Acute myocarditis is an acute inflammatory syndrome characterized by myocardial damage and dysfunction often due to a viral infection followed by a variable development over time. There are currently no specific treatments and standard treatments for heart failure are generally applied. The inflammasome is a recently identified macromolecular structure that occupies a central role in the amplification of the inflammatory response and promotion of cell death during acute and chronic infections. We hypothesized the formation of the inflammasome in acute myocarditis. To investigate, samples of patients were collected from the Cardiomyopathy Registry in Trieste, with 12 cases of biopsy-proven myocarditis and 11 cases of autopsy-proven myocarditis stained for major components of the inflammasome through immunofluorescence; 10 of the 12 (83.3%) biopsy cases and 8 of the 11 (72.7%) autopsy cases presented formation of the inflammasome in a variety of cells including resident cells (i.e. cardiomyocytes, endothelial cells, fibroblasts) and infiltrating cells (i.e. leukocytes) while varying in intensity and distribution. Control samples of 5 subjects not presenting with any acute cardiac events showed no formation of the inflammasome. While further studies should look to elucidate the correlation of inflammasome-formation and progression of disease, this finding paves the way for further insight into the pathophysiology of acute myocarditis.

Em diferentes mopdelos, o aumento dos níveis séricos de glicose pode ativar cascatas de sinalização envolvendo receptores semelhantes a Toll, o fator de transcrição NF-kB, moléculas da familia NLRP, ASC e caspase-1, que culminam na produção, ativação e liberação de moléculas inflamatórias. Investigamos o perfil inflamatório sérico e placentário em gestantes com distúrbios glicêmicos e a possível participação da via do inflamassoma NLRP1 e NRLP3 para o estabelecimento desse perfil. Para avaliar o papel da hiperglicemia no estabelecimento desse perfil, a via do inflamassoma foi também estudada em explantes de vilos coriônicos tratados com 100, 200, 300 e 400 mg/dL de glicose. Nossos resultados mostraram aumento na ativação de NF-kB e da expressão de NLRP1, NLRP3, ASC e Caspase-1 nas placentas de gestantes hiperglicemicas e nas culturas tratadas com doses de 300 r 400 mg/dL de glicose. Esses dados sugerem que a ativação da via do inflamassoma na porção fetal da placenta contribui para o processo inflamatório nessas pacientes. / In different models, the increase in serum glucose levels can activate signaling cascades involving Toll-like receptors, the transcription factor NF-kB, NLRP proteins, ASC and caspase-1, which culminate in the production, activation and release of inflammatory molecules . We investigated the serum and placental inflammatory profile in pregnant women with glucose disorders and the possible participation of via the NLRP1 and NRLP3 inflammasome to establish this profile. To evaluate the role of hyperglycemia, molecules of the inflammasome pathway was also studied in explants of chorionic villi treated with 100, 200, 300 and 400 mg / dL glucose. Our results showed an increase in NF-kB activation and expression of NLRP1, NLRP3, ASC and Caspase-1 in placentas of pregnant hyperglycemic women and in cultures treated with 300 and 400 mg / dL glucose. These data suggest that the inflammasome activation of the fetal portion of the placenta contributes to the inflammatory process in these patients.

O reconhecimento eficiente do fungo Paracoccidioides brasiliensis pelos receptores do sistema imune inato do hospedeiro é essencial para a proteção contra a paracoccidioidomicose (PCM), micose sistêmica prevalente na América Latina. Diferente dos receptores do tipo Toll (TLRs),os receptores do tipo Nod-like (NLRs) são proteínas citoplasmáticas com capacidade de formar uma plataforma molecular denominada inflamassoma. Esta plataforma ativa caspase-1 e desencadeia a produção de IL-1? e IL-18. O inflamassoma de NLRC4 regula a resposta imune contra bactérias intracelulares através do reconhecimento de flagelina. No entanto, mesmo na ausência desta molécula, a bactéria Shigella flexneri é capaz de ativar o inflamassoma de NLRC4, o que sugere a existência de outros agonistas. Trabalhos recentes mostraram que o inflamassoma de NLRC4 tem papel importante no controle da infecção por Candida albicans. Contudo, sua participação na infecção por P. brasiliensis é uma incógnita. Portanto, o presente estudo teve como objetivo avaliar a participação do inflamassoma de NLRC4 na paracoccidioidomicose experimental. Os resultados obtidos demonstram que durante a infecção experimental por P. brasiliensis,macrófagos derivados de medula óssea (BMDM) provenientes de animais Nlrc4-/- produzem mais IL-1? do que BMDM de animais controles. Não foram detectadas diferenças significativas quanto aos níveis de TNF- ? em ambos os grupos. Para investigar o mecanismo responsável pelo aumento da produção de IL-1? por BMDM de camundongos Nlrc4-/-, as expressões do gene Nlrp3 e Nlrc4 foram avaliadas por qPCR durante, 0, 2, 6, 12, e 24 horas após a infecção. Comparado aos camundongos WT, BMDM de animais Nlrc4-/- exibiram um aumento na expressão de Nlrp3 durante as primeiras 12 horas de infecção. Surpreendentemente, 24 horas após a infecção, um aumento na expressão do gene de Nlrc4 foi detectado em BMDM de animais Nlrp3-/-, comparado ao controle. Estes resultados sugerem que o inflamassoma de NLRC4 reprime a atividade do inflamassoma de NLRP3, e consequentemente, anula a produção de IL-1? em macrófagos estimulados por P. brasiliensis. Além disso, camundongos Nlrc4-/- exibiram um aumento na produção de IL-1? e redução na produção de IL-18 aos7 dias após a infecção in vivo. Em contraste, a produção de IL-1? foi menor e a produção de IL-18 aumentou aos 30 dias após a infecção.Comparado ao grupo controle observamos menor carga fúngica, formação de granulomas bem definidos e compactos e redução na fibrose em pulmões de camundongos Nlrc4-/- aos 30 dias após infecção. Adicionalmente, observamos aumento na produção de citocinas do perfil Th1 (IFN-?, IL-12p40) e níveis reduzidos de IL-10. Em conclusão, estes dados mostram que o inflamassoma NLRC4 tem papel importante na suscetibilidade do hospedeiro durante a infecção por P. brasiliensis. / The efficient recognition of P. brasiliensis fungal cells by immune system receptors is essential for protection against paracoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America. Different from the Toll-like receptors (TLRs), NOD-like receptors (NLRs) are cytoplasmic proteins able to form the molecular platform, denominated inflammasome. This platform activates caspase-1 and triggers the production of IL-1? and IL-18. The NLRC4 inflammasome regulates the immune response against intracellular bacteria through recognition of flagellin. However, even in the absence of this structure, the bacteria Shigella flexneri activates the NLRC4 inflammasome, suggesting the existence of other agonists. It is known that NLRC4 control the Candida albicans infection. Therefore, in this study we evaluated the participation of NLRC4 inflammasome in experimental paracoccidioidomycosis. Our data demonstrated that Nlrc4-/- BMDMs produces more IL-1? than WT BMDMs during P. brasiliensis infection. No differences were detected regarding to TNF-? levels in both groups. To investigate the mechanism responsible to improvement of IL-1? production by Nlrc4-/- BMDMs, the nlrp3 and nlrc4 gene expression were evaluated by qPCR during 2, 6, 12 and 24 hours post infection. Compared to WT mice, the expression of nlrp3 was increased in Nlrc4-/- BMDMs during the first 12 hours of infection. Surprisingly, at 24 hour post infection and nlrc4 exhibited increased expression in Nlrp3-/- BMDMs, compared to WT controls. These data suggest that NLRC4 represses NLRP3 inflammasome activity, and consequently, abrogates the IL-1B production by P.brasiliensis estimuled macrophages. Furthermore, Nlrc4-/- mice exhibited an increased production of IL-1? and decreased production IL-18 at 7 days post infection in vivo. In contrast, there was a decrease in the production of IL-1? and increased production of IL-18 at 30 days post infection. Compared to controls, we observed lower fungal load, the formation of well-defined and compact lung granulomas, and decreased fibrosis in the lungs of Nlrc4-/- mice after 30 days post infection. In addition, we observed increased production of Th1 cytokine profile (IFN-?, IL-12p40) and reduced levels of IL-10. In conclusion, these data show that NLRC4 inflammasome play an important role on the host susceptibility during P. brasiliensis infection.

Orientador: James Venturini / Resumo: A paracoccidioidomicose (PCM) é micose sistêmica causada por fungos do gênero Paracoccidioides. As principais formas clínicas da doença são aguda/subaguda e crônica (FC), sendo que nessa última, a maioria dos pacientes desenvolvem fibrose pulmonar e enfisema. Estudos prévios demonstraram que pacientes FC, na forma ativa da doença, apresentam elevada produção de mediadores inflamatórios, incluindo a IL-1β. Essa citocina, diferente das demais, é produzidas por uma plataforma protéica intracelular denominada inflamassoma que pode ser ativadas por patógenos e sinais de dano do hospedeiro. Considerando-se que os mecanismos de ativação do inflamassoma em pacientes com PCM não são conhecidos, o presente estudo teve por objetivo determinar a expressão de genes envolvidos na ativação do inflamassoma e a produção de citocinas por monócitos e macrófagos alveolares de pacientes com PCM em diferentes momentos do tratamento antifúngico.Nossos resultados demonstram a ativação do NLRP3-inflamassoma, caracterizada pela elevada expressão de NLRP3, CASP1 e IL1B por monócitos. Esses achados corroboram a contribuição do NLRP3-inflamassoma na patogênese da PCM também em pacientes. / Abstract: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by species of the genus Paracoccidioides . The main clinical forms of the disease are acute/subacute (AF) and chronic (CF), and in the latter, most patients develop pulmonary fibrosis and emphysema. Previous studies showed that CF patients in active disease, exhibit elevated production of inflammatory mediators, including IL-1β. This cytokine, different from the others, are produced by an intracellular multiprotein platform called inflammasome that can be activated by pathogens and host signs of damage. Considering that the activation mechanisms of the inflammasome in patients with PCM are not know, this study aimed to determine the expression of genes involved in inflammasome activation and cytokine production by monocytes and alveolar macrophages from PCM patients at different times of antifungal treatment. Our results demonstrate the activation of the NLRP3 inflammasome-characterized by high expression of NLRP3, CASP1 and IL1B by monocytes. These findings corroborate the contribution of NRLP3-inflamassome in pathogenesis of PCM also in patients. / Mestre

The mechanisms by which the facultative intracellular pathogen Francisella tularensis is recognized by the innate immune system and the strategies that F. tularensis uses to avoid this recognition are not well understood. We have identified the basic components of the inflammasome that assemble in response to F. tularensis Live Vaccine Strain (LVS) challenge as containing the cysteine protease caspase-1, the adaptor protein ASC and the PYHIN molecule AIM2. We have also shown here that the nucleotide-binding domain leucine-rich repeat containing receptors (NLRs), NLRC4, NLRP3, NLRP6, NLRP10, and NLRP12 were not necessary for activation of caspase-1 and subsequent IL-1β secretion in response to challenge with F. tularensis LVS in vitro. In vivo, NLRC4, NLRP3, NLRP6, NLRP10, and NLRP12 did not appear to enhance survival. However, caspase-1- and ASC-deficient mice succumbed more rapidly to infection, indicating that the inflammasome played a role in defense against F. tularensis LVS. Additionally, we identified a gene with homology to Escherichia coli mviN, a putative lipid II flippase, that functions as a F. tularensis virulence factor. In vivo infection of mice with a F. tularensis LVS mviN transposon mutant (mviN::Tn5) resulted in improved host survival and decreased bacterial burdens compared to infection with wild-type F. tularensis LVS. Wild-type F. tularensis LVS and the mviN::Tn5 mutant replicated at a similar rate in both macrophages and liquid broth culture. Additionally, the ability to induce the production of TNF-α or IL-6 was also similar between WT F. tularensis and the mviN::Tn5 mutant. In contrast to the similar levels of production of IL-6 and TNF-α, the mviN mutant induced increased AIM2 inflammasome-dependent IL-1β secretion and cytotoxicity in macrophages compared to wild-type F. tularensis. The compromised in vivo virulence associated with the mutation of mviN was dependent upon inflammasome activation, as caspase-1- and ASC-deficient mice did not exhibit preferential survival following infection. These data show that F. tularensis LVS activation of the inflammasome is caspase-1-, ASC-, and AIM2-dependent. These data also identify mviN as a novel F. tularensis virulence factor that enables F. tularensis LVS to evade some AIM2 inflammasome activation.

Pattern recognition receptors coordinate innate immune responses by sensing infection or injury. Nucleotide-binding, leucine rich repeat, and pyrin domain-containing protein 3 (NLRP3) is a cytosolic PRR which perceives diverse pathogenic and sterile insults. NLRP3 orchestrates inflammatory signaling responses by forming inflammasomes with the adaptor protein apoptosis-associated speck like protein with a caspase recruitment domain (ASC) and the cysteine protease caspase-1. Assembly of the intracellular macromolecular inflammasome complex culminates in proximity-induced autocatalysis of caspase-1. Caspase-1 activation promotes cell death by pyroptosis and activation and secretion of proinflammatory cytokines interleukin (IL)-1β and IL-18. While NLRP3-mediated inflammation protects against bacterial, fungal, viral, and parasitic infections, aberrant NLRP3 activation is implicated in numerous inflammatory diseases and heritable syndromes. Mechanistically, inflammasome activation requires a preliminary NF-κB-activating priming step (signal 1) and a subsequent NLRP3-specific stimulus (signal 2). While there is enormous molecular diversity among NLRP3-specific agonists, this second signal appears to engage a common pathway involving cation flux. Furthermore, NLRP3 associates with mitochondria and mitochondrial damage is implicated in NLRP3 activation, although the precise role for mitochondria in inflammasome assembly remains controversial. We previously demonstrated that the mitochondrial phospholipid cardiolipin binds to NLRP3 and is critical for NLRP3 inflammasome activation. Here, we further investigated how mitochondria contribute to NLRP3 activation. We found that liposomes containing molar concentrations of cardiolipin that resemble mitochondrial cardiolipin levels can induce NLRP3-dependent caspase-1 autoactivation. Unexpectedly, we discovered that caspase-1 binds directly to cardiolipin, causing inflammasome-independent caspase-1 complex formation and autocatalysis at higher cardiolipin densities. Finding that caspase-1 and NLRP3 are independently capable of binding to cardiolipin, we more thoroughly examined the association of inflammasome components with mitochondria. Normally confined within mitochondrial inner membranes, cardiolipin relocates to outer membranes of stressed mitochondria. We found that reactive oxygen species (ROS) produced in response to signal 1 facilitate cardiolipin externalization to the outer membrane during priming. We also determined that this coincides with ROS-dependent recruitment of NLRP3 and caspase-1 to the outer membrane of mitochondria at priming. In contrast, we found that NLRP3 activation by the signal 2 agonist nigericin induces calcium-dependent recruitment of the adaptor ASC to mitochondria and caspase-1 activation. Finally, to determine what type of mitochondrial damage was necessary to promote NLRP3 inflammasome activation, we examined how different NLRP3 agonists affect mitochondria. We found substantial variability in the extent of mitochondrial damage induced among different NLRP3 agonists. Collectively, our findings illustrate that mitochondria serve as innate immune signaling platforms through multiple stages of NLRP3 inflammasome activation. Further, paralleling lipid A interactions with caspase-11, we have demonstrated that caspase-1 is capable of binding to the phospholipid cardiolipin.

Le laboratoire a établi un modèle de souris α6ΔIEC qui développe une inflammation chronique intestinale associée à la formation d’adénocarcinomes colorectaux. Ce modèle correspond à une délétion ciblée à l’épithélium intestinal de l’intégrine α6β4. Mon projet de thèse a consisté à définir les mécanismes qui influencent la transformation de lésions inflammatoires en adénocarcinomes. La caractérisation du modèle α6ΔIEC a permis de mettre en évidence plusieurs altérations : détachement de l’épithélium, régénération du tissu, prolifération, augmentation de la perméabilité intestinale, hypersécrétion du mucus, ségrégation anormale des bactéries, inflammation chronique et formation de tumeurs.Pour étudier la séquence et la cinétique des mécanismes, j’ai développé un modèle de souris inductible (α6ΔIECTAM). Cette lignée présente, deux semaines après l’invalidation de l’intégrine α6,les mêmes signes d’inflammation que les souris α6ΔIEC. Mon approche a consisté à dissocier les processus impliqués dans chacune des étapes-clés de la pathologie afin de définir la contribution respective de l’infection par les bactéries et du stress mécanique. / We generated a new mouse model, α6ΔIEC, in which the genetic ablation of α6 integrin from intestinal epithelial cells triggered the development of spontaneous colitis and colorectal cancer. My main goal was to define the mechanisms by which inflamed lesions degenerate into infiltrating adenocarcinomas. Loss of α6 integrin in this model resulted in epithelial barrier damage, enhanced permeability, altered mucus layers, abnormal bacterial segregation, chronic inflammation and tumor development.In order to define the sequence of events and the mechanisms involved at each stage of the disease, from inflamed to tumor lesions, I developed an inducible mouse model, α6ΔIECTAM, in which α6 integrin ablation was induced by tamoxifen treatment. This line recapitulates all aspects of inflammation observed in the α6ΔIEC model, as early as two weeks after tamoxifen treatment. In particular, I tried to define the respective contribution of infection by bacteria and mechanical stress during disease progression.

<p>Innate immunity has received impressive attention in the past decade owing to the discovery of the Toll like receptors (TLRs) and the NOD-like receptors (NLRs). While the TLRs specialize in fighting microbes at the cell surface, the NLRs complement by detecting and responding to intracellular microbes. Recently, the non-microbe sensing NLR called inflammasomes, have been identified, which senses metabolic stress as well as certain pathogenic microbes and elicits host’s inflammatory response. <strong></strong></p><p>The NLR, NALP3 (formerly known as cryopyrin) forms a large cytoplasmic complex called the ‘inflammasome’ when NALP3, activated by a stimuli, associates with the adaptor proteins ASC and CARD-8. This interaction leads to the activation of pro-inflammatory caspase-1 which subsequently results in the formation of Interleukin (IL)-1β and IL-18. Mutations in the gene encoding NALP3, termed <em>NLRP3</em> can lead to its constitutive activation resulting in an uncontrolled production of IL-1β. These mutations have been implicated in hereditary inflammatory diseases, often grouped under cryopyrin associated periodic syndromes (CAPS).</p><p>This thesis describes a patient with a long history of arthritis and antibiotic resistant fever, but without the typical symptoms of CAPS. The patient was found to be a heterozygous carrier of two common polymorphisms Q705K in <em>NLRP3 </em>and C10X in the <em>CARD-8</em>. Experimental studies showed elevated levels of caspase-1 and IL-1β in the patient, and a total clinical remission was achieved by IL-1β blockade. These two polymorphisms combined, were found to occur in approximately 4% of the control population, suggesting the possibility of a genetic predisposition for inflammation in these individuals. Therefore, a cohort of rheumatoid arthritis (RA) patients, where elevated IL-1β could be one of the reasons behind chronic inflammation, was investigated. We found that carrying the combined polymorphisms resulted in increased RA susceptibility and a more severe disease course. Hypothetically, this subgroup of patients might benefit from IL-1β blockade. Additional studies are warranted to elucidate the functional effects of the two polymorphisms and to determine whether they identify a subgroup of patients that could benefit from IL-1 targeted therapy. Given the structural similarity of NALP3 to other NALPs, the possibility of involvement of the alternative, homologous genes cannot be eliminated.</p>

Age-related macular degeneration (AMD) is a leading cause of visual impairment and blindness, affecting over 30 million people worldwide. It is characterized by the appearance of insoluble deposits known as drusen in the outer retina, between the retinal pigment epithelium (RPE) and Bruch's membrane. Drusen are heterogeneously composed of many compounds, including cholesterol, amyloid-β, and complement proteins. AMD also involves the accumulation of pigments collectively termed lipofuscin in RPE lysosomes. The underlying causes of AMD are unknown, but studies have implicated inflammatory processes in its pathogenesis.

Em diferentes mopdelos, o aumento dos níveis séricos de glicose pode ativar cascatas de sinalização envolvendo receptores semelhantes a Toll, o fator de transcrição NF-kB, moléculas da familia NLRP, ASC e caspase-1, que culminam na produção, ativação e liberação de moléculas inflamatórias. Investigamos o perfil inflamatório sérico e placentário em gestantes com distúrbios glicêmicos e a possível participação da via do inflamassoma NLRP1 e NRLP3 para o estabelecimento desse perfil. Para avaliar o papel da hiperglicemia no estabelecimento desse perfil, a via do inflamassoma foi também estudada em explantes de vilos coriônicos tratados com 100, 200, 300 e 400 mg/dL de glicose. Nossos resultados mostraram aumento na ativação de NF-kB e da expressão de NLRP1, NLRP3, ASC e Caspase-1 nas placentas de gestantes hiperglicemicas e nas culturas tratadas com doses de 300 r 400 mg/dL de glicose. Esses dados sugerem que a ativação da via do inflamassoma na porção fetal da placenta contribui para o processo inflamatório nessas pacientes. / In different models, the increase in serum glucose levels can activate signaling cascades involving Toll-like receptors, the transcription factor NF-kB, NLRP proteins, ASC and caspase-1, which culminate in the production, activation and release of inflammatory molecules . We investigated the serum and placental inflammatory profile in pregnant women with glucose disorders and the possible participation of via the NLRP1 and NRLP3 inflammasome to establish this profile. To evaluate the role of hyperglycemia, molecules of the inflammasome pathway was also studied in explants of chorionic villi treated with 100, 200, 300 and 400 mg / dL glucose. Our results showed an increase in NF-kB activation and expression of NLRP1, NLRP3, ASC and Caspase-1 in placentas of pregnant hyperglycemic women and in cultures treated with 300 and 400 mg / dL glucose. These data suggest that the inflammasome activation of the fetal portion of the placenta contributes to the inflammatory process in these patients.