Orientador: Alexandrina Sartori / Resumo: A leishmaniose visceral (LV) é uma doença negligenciada causada por Leishmania donovani na Índia e África ou Leishmania infantum na Europa e América Latina. O desenvolvimento de resposta imune eficaz e subsequente eliminação destes patógenos, requer o reconhecimento inicial da Leishmania, o qual é intermediado por receptores de reconhecimento padrão expressos por células da imunidade inata, entre eles os receptores de ligação a nucleotídeo (NLRs). Alguns NLRs ativam uma plataforma de proteínas, os inflamassomas, responsáveis pela ativação da caspase-1 e maturação de IL-1β e IL-18 e outra classe de NLRs, chamada NODs, ativam vias que culminam na ativação de NF-κB e produção de mediadores inflamatórios. O envolvimento desses receptores na LV ainda é pouco elucidado. Mesmo diante dos mecanismos de defesa do hospedeiro, esses parasitas conseguem sobreviver dentro dos macrófagos utilizando várias estratégias para escapar da resposta imune. Para um melhor entendimento dos mecanismos imunes envolvidos na LV, caracterizamos o perfil transcricional e a formação de inflamassomas e NODsomas de células THP-1 infectadas com L. infantum. Os resultados mostram que a L. infantum não induziu produção de TNF-α, IL-6 e IL-1β e nem ativação de caspase-1 após 8, 24 e 48 horas de infecção. Além disso, a infecção resultou em padrão de expressão gênica similar às células sem estímulo e distinto de células estimuladas com LPS, indicando que os parasitas entram nas células de forma mais silenciosa. Ap... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Visceral leishmaniasis (VL) is a neglected infectious disease caused by Leishmania donovani in India and Africa or Leishmania infantum in Europe and Latin America. The development of an effective immune response and subsequent elimination of these pathogens requires the initial recognition of the Leishmania that is mediated by pattern recognition receptors expressed in innate immunity cells, such as nucleotide-binding receptors (NLRs). Some NLRs activate a multiprotein platform named inflammasomes, responsible for the activation of caspase-1 and consequent maturation of IL-1β and IL-18; and another class of NLRs, the NODs, activate pathways that trigger NF-κB activation and production of inflammatory mediators. The involvement of NLRs in LV is poorly elucidated. Even in the presence of host defense mechanisms, these parasites can survive within the macrophages by employing successful strategies to escape from immune response. For a better understanding of the immune mechanisms involved in LV, we characterized the transcriptomic profiling and assembly of inflammasomes and NODsomas during infection with L. infantum in THP-1 cells. The results show that L. infantum did not induce the production of TNF-α, IL-6 and IL-1β nor activation of caspase-1 after 8, 24 and 48 hours of infection. In addition, the infection resulted in a pattern of gene expression similar to the non-stimulated cells and distinct from LPS-stimulated cells, indicating that the parasites enter inside cells in a... (Complete abstract click electronic access below) / Doutor

Activation of the cysteine protease caspase-1 and the subsequent processing and secretion of the pro-inflammatory cytokines IL-1Β and IL-18 is central to the inflammatory response as well as the induction of adaptive immune responses. Caspase-1 is activated as a part of a high-molecular weight multi-protein complex termed the inflammasome. The NLRP3 inflammasome is by far the best studied of these complexes, and it is the most promiscuous in terms of activating signals. The diversity of NLRP3 activating signals makes it likely that NLRP3 does not recognize each agonist directly, rather it detects a molecule that is generated, revealed, or altered by cellular stress. Recent studies have indicated that mitochondrial dysfunction is crucial for NLRP3 inflammasome activation, yet the activating ligand has not yet been identified. Appropriate and timely activation of this inflammatory pathway is required for host immunity to a variety of pathogens, however dysregulated activation leads to autoinflammation and potentially autoimmunity. Hence it is important to identify mechanisms for inflammasome activation and regulation. Therefore, this dissertation has focused on investigating the mechanisms for activation and regulation of the NLRP3 inflammasome, and the biological consequences of these changes. We show that the mitochondrial lipid cardiolipin is required for NLRP3 inflammasome activation. We have also identifying a novel mechanism by which inflammasome activation is regulated. Data presented in this dissertation shows that IgG immune complexes effectively suppress inflammasome activation and the subsequent processing and secretion of IL-1Α and IL-1Β. Furthermore we show that immunization with IgG immune complexes suppresses both Th2 and Th17 immune responses. Together these data provide novel insights into the activating and regulatory pathways of both the innate and adaptive immune systems.

Interleukin-1 alpha (IL-1a) is a powerful inflammatory cytokine that modulates both innate and adaptive immunity. As such, IL-1a is implicated in the development of multiple inflammatory and autoimmune diseases including atherosclerosis, arthritis and cancer. Therefore, understanding the mechanisms that regulate IL-1a activity is extremely important. For many years, pro-IL-1a was considered to be a fully active alarmin. However, we have previously shown that the removal of the pro-domain by calpain, a protease that is activated upon necrosis, significantly increases IL-1a bioactivity. The work presented in this thesis demonstrates that multiple proteases from diverse biological systems cleave and activate IL-1a. We therefore suggest that IL-1a is an important signalling hub that integrates diverse proteolytic danger signals to alert the immune system. In particular we have identified the inflammatory caspase, caspase-5, as a novel and potent activator of IL-1a. We show that caspase-5 directly cleaves pro-IL-1a during the activation of the non-canonical inflammasome by cytosolic LPS, which mimics intracellular bacterial infection. We also demonstrate that caspase-5-cleaved IL-1a mediates the senescence-associated secretory phenotype (SASP), which drives the deleterious effects of senescent cells in multiple age-related diseases. Therefore, therapeutically targeting caspase-5 may be of interest for pathologies mediated by the non-canonical inflammasome and/or senescent cells. Finally we find that rs17561, a common IL1A polymorphism, reduces active IL-1a release. We find that blood from minor allele homozygotes releases significantly less IL-1a than major allele homozygotes upon LPS stimulation. Therefore, genotyping patients under consideration for anti-IL-1a therapy could predict who would be likely to respond well to the treatment. In conclusion, the work presented in this thesis enhances our understanding of how IL-1a activity is regulated. The identification of both the caspase-5-mediated pathway of IL-1a activation and the defect conferred by the rs17561 SNP could have important clinical implications for the treatment of multiple inflammatory diseases.

Submitted by Mariana Gatto (marianagatto11@hotmail.com) on 2018-05-21T17:47:50Z No. of bitstreams: 1 Tese Mariana Gatto.pdf: 3235524 bytes, checksum: b7c9938cd744aff0ff8a8ee5f858831e (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-05-22T14:28:44Z (GMT) No. of bitstreams: 1 gatto_m_dr_bot.pdf: 3235524 bytes, checksum: b7c9938cd744aff0ff8a8ee5f858831e (MD5) / Made available in DSpace on 2018-05-22T14:28:44Z (GMT). No. of bitstreams: 1 gatto_m_dr_bot.pdf: 3235524 bytes, checksum: b7c9938cd744aff0ff8a8ee5f858831e (MD5) Previous issue date: 2018-04-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A leishmaniose visceral (LV) é uma doença negligenciada causada por Leishmania donovani na Índia e África ou Leishmania infantum na Europa e América Latina. O desenvolvimento de resposta imune eficaz e subsequente eliminação destes patógenos, requer o reconhecimento inicial da Leishmania, o qual é intermediado por receptores de reconhecimento padrão expressos por células da imunidade inata, entre eles os receptores de ligação a nucleotídeo (NLRs). Alguns NLRs ativam uma plataforma de proteínas, os inflamassomas, responsáveis pela ativação da caspase-1 e maturação de IL-1β e IL-18 e outra classe de NLRs, chamada NODs, ativam vias que culminam na ativação de NF-κB e produção de mediadores inflamatórios. O envolvimento desses receptores na LV ainda é pouco elucidado. Mesmo diante dos mecanismos de defesa do hospedeiro, esses parasitas conseguem sobreviver dentro dos macrófagos utilizando várias estratégias para escapar da resposta imune. Para um melhor entendimento dos mecanismos imunes envolvidos na LV, caracterizamos o perfil transcricional e a formação de inflamassomas e NODsomas de células THP-1 infectadas com L. infantum. Os resultados mostram que a L. infantum não induziu produção de TNF-α, IL-6 e IL-1β e nem ativação de caspase-1 após 8, 24 e 48 horas de infecção. Além disso, a infecção resultou em padrão de expressão gênica similar às células sem estímulo e distinto de células estimuladas com LPS, indicando que os parasitas entram nas células de forma mais silenciosa. Após a infecção houve aumento da expressão de alguns genes como ACTG1, ACTB, CD36 e DUSPs relacionados com vias de motilidade celular e regulação de MAPKs. Os genes CSF1 e CDC20 foram dois dos 30 mais expressos após infecção e estão relacionados com ciclo celular e diferenciação de macrófagos para um perfil anti-inflamatório. O gene GBP1, associado com ativação de inflamassomas, foi sub expresso após a infecção. Além disso, infecção com L. infantum resultou na expressão de poucos genes relacionados com a via dos NLRs, destacando-se entre esses o TNFAIP3 e IL1RN, referentes à modulação negativa dessa via. Os resultados obtidos indicam que a L. infantum entra nas células THP-1 de forma mais silenciosa, desativa vias inflamatórias, entre essas a via de receptores NLRs e evita a montagem de uma resposta imunológica efetora. Provavelmente o parasita usa esses recursos como mecanismos adicionais de escape para garantir sua sobrevivência dentro das células. / Visceral leishmaniasis (VL) is a neglected infectious disease caused by Leishmania donovani in India and Africa or Leishmania infantum in Europe and Latin America. The development of an effective immune response and subsequent elimination of these pathogens requires the initial recognition of the Leishmania that is mediated by pattern recognition receptors expressed in innate immunity cells, such as nucleotide-binding receptors (NLRs). Some NLRs activate a multiprotein platform named inflammasomes, responsible for the activation of caspase-1 and consequent maturation of IL-1β and IL-18; and another class of NLRs, the NODs, activate pathways that trigger NF-κB activation and production of inflammatory mediators. The involvement of NLRs in LV is poorly elucidated. Even in the presence of host defense mechanisms, these parasites can survive within the macrophages by employing successful strategies to escape from immune response. For a better understanding of the immune mechanisms involved in LV, we characterized the transcriptomic profiling and assembly of inflammasomes and NODsomas during infection with L. infantum in THP-1 cells. The results show that L. infantum did not induce the production of TNF-α, IL-6 and IL-1β nor activation of caspase-1 after 8, 24 and 48 hours of infection. In addition, the infection resulted in a pattern of gene expression similar to the non-stimulated cells and distinct from LPS-stimulated cells, indicating that the parasites enter inside cells in a more silent way. After infection, there was increased expression of some genes, such as ACTG1, ACTB, CD36 and DUSPs related to cellular motility and regulation of MAPKs pathways. The CSF1 and CDC20 genes were two of the 30 most expressed after infection and were related to cell cycle pathway and macrophage differentiation to an anti-inflammatory profile. The GBP1 gene, associated with inflammasome activation, was downregulated after infection. In addition, infection with L. infantum resulted in the expression of few genes related to the NLRs pathway, such as TNFAIP3 and IL1RN that are related to down modulation of this pathway. The results indicate that L. infantum enters inside the THP-1 cells more quietly, deactivates inflammatory pathways, including the NLR receptor pathway, and avoids the assembly of an effector immune response. Probably the parasite uses these strategies as additional escape mechanism to ensure its survival within host cells.

A resposta inflamatória está envolvida em muitos aspectos da biologia do câncer. O inflamassoma é um complexo multiprotéico intracelular compostos por três elementos: um receptor de reconhecimento de padrões moleculares (PRR), uma proteína ligadora ASC (proteína speck-like associada à apoptose com domínio de recrutamento de caspase) e o zimogênio pró-caspase-1. A ativação da caspase-1 é responsável pela síntese de IL-18 e, principalmente, de IL-1?. A ativação da caspase-1 é ainda capaz de induzir a piroptose, um tipo de morte celular inflamatória. O papel dos inflamassomas no câncer ainda é mal definido, devido às suas funções contrastantes na oncogênese, variando a depender do tipo de tecido e do estágio da tumorigênese em que são ativados. Estudos recentes mostraram uma ativação do inflamassoma à medida que o melanoma progride. Avaliamos a expressão de componentes do inflamassoma, incluindo dois tipos de PRR (NLRP1 e NLRP3), da enzima caspase-1, e da IL-1beta em neoplasias melanocíticas benignas e malignas, por meio de técnica de imunohistoquímica. Foram analisadas amostras de tecidos embebidos em parafina de 25 pacientes com melanoma (16 melanomas finos e 9 melanomas intermediários-espessos) e 22 pacientes com nevo melanocítico (12 nevos intradérmicos e 10 nevos displásicos). Todas as amostras de pele foram recuperadas dos arquivos do Departamento de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Evidenciamos uma maior expressão de NLRP1, NLRP3 e caspase-1 nos melanomas quando comparados aos nevos. Não houve diferença estatisticamente significativa quanto a expressão de IL-1beta entre os grupos. De forma inesperada, a descoberta mais interessante foi uma maior expressão de NLRP1 em melanomas finos do que nos tumores mais espessos. Esses achados sugerem que um aumento de NLRP1 poderia representar um evento promotor na transformação de melanócitos, mas que não estaria envolvido na progressão tumoral. Estudos adicionais são necessários para esclarecer esta complexa relação entre as proliferações melanocíticas e o inflamassoma / Inflammatory response is involved in many aspects of cancer biology. Inflammasomes are a group of cytosolic multiprotein complexes, classically consisting of an upstream sensor protein of the NOD-like receptor (NLR) family, the adaptor protein ASC, and the downstream effector caspase-1. Its activation leads to the production of biologically active IL-1beta and IL-18, and consequently contributing to the inflammatory process. Caspase-1 activation can also induce pyroptotic cell death, that is accompanied by the release of cytosolic contents to the extracellular space eliciting local inflammation. The roles of the inflammasomes in cancer are still ill defined, due to their contrasting roles in oncogenesis. Recent studies have shown an activation of the inflammosome as melanoma progresses. We evaluated the expression of inflamassome components (NLRP1, NLRP3, caspase-1) and of IL-1beta in melanocytic neoplasms, by immunohistochemistry. Formalin-fixed, paraffin-embedded tissue samples from 25 patients with melanoma (16 thin melanomas and 9 intermediate-thick melanomas), and 22 patients with melanocytic nevus (12 intradermal nevi and 10 dysplastic nevi) were analyzed. All skin samples were retrieved from the files of the Department of Dermatology at Clinics Hospital of Faculty of Medicine, University of São Paulo. Comparing all nevi with all melanomas, we found a higher expression of NLRP1, NLRP3 and caspase-1 in melanomas. There was no difference of IL-1beta expression between the groups. For the first time, to our knowledge, we reported an increasing expression of NLRP1 in melanoma compared to melanocytic nevus. Unexpectantly, NLRP1 expression resulted augmented NLRP1 in thin melanomas compared with intermediate-thick melanomas. These data may suggest a role of NLRP1 in oncogenesis, but that its expression decreases as disease progresses. We can hypothesize that an increase of NLRP1 could represent a promoter event in melanocyte transformation, but it does not be involved in tumour progression. The association between nevus, melanoma and inflammasomes seems to be complex and further studies are necessary to clarify this

A flagelina é um agonista natural do sensor TLR5 e do inflamassoma NAIP/NLRC4 que é responsável pela secreção de IL-1β e IL-18 e pela indução de morte celular necrótica, via ativação da caspase-1. Entretanto, nós observamos que a inserção da flagelina de B. subtilis no citosol celular por meio de vesículas lipídicas, induz um processo atípico de morte nos macrófagos peritoneais (PMs) deficientes em NLRC4, ASC e caspase-1/11. A morte dos PMs manteve seu resultado antimicrobiano, sendo acompanhada da liberação de IL-1α. A morte celular e a secreção das citocinas IL-1α e IL-1β, foi mediada por catepsinas lisossomais, sugerindo uma cooperação entre a via lisossomal e os inflamassomas nas respostas induzidas pela flagelina. Além disso, a flagelina de S. typhimurium foi capaz de induzir dano lisossomal e secreção de IL-1α e IL-1β mediada pelo eixo caspase-catepsinas, na ausência de carreadores, e estas citocinas tiveram um impacto na imunidade adaptativa induzida pela flagelina, no modelo de ativação de linfócitos T específicos por células dendríticas, in vitro. / Flagellin is a natural agonist of TLR5 and NAIP/NLRC4 inflammasome that is responsible for IL-1β and IL-18 secretion and for the induction of a necrotic cell death, both mediated by caspase-1. However, we observed that flagellin from B. subtilis inserted into lipid vesicles, induced an atypical cell death in peritoneal macrophages (PMs) in the absence of NLRC4, ASC and caspase-1/11. This inflammasome-independent cell death retained its antimicrobial outcome, being accompanied with IL-1α secretion. Importantly, cell death and caspase-1-dependent IL-1α and IL-1β secretion were regulated by lysosomal cathepsins, suggesting a cooperation between the inflammasome and lysosomal pathway in response to flagellin. We also observed that flagellin from S. typhimurium is able to induce lysosomal damage and IL-1α and IL-1β secretion by PMs in the absence of a carrier, through a caspase-catepsins-dependent manner, and that cytokines were important to the ability of flagellin in to induce adaptive immune response by antigen-specific T cells.

L'inflammasome est complexe protéique intracellulaire de l'immunité innée permettant la reconnaissance de pathogènes intracellulaires. NLRC4, un Nod-like récepteur permettant l'activation de l'inflammasome est impliqué dans la reconnaissance du flagelle ainsi que du système de sécrétion de type 3 (SST3) de Pseudomonas aeruginosa, une bactérie majoritairement extracellulaire. Nous avons donc déterminer l'impact de la reconnaissance de P. aeruginosa par le NLRC4-inflammasome in vivo dans un modèle murin de pneumonie aiguë. De façon surprenante, l'activation du NLRC4-inflammasome par le SST3 de P. aeruginosa contribue à diminuer la survie de l'hôte en diminuant la clairance bactérienne pulmonaire et en augmentant la lésion pulmonaire induite. En effet, la perte de l'activation de l'inflammasome chez les souris NLRC4/- permet d'une part, une réponse précoce méfiée par l'IL-17A. Cette réponse dépendant de l'IL-17A conduit à une expression majeure de peptides antimicrobiens par l'épithélium pulmonaire et diminue la lésion pulmonaire en diminuant le recrutement des cellules immunitaires inflammatoires. L'administration d'IL-18 recombinante murine ou l'inhibition de cette voie par un anticorps anti-IL-17A inhibe cette réponse IL-17A dépendante. Ces résultats mettent en évidence un nouveau rôle du SST3 de P. aeruginosa, qui en plus de son effet cytotoxique et de la translocation d'exotoxines, permet d'activer l'inflammasome pour échapper à la réponse immunitaire innée de l'hôte en inhibant une voie IL-17 dépendante. / The inflammasome is thought to function as a cytosolic surveillance system against intracellular pathogens. However, we report that Pseudomonas aeruginosa, an extracellular pathogen responsible for acute lung infection, relies upon inflammasome activation to persist and worsen disease. Specifically, we show that loss of either NLRC4 expression or type-3 secretion system (T3SS)-driven activation of NLRC4, surprisingly, resulted in a robust Th-17-like immune response that enhanced bacterial clearance and attenuated virulence independently of exotoxins. This Th-17-like response correlated with marked upregulation of antimicrobial peptides and was suppressed by either neutralization of IL-17A or exogenous IL-18 administration in vivo. Together, these results unveil an adaptation mechanism through which the problem pathogen manages to evade Th17-mediated immunity and invade the lung, providing a potential mechanism for infectious complications of anti-IL17 therapy in inflammatory diseases. The T3SS exploitation of NLRC4-coupled inflammasome response may therefore represent a novel gene-for-gene model of pathogen evolution alongside host immunity.

La septicémie est un syndrome complexe au cours duquel une infection systémique conduit à une inflammation exacerbée et une immunosuppression pouvant mener au développement d’infections secondaires. La progression de ce syndrome est corrélée avec une diminution marquée du nombre de cellules immunitaires protectrices, notamment les cellules dendritiques (DC). Des études sur des modèles précliniques murins ont montré que restaurer les effectifs ou la fonction des DC permettait de diminuer l’immunosuppression et la susceptibilité aux infections. Les DC conventionnelles (cDC) et les monocytes font partie du système des phagocytes mononucléés, cruciaux à l’homéostasie et en conditions pathologiques. Les cDC ont un rôle de présentation des antigènes et d’orchestration globale du système immunitaire. Les monocytes et leurs dérivés ont un rôle d’assainissement des tissus et de régulation locale de l’immunité. Les monocytes peuvent également développer des fonctions homologues à celles des cDC dans certaines conditions. Cependant, de récentes études ont montré que les DC pouvaient avoir des rôles différents voire antagonistes selon leur ontogénie.Ces cellules proviennent chez l’adulte d’un précurseur hématopoïétique bipotent restreint à ces deux lignées, le MDP (Monocyte and DC progenitor). Il est connu qu’une infection systémique induit la génération massive de cellules myéloïdes au détriment des lymphocytes. Ce mécanisme est parfois appelé myélopoïèse d’urgence. Historiquement, la distinction des cDC des cellules dérivées de monocytes a été compliquée par le phénotype très proche de ces différentes populations. C’est pourquoi beaucoup d’études analysant l’impact des septicémies sur les DC n’ont pas évalué en détail les sous populations impliquées. De récentes avancées ont été réalisées concernant l’identification cDC et des cellules dérivées de monocytes ainsi que de leurs précurseurs hématopoïétiques. Nous avons alors cherché à évaluer si le processus de myélopoïèse d’urgence favorisait certaines populations de phagocytes mononucléés, dans un modèle murin d’endotoxémie.Au cours de cette étude, nous avons montré que la présence systémique de lipopolysaccharides (LPS) induit la génération de cellules présentatrices d’antigènes d’origine monocytaire (Mo-APC) dans la rate. Ce phénomène est corrélé avec une diminution du nombre des cDC splénique. Contrairement à la diminution des cDC, la génération des Mo-APC est dépendante de la signalisation des IFN de type I. De plus, nous avons observé que le LPS permet l’induction d’une monopoïèse active dans la moelle osseuse, dépendamment des IFN de type I.En parallèle, nous avons souhaité étudier une voie de détection du LPS intracellulaires récemment mise en évidence et potentiellement impliquée dans la diminution du nombre des cDC suite à l’injection de LPS. Cette voie de détection impliquée dans la létalité de l’endotoxémie active l’inflammasome non canonique induisant la mort cellulaire de type pyroptose et la production de cytokines pro-inflammatoires. Nous avons observé que cette voie de signalisation ne semblait pas impliquée dans la diminution de l’effectif des cDC spléniques suite à l’injection de LPS. Cependant, un criblage à haut débit a révélé des régulateurs potentiels de l’inflammasome non canonique, et en particulier au niveau de la production des cytokines pro-inflammatoires.Dans leur ensemble, ces travaux de thèse contribuent à l’amélioration des connaissances concernant la plasticité des précurseurs hématopoïétiques du système des phagocytes mononucléés en condition pathologique. A l’avenir, ces connaissances pourraient permettre le développement de nouvelles stratégies thérapeutiques visant à limiter l’immunosuppression des patients via la manipulation de la myélopoïèse et des phagocytes mononucléés. / Sepsis is a complex syndrome in which systemic infection leads to exacerbated inflammation and immunosuppression that can lead to the development of secondary infections. The progression of this syndrome is correlated with a marked decrease in the number of protective immune cells, especially dendritic cells (DCs). Studies on preclinical mouse models have shown that restoring the numbers or function of DCs decreased immunosuppression and susceptibility to infections.Conventional DC (cDCs) and monocytes are part of the system of mononuclear phagocytes, important in homeostasis and pathological conditions. Conventional DCs have a role of antigen presentation and overall orchestration of the immune system. Monocytes and their derivatives have a role of tissue cleaning and local regulation of immunity. Monocytes can also develop functions homologous to those of cDCs under certain conditions. However, recent studies have shown that DC may have different or even antagonistic roles depending on their ontogeny.These cells originate from a bipotent hematopoietic precursor restricted to these two lineages, the MDP (Monocytes and DCs progenitor). It is known that systemic infection induces the massive generation of myeloid cells at the expense of lymphocytes. This mechanism is sometimes called emergency myelopoiesis. Historically, the distinction of cDCs from cells derived from monocytes has been complicated by the very similar phenotype of these different populations. This is why many studies analysing the impact of septicemia on DCs have not evaluated the subpopulations involved in details. Recent advances have been made in the identification of cDCs and cells derived from monocytes as well as their respective haematopoietic precursors. We then sought to re-evaluate whether the emergency myelopoietic process favoured certain populations of mononuclear phagocytes in a murine model of endotoxemia.In this study, we have shown that the systemic injection of lipopolysaccharides (LPS) induces the generation of antigen-presenting cells of monocytic origin (Mo-APC) in the spleen. We observed a parallel decrease in splenic cDCs numbers. In contrast to the decrease of cDCs numbers, Mo-APC generation is dependent on type I IFNs signalling. Moreover, we observed that LPS allows the induction of active monopoiesis in the bone marrow, depending on type I IFNs signalling.In parallel, we studied a recently discovered detection pathway for intracellular LPS potentially implicated in the reduction of cDCs number following LPS injection. This detection pathway is implicated in the lethality of LPS induced endotoxic shock. It activates the non-canonical inflammasome inducing pyroptotic cell death and the production of pro-inflammatory cytokines. We observed that this signaling pathway did not appear to be involved in the reduction of the splenic cDCs following LPS injection. However, high-throughput screening revealed potential regulators of non-canonical inflammasome, and in particular in the production of pro-inflammatory cytokines.Overall, this work contributes to the improvement of our knowledge concerning the plasticity of haematopoietic precursors of mononuclear phagocytes system in pathological conditions. In the future, this knowledge could allow the development of new therapeutic strategies aimed at limiting the immunosuppression of patients through the manipulation of myelopoiesis and mononuclear phagocytes.

Background: Systemic hypertension is a risk factor of age-related macular degeneration (AMD), a chronic inflammatory disease. Acute hypertension is caused by increased extracellular osmolarity after intake of dietary salt (NaCl). We determined in cultured human retinal pigment epithelial (RPE) cells whether high extracellular NaCl alters the gene expression of inflammasome-associated proteins, and whether autocrine/paracrine purinergic (P2) receptor signaling contributes to the NaCl-induced NLRP3 gene expression. Methodology/Principal Findings: Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Gene and protein expression levels were determined with real-time RT-PCR and Western blot analysis, respectively. IL-1β and IL-18 levels were evaluated with ELISA. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. High extracellular NaCl induced NLRP3 and pro-IL-1β gene expression, while the gene expression of further inflammasome-associated proteins (NLRP1, NLRP2, NLRP6, NLRP7, NLRP12, NLRC4, AIM2, ASC, procaspase-1, pro-IL-18) was not altered or below the detection threshold. The NaCl-induced NLRP3 gene expression was partially dependent on the activities of phospholipase C, IP3 receptors, protein kinase C, the serum and glucocorticoid-regulated kinase, p38 MAPK, ERK1/2, JNK, PI3K, and the transcription factors HIF-1 and NFAT5. Pannexin-dependent ATP release and P2Y1 receptor activation is required for the full induction of NLRP3 gene expression. High NaCl induced a transient increase of the NLRP3 protein level and a moderate NLRP3 inflammasome activation, as indicated by the transient increase of the cytosolic level of mature IL-1β. High NaCl also induced secretion of IL-18. High extracellular NaCl induces priming of the NLRP3 inflammasome in RPE cells, in part via P2Y1 receptor signaling. The inflammasome priming effect of NaCl suggests that high intake of dietary salt may promote local retinal inflammation implicated in the development of AMD.

Chitin is an abundant polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons. The immunological properties of both chitin and its deacetylated derivative chitosan are of relevance due to frequent natural exposure and their increasing use in translational applications. Depending on the preparation studied and the endpoint measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. Highly purified chitosan and chitin were prepared and the capacity of these glycans to stimulate the release of the inflammasomeassociated cytokine IL-1β was examined. Chitosan was shown to be a potent inflammasome activator in mouse bone marrow macrophages, macrophages polarized towards a M1 or M2 phenotype, dendritic cells, peritoneal cells, and human PBMCs. Acetylation of the chitosan to chitin resulted in a near total loss of IL-1β activity in all cell types tested. The size of the chitosan particles played an important role, with small particles eliciting the greatest activity. An inverse relationship between size and stimulatory activity was demonstrated using chitosan passed through size exclusion filters as well as with chitosan-coated beads of defined size. Partial digestion of chitosan with pepsin resulted in a larger fraction of small phagocytosable particles and more potent inflammasome activity. Inhibition of phagocytosis with cytochalasin D abolished the IL- 1β stimulatory activity of chitosan, offering an explanation for why the largest particles were nearly devoid of activity. Thus, the deacetylated polysaccharide chitosan potently activates the NLRP3 inflammasome in a phagocytosis-dependent manner. The reason for chitin’s inability to elicit IL-1β is unknown, but it does not appear to be due to active inhibition of the inflammasome and while chitin appears to be more readily digested by macrophage cell lysates, it does not occur at a rate which would likely impact inflammasome activation. There are three proposed mechanisms for NLRP3 inflammasome activation: K+ efflux, ROS, and lysosomal destabilization. The contributions of these mechanisms were tested and it was revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, the laminin receptor was evaluated as a potential chitin receptor. These studies provide insight into the activating properties of chitin and chitosan and highlight the importance of matching particle size and degree of acetylation to the level of activity desired for translational applications.