericidal activity of bacteriocins that inhibits the growth of S. aureus, as observed in the Table 3. Varella et al (2017) described the inhibitory activity of seven bacteriocins in 165 strains of S. aureus in cases of bovine mastitis, showing a strong inhibition by epidermin (>85%) and a medium inhibition by aureocin A53 (>67%) (Varella Coelho et al., 2017). However, a combination of aureocin 70 and A53 showed a greater inhibitory potential compared to previous results (>91%). Still exploring cases of bovine mastitis, another study [START_REF] Barboza-Corona | Activity of bacteriocins synthesized by Bacillus thuringiensis against Staphylococcus aureus isolates associated to bovine mastitis[END_REF] highlighted five bacteriocins derivate from B. thuringiensis that were tested against 50 strains of S. aureus recovered from milk of lactating cows. The results of the study presented data on the resistance of these strains to penicillin, dicloxacillin, ampicillin and erythromycin. However, all strains were susceptible to the five tested bacteriocins, showing them to be useful as an alternative approach to control bovine mastitis.

Currently, one bacteriocin has shown strong relevance in the treatment of bovine mastitis [START_REF] Ceotto-Vigoder | Nisin and lysostaphin activity against preformed biofilm of Staphylococcus aureus involved in bovine mastitis[END_REF]. The bacteriocin lysostaphin shows a minimal inhibitory concentration of 3.9 to 50 µg ml-1 compared to the usually used bacteriocin nisin, which was 15.6 to 500 µg ml-1. This study concludes that the treatment using lysostaphin alone or associated with nisin was efficient in promoting bacterial cell lysis.

Others studies investigating the role of biofilm formation in methicillin-resistant S. aureus (MRSA) strains, which has been observed to be able to alternate the resistance phenotype and thus attenuate the virulence [START_REF] Pozzi | Methicillin Resistance Alters the Biofilm Phenotype and Attenuates Virulence in Staphylococcus aureus Device-Associated Infections[END_REF], noted the effects of three bacteriocins. In this study, nisin A showed the highest bactericidal activity against planktonic and biofilm cells, while Lacticin Q showed lower activity. However, the Nukacin ISK-1 just showed bacteriostatic activity against planktonic cell [START_REF] Nissen-Meyer | Structure-function relationships of the non-lanthionine-containing peptide (class II) bacteriocins produced by gram-positive bacteria[END_REF]. Despite this, the results show that the bacteriocins used stand out as potent molecules effective in the treatment of MRSA infections [START_REF] Okuda | Effects of bacteriocins on methicillin-resistant Staphylococcus aureus biofilm[END_REF]. Recently, cases of MRSA have been achieving prominence in the search for new bacteriocins. [START_REF] Jiang | Purification, Characterization, and Mode of Action of Pentocin JL-1, a Novel Bacteriocin Isolated from Lactobacillus pentosus , against Drug-Resistant Staphylococcus aureus[END_REF] observed the role of pentocin JL-1 bacteriocin showing effectiveness in both gram negative and positive bacteria [START_REF] Jiang | Purification, Characterization, and Mode of Action of Pentocin JL-1, a Novel Bacteriocin Isolated from Lactobacillus pentosus , against Drug-Resistant Staphylococcus aureus[END_REF]. Moreover, the authors also explored the ability of this bacteriocin to target the cell membrane of MRSA strains leading to the cell death. At the same time, another study devoted to the investigation of the pattern of bacterial strains that are capable of exhibiting antibacterial activity against MRSA, revealed one bacteriocin-like protein with a molecular mass of ~10 kDa produced by P. aeruginosa TA6 strain [START_REF] Arumugam | Inhibition of Methicillin Resistant Staphylococcus aureus by Bacteriocin Producing Pseudomonas aeruginosa[END_REF]. Several analysis have highlighted the fact that, in addition to resistance to high temperature and various chemical compounds, it is a potent antimicrobial efficient against MRSA and a strong candidate for higher yielding and enhancement of bacteriocin production. In a different study trying to comprise the synergy of antimicrobials, a group of bacteriocins and some antibiotics were evaluated considering the two subtypes in order to understand the efficacy of bacteriocins vs antibiotics; these results show the effectiveness of nisin Z, pediocin pa-1, duracin 61 and reuterin in inhibition of MRSA [START_REF] Hanchi | Inhibition of MRSA and of Clostridium difficile by durancin 61A: synergy with bacteriocins and antibiotics[END_REF]. However, in the set of antibiotics used, only vancomycin displays effects against MRSA. Evaluation of synergistic activities of antimicrobial agents showed that the combination of duracin with nisin or pediocin is a strong strategy in the control of bacterial growth, thus demonstrating the important role of duracin 61A

as an active bacteriocin against clinical drug-resistant MRSA.

Bacteriocins production: Perspective at the industrial scale

Interest in bacteriocins as well as their production has been growing over the years due to their use in food preservation, which exhibits antimicrobial activity as being an alternative to the use of chemical preservatives. In view of the vast amount of methods capable of purifying these biomolecules, two methods have been gaining more interest and becoming more effective in purifying bacteriocins: the Aqueous two-phase system (ATPS) and the Aqueous micellar twophase system (AMTPS) [START_REF] Jamaluddin | Novel approaches to purifying bacteriocin: A review[END_REF]. These systems can be formed by mixing in a solution with various components. ATPS are generally formed when two polymers that are inconsistent, i.e. polyethyleneglycol (PEG) and dextran or sodium sulfate, are diluted in water (Telis, 2012) (Figure 4). There are other types including, ionic liquids and short-chain alcohols.

The result of purification from ATPS displays a higher yield (~70%) (Md Sidek et al., 2016) compared to the conventional method, for example the single gel filtration chromatography

Conclusions

Our work concludes that bacteriocins are becoming increasingly important in the fight against infections by several microorganisms, especially S. aureus. The mechanisms of resistance to antibiotics explored here show the need to obtain new strategies to combat this pathogen. A number of bacteriocins having latent action spectrum against S. aureus are shown to be as effective as or even more so than conventional antibiotics as discussed herein. We believe that the information shown here is remarkably important for animal and human health, as well as to provide a means for production at an industrial scale besides creating new possibilities for future applications such as therapeutic methods.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Table 1. Classification of bacteriocins produced by gram-positive (Bierbaum and Sahl, 2009;[START_REF] Nissen-Meyer | Structure-function relationships of the non-lanthionine-containing peptide (class II) bacteriocins produced by gram-positive bacteria[END_REF]Coelho et al., 2017) and gram-negative bacteria (Rebuffat, 2011).

Classification Features Subclasses

Gram