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.

Orientador: Iracilda Zeppone Carlos / Banca: Fernanda Freitas Anibal / Banca: Luciana Simon Pereira Crott / Banca: Angela Maria Victoriano de Campos Soares / Banca: Cleni Mara Marzocchi Machado / Resumo: A esporotricose é uma infecção fúngica causada pelas espécies do complexo Sporothrix, incluindo Sporothrix schenckii sensu stricto. Os receptores de reconhecimento padrão (PRRs), pertencentes às células da resposta imune inata, reconhecem os padrões moleculares associados a patógenos (PAMPs). Uma classe de PRRs que tem sido associada ao reconhecimento de fungos é a dos receptores Nod-like (NLR). Após o reconhecimento de PAMPs, os receptores NLR family pyrin domain-containing 3 (NLRP3), em conjunto com a molécula adaptadora Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) e com a caspase-1, formam o inflamassoma NLRP3. Quando ativado, este complexo protéico promove a maturação das citocinas pró-inflamatórias IL-1β e IL-18, que influenciam no desenvolvimento das respostas imune Th17 e Th1, respectivamente. O inflamassoma NLRP3 também é responsável pela piroptose, uma morte celular inflamatória regulada por caspase-1. Neste contexto, o objetivo deste estudo foi avaliar o papel do inflamassoma NLRP3 em modelo de infecção sistêmica por S. schenckii, utilizando um camundongo selvagem (WT) e três diferentes camundongos knockout (KO): NLRP3-/-, ASC-/- e Casp-1-/-. Todos os animais KO foram mais suscetíveis que os WT na infecção por S. schenckii. A ativação do inflamassoma NLRP3 foi essencial para a liberação ex vivo de NO, IL-1β, IL-17, IL-18, mas não para IFN-γ. Além disso, S. schenckii desencadeou maior expressão de caspase-1 ativa e maior porc... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Sporotrichosis is a mycosis caused by fungi from the Sporothrix schenckii species complex, whose prototypical member is Sporothrix schenckii sensu stricto. Pattern recognition receptors (PRRs) recognize and respond to pathogen-associated molecular patterns (PAMPs) and shape the following adaptive immune response. A family of PRRs most frequently associated to fungal recognition is the nucleotide-binding oligomerization domain-like receptor (NLR). After recognition of a PAMP, NLR family pyrin domain-containing 3 (NLRP3) binds to apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 to form the NLRP3 inflammasome. When activated, this complex promotes maturation of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 that influence the development of the Th17 and Th1 immune responses, respectively. In addition, the NLRP3 inflammassome is responsible for pyroptosis, a highly inflammatory cell death regulated by caspase-1. In this work, we aimed to evaluate the role of the NLRP3 inflammasome to the outcome of the S. schenckii infection using a wild-type mice (WT) and three different knockout mice (KO): NLRP3-/-, ASC-/-, and Casp-1 -/- . All the KO mice were more susceptible to the infection than the WT during the S. schenckii infection. Furthermore, the NLRP3 inflammasome seems to be critical to the ex vivo release of NO, IL-1β, IL-18, and IL-17, but not IFN-γ. The S. schenckii infection led to increased activation of caspase-1 and cell death by pyroptosis in WT mice. Also, a role for the inflammasome in shaping the adaptive immune response was suggested by lower frequencies of type 17 helper T (Th17) and Th1/Th17 cells in S. schenckii-infected KO mice. On the other hand, absence of any of the inflammasome components resulted in increased frequency of cytotoxic T cells in this infection... (Complete abstract click electronic access below) / Doutor

The immune system rapidly mounts an innate immune response to invading pathogens that is accompanied by antigen-presentation, to promote the development of the adaptive immune response. These responses orchestrate through signal transduction by PRRs that recognize PAMPs, which results in the expression of various cytokines and mediators to promote pathogen control. Herein, we investigated the role of the type I interferon (IFN)- and the p38MAPK- pathways in response to infection with Salmonella Typhimurium (ST). We delved into the mechanisms through which IFNAR1-signaling results in host susceptibility against ST and show that while STAT2 and IRF9 promote susceptibility against ST, this is antagonized by STAT1. Our results indicate that IFNAR1-signaling induces IL-10 production through the ISGF3 complex, which indeed inhibits the production of IL-1β (via NLRP3 and caspase-1) resulting in a state of resistance against ST. Furthermore, our work elucidates that MK2, which is a p38MAPK substrate promotes host resistance, which is contradictory to type I IFNs despite the fact that MK2 regulates cytokine expression in a similar pattern to IRF9. We demonstrate that MK2 inhibits inflammasome signaling via NLRP3, caspase-1 and caspase11. We also reveal a role for MK2 in regulating IL-1β production via distinct signaling pathways including inhibition of MSK1/2 besides activation of the autophagic machinery; which also contribute to the enhanced inflammasome activation seen in Mk2- deficient cells. Thus, our observations illuminate the fact that the type I IFN pathway and the p38MAPK pathway are only dependent on each other to a certain extent in modulating the innate immune response to Salmonella infection, thereby bringing about varied outcomes in the infected host.

La goutte est une maladie inflammatoire particulièrement prévalente causée par la formation de dépôts articulaires et péri-articulaires de cristaux d’urate monosodique (UMS ou MSU) et dépendante de la cytokine interleukine-1β (IL-1β). En 2006, l’inflammasome NLRP3 a été montré comme nécessaire pour la maturation de l’IL-1β, in vitro, en réponse aux cristaux de MSU. Néanmoins, sa nécessité in vivo est un sujet de controverse. Mon travail de thèse a porté sur la caractérisation d’un modèle murin d’inflammation aiguë uratique et l’analyse de la contribution des inflammasomes dans cette pathologie. J’ai d’abord montré que notre modèle par injection sous-cutanée de cristaux de MSU donne lieu une forte inflammation des tissus mous comme cela est souvent observé lors des crises de goutte chez l’Homme. L’emploi de souris invalidées génétiquement et d’inhibitions pharmacologiques m’a permis de décrire son indépendance vis-à-vis de plusieurs composants des inflammasomes et confirme le rôle majeur de l’IL-1β. De manière intéressante, j’ai ensuite montré qu’il est possible de réduire fortement l’inflammation dans ce modèle par un traitement topique à base d’imiquimod (crème ALDARA®), un ligand synthétique de TLR7. Des expériences réalisées in vivo et in vitro m’ont permis de relier l’effet de l’imiquimod à une baisse importante de l’Il1b au niveau transcriptionel, via une signalisation faisant probablement intervenir les interférons de type I et possiblement le facteur RUNX3. Mes données montrent donc que la production d’IL-1β, dans ce modèle, est visiblement indépendante de NLRP3 mais peut être fortement abaissée par l’application topique d’imiquimod. L’imiquimod pourrait ainsi représenter une piste thérapeutique attractive. / Gout is a prevalent inflammatory disease caused by the deposition of monosodium urate crystals (MSU) in articular/periarticular areas, which strongly depends on interleukine-1β (IL-1β). In 2006, the NLRP3 inflammasome has been shown to perform IL-1β maturation in vitro after MSU crystal exposure. However, its in vivo dependence is still matter of controversy. In my thesis project, I focused on the characterization of a murine acute uratic inflammation and analysed the contribution of inflammasome components. I first showed that the subcutaneous injection of MSU crystals in mice generate a strong soft tissue inflammation as observed in human gouty crises. Then, by using genetically-modified mouse lines and pharmacological inhibitions, I demonstrated that this model is inflammasome-independent, while still requiring IL-1β secretion. Interestingly, I observed that the topical application of imiquimod (ALDARA® cream), which is a synthetic TLR7 ligand, strongly dampens inflammation. In vivo and in vitro experiments further demonstrated that this effect is linked to reduced Il1b gene expression, which linkely involves type I interferon signaling and eventually the transcription factor RUNX3. Altogether, my results show that IL-1β production is NLRP3-independent in this mouse model but can be strongly decreased by topical application of imiquimod. Therefore, imiquimod might be an attractive therapeutic option for gouty patients.

O processo inflamatório pode ser iniciado pela presença de patógenos (PAMPs) ou de moléculas associadas ao dano tecidual (DAMPs), estes sinais são reconhecidos por células do sistema imune via receptores de reconhecimento de padrão localizados na membrana ou no citoplasma destas células. A ativação da família de receptores intracitoplasmáticos do tipo NOD (NLR) pode levar à formação de complexos proteicos denominados inflamassomas, entre eles, o inflamassoma NLRP3, formado por três estruturas básicas: receptores do tipo NOD, proteína adaptadora (ASC) e pró-caspase 1. Essas caspases ativas são capazes de induzir a maturação proteolítica de interleucina-1 beta (IL-1b) e IL-18, que então são liberadas para o meio extracelular. É sabido que a hipertensão é uma doença inflamatória associada à presença de DAMPs em regiões de controle da pressão arterial, como o núcleo paraventricular do hipotálamo (PVN) bem como, que o treinamento aeróbio é uma conduta eficaz em reverter muitos dos efeitos deletérios da hipertensão. Desta forma, é nossa hipótese de trabalho que inflamassomas NLRP3 possam estar ativados na hipertensão e que o treinamento aeróbio corrija muitos déficits do controle cardiovascular por modular a resposta imune inata. Em ratos machos adultos Wistar Kyoto e ratos espontaneamente hipertensos (SHR), foram feitas análises de parâmetros cardiovasculares, além da expressão gênica dos constituintes do inflamassoma e citocinas, e proteica de citocinas no PVN de animais sedentários ou treinados por quatro semanas de ambas as linhagens. Nossos resultados mostram que animais hipertensos se caracterizam por valores aumentados de pressão arterial e frequência cardíaca de repouso, associados à disfunção autonômica ao coração e vasos. SHR apresentam inflamação no PVN, o que foi evidenciado pelo elevado perfil de expressão dos constituintes da via do inflamassoma NLRP3, TLR-4 e citocinas pró-inflamatórias. O treinamento aeróbio de baixa a moderada intensidade reduziu a expressão gênica desses parâmetros e proteica de citocinas pró-inflamatórias, bem como a disfunção autonômica, além de determinar a instalação da bradicardia de repouso. Nossos dados sugerem que a elevada expressão de citocinas pró-inflamatórias no PVN de SHR seja devido à ativação do inflamassoma NLRP3, numa via dependente de TLR-4. Nosso trabalho é o primeiro a investigar o papel de inflamassomas em regiões autonômicas em hipertensos, bem como o efeito do treinamento físico sobre este perfil inflamatório. / The inflammatory process can be triggered by the presence of pathogens (PAMPs) or damaged-associated molecules pattern (DAMPs), this danger signals are recognized by immune system via pattern recognition receptors (PRR) in membrane or in the cytoplasm cells. The activation of NOD like receptors in the cytoplasm can trigger the formation of protein complex called inflammasomes, such as the NLRP3 inflammasome, which is comprised of three basics structures: NOD like receptor, adaptor protein and pro-caspase-1. This complex can induce the proteolytic maturation of pro-inflammatory cytokines (PICs), like interleukin 1 beta (IL-1b) and IL18, which are then released into the extracellular medium. It is known that hypertension is an inflammatory disease associated with DAMPs in blood pressure controlling areas in hypothalamus, like paraventricular nucleus (PVN) and that aerobic training can reverse the deleterious effects of hypertension. So, is our hypothesis that NLRP3 inflammasomes can be active in hypertension and that the exercise training restored the cardiovascular deficits by modulate the innate response. In adult male rats Wistar Kyoto and spontaneously hypertensive rats (SHR), cardiovascular parameters, gene expression of inflammasomes members or cytokines and cytokines protein contents were measured in the PVN of sedentary or four weeks trained groups from both lineages. Our results show that hypertensive animals are characterized by enhanced blood pressure and resting heart rate values, associated with autonomic dysfunction to heart e vessels. SHR had PVN inflammation, what is evidenced by the enhanced inflammasome NLRP3, TLR-4 and PICs expression in this area. The aerobic training with low or moderate intensity reduce the gene expression of these parameters and the protein expression of PICs, as well as, the autonomic dysfunction, in addition to determining the installation of resting bradycardia. Our data suggest that the elevated expression of PICs in SHR PVN is due to the activation of NLRP3 inflammasomes, in a TLR-4 dependent pathway. Our work is the first to investigate the role of inflammasomes in autonomic brain areas in hypertensive rats, as well as, the effect of exercise training on this inflammatory profile.

Inflammasome signaling during infections results in cell death and processing and secretion of cytokines from the IL-1 family, which facilitates control over an infection. Pseudomonas aeruginosa and Salmonella Typhimurium are opportunistic bacterial pathogens which may induce acute infections and activate various innate immune signaling pathways, including inflammasomes. However, under favourable conditions these pathogens may evade immune clearance resulting in the establishment of a chronic infection. In this study, I evaluated the modulation of host inflammasome signaling induced by P. aeruginosa and S. Typhimurium during chronic infections. I used a collection of P. aeruginosa clinical isolates obtained from the sputum of cystic fibrosis patients collected during stable and exacerbation periods of disease. I demonstrated that the majority of isolates displayed poor inflammasome signaling and only a small proportion of isolates retained their ability to induce inflammasome activation, which may be associated with pulmonary exacerbations in cystic fibrosis. Sequencing and bioinformatics revealed genetic variations within the type III and type VI secretion systems of P. aeruginosa. While an inactivation of the type III secretion system is expected to impair inflammasome signaling, my results indicate that the type VI secretion system inhibits inflammasome signaling in eukaryotic cells. Due to the lack of chronic animal models for P. aeruginosa, I utilized a murine model of chronic S. Typhimurium infection to assess the modulation of inflammasome signaling throughout the course of a chronic infection. I observed that S. Typhimurium isolated during the acute phase of infection displayed an increased potential to activate inflammasome signaling and this ability progressively declined during the chronic phase of infection. This reduction in inflammasome activation was associated with reduced expression of bacterial virulence factors, such as flagella and the type III secretion system, and was dependent on the NLRP3 inflammasome. Overall, these results reveal that the expression of virulence factors is modulated during chronic bacterial infections, which results in a reduction of inflammasome activation leading to co-survival of the pathogen and host.

L'inflammasome est une plateforme moléculaire composée d'un récepteur de l'immunité innée tels que NLRP3 ou NLRP1b, de la protéine adaptatrice ASC et de la caspase-1. Il joue un rôle essentiel dans le déclenchement de la réponse inflammatoire via l'activation de la caspase-1 qui mène à la sécrétion de cytokines pro-inflammatoires telle que l'IL-1ß. Dans un premier axe de recherche, nous avons mis en évidence que les macrophages de souris déficientes pour ASC traités avec l'activateur de l'inflammasome NLRP1b, la toxine létale de bacillus anthracis, étaient capables de sécréter la forme mature de l'IL-1ß en absence de clivage de la caspase-1 pourtant décrit comme un événement indispensable à son activité. En reconstituant des macrophages caspase-1 KO avec une forme mutante non-clivable de la caspase-1, nous avons démontré que la forme entière de la caspase-1 est capable d'induire la sécrétion d'IL-1ß en réponse à la toxine létale alors qu'elle n'est pas fonctionnelle au sein de l'inflammasome NLRP3.Dans un second axe de recherche, mon travail de thèse s'est intéressé à comprendre le rôle de l'inflammasome au cours de la progression tumorale. En effet, l'IL-1ß est une cytokine fréquemment retrouvée dans le microenvironnement tumorale mammaire suggérant donc l'activation de l'inflammasome au sein des tumeurs. Au moyen d'un modèle de tumeurs in vivo, nous avons montré que l'absence de la caspase-1 et de ASC dans les cellules immunitaires chez la souris conduit à une réduction de la croissance tumorale. De plus, l'absence de ces deux protéines provoque également un plus fort recrutement et une meilleure activité des cellules NK au sein des tumeurs.En plus d'identifier un nouveau mécanisme d'activation de la caspase-1, mon travail de thèse a permis de mettre en évidence le rôle de l'inflammasome dans l'altération des cellules NK au cours de la progression tumorale mammaire. Ces données permettent d'envisager l'inflammasome comme une cible thérapeutique dans le cancer / The inflammasome is a molecular platform composed of an innate immune receptor such as NLRP3 or NLRP1b, of the adaptor protein ASC and of the caspase-1. It plays an essential role in triggering inflammatory response through the activation of caspase-1 that leads to the secretion of pro-inflammatory cytokines such as IL-1ß.In a first research axis, we showed that ASC deficient mice macrophages treated with an NLRP1b inflammasome activator, the lethal toxin of Bacillus anthracis, were able to secrete the mature form of IL-1ß in absence of any cleavage of caspase-1 previously described as an essential event for its function. By reconstituting caspase-1 KO macrophages with an uncleavable mutant form of caspase-1, we showed that the entire form of the protein is able to induce IL-1ß secretion upon lethal toxin treatment but is nonfunctional upon NLRP3 inflammasome activation.In a second research axis, my PhD work focused on underlying the inflammasome role during tumor progression. Indeed, IL-1ß is frequently found within breast tumor microenvironment suggesting that inflammasome is activated in tumors. Using in vivo tumor model, we showed that the absence of caspase-1 of ASC in immune cells lead to a delay tumor growth. In addition, the absence of these two proteins also causes a stronger recruitment and an enchanced activity of NK cells within mammary tumors

Malignant Mesothelioma (MM) is a fatal disease with a low median survival between 8 to 12 months after diagnosis. MM has a long latency period (10-60 years), is causally related to asbestos exposure, and is refractory to all available modes of therapy. Despite the causal association between asbestos exposure and MM however, the mechanisms by which asbestos induces this deadly disease remain unclear. Chronic inflammation due to the presence of asbestos fibers is believed to play an important role in all aspects of MM pathogenesis, from development to progression and resistance. Chronic inflammation has been shown to promote dysregulated wound repair, fibrosis and epithelial to mesenchymal transition (EMT). One of the inflammatory pathways that asbestos activates is the inflammasome (a multiprotein scaffold that assembles in response to various stimuli to facilitate the activation of caspase-1), which has been implicated in several chronic inflammatory diseases and disorders. The nucleotide binding oligomerization domain (NOD) - like receptor containing a pyrin domain 3 (NLRP3) inflammasome, both as a whole or via its components [NLRP3, apoptosis related speck-like protein containing a CARD (caspase activating and recruitment domain) (ASC) and caspase-1] as well as its products, IL-1β and IL-18, has been implicated in the development of EMT during chronic inflammation. Asbestos fibers, especially the amphiboles, are non-biodegradable and thus persist in tissues of the body for years after exposure. In mesothelial cells, the squamous epithelial-like cells that line the serosal cavities of the body, from which MM originates, asbestos chronically activates the NLRP3 inflammasome. Asbestos also activates the NLRP3 inflammasome in human macrophages that can lead to the establishment of a chronic inflammation environment. We therefore hypothesized that asbestos dependent regulation of the inflammasome played a role in mesothelial to fibroblastic transition to facilitate eventual neoplastic transformation of the mesothelial cells. Using in vitro models, siRNA knockdown approaches as well as in vivo models of asbestos exposure utilizing inflammasome component knockout mice, we demonstrate that asbestos-induced reactive oxygen species generation modulates the redox state of the endogenous antioxidant, thioredoxin, causing its dissociation from thioredoxin interacting protein to promote activation of the inflammasome. We also show that the inflammasome plays a role in asbestos-induced mesothelial to fibroblastic transition (MFT) (a form of EMT occurring in the mesothelial cells) both in vitro and in vivo with a requirement for caspase-1 in vivo to promote thickening of the submesothelium. Through our studies, we have identified tissue factor pathway inhibitor 2 (TFPI2) and fibroblast growth factor 2 (FGF2) as molecules that are upregulated in response to asbestos exposure with potential roles in the progression of asbestos-induced MFT. There is a dearth of diagnostic biomarkers that enable early detection of MM, thus with further studies these two molecules could be explored as biomarkers of asbestos exposure/disease progression. TFPI2 levels were downregulated in response to blockage of IL-1β signaling and thus could be harnessed as a potential marker for therapy efficiency with further studies.

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.