AIM2 (absence in melanoma 2) can sense foreign cytosolic double-stranded DNA (dsDNA) through the dsDNA binding HIN-200 domain at the C-terminus. Once dsDNA is bound to HIN-200 domain, AIM2 can activate the AIM2 inflammasome, resulting in maturation of pro-interleukin-1β by activation of caspase-1. To investigate the mechanism of DNA binding, HIN-200 domain of mouse AIM2 bound to 15bp, 18bp, 20bp and 30bp dsDNAs were purified and crystallized. Diffraction data for four different crystals were collected to about 4Å resolution. Interestingly, all the crystals were in the same cubic space group I23 or I213 with almost same unit-cell parameters. Mutagenesis of HIN-200 domain of mouse AIM2 and DNA binding studies revealed amino acid residues involved in DNA binding. These residues were compared with dsDNA binding residues identified from the structure of HIN-200 domains of human AIM2, which suggested mouse AIM2 uses a similar dsDNA binding surface as human AIM2. Polynucleotidyl transferase (PNT1) was discovered in the proteomics study of Argonaute10 (AGO10). AGO10 was known as a key regulator of shoot apical meristem (SAM) maintenance in Arabidopsis. A recent study reported that AGO10 can regulate the developmental processes in SAM by repressing two miRNAs, miR166/165, and bind to these two miRNAs. Sequence analysis of PNT1 showed that it belongs to DnaQ-like 3'-5' exonuclease family, and hypothesized to be involved in RNA regulation with AGO10. PNT1 was purified and strong 3´-5´ exonuclease activity was revealed by activity test. Purified protein was crystallized, and the solved structure showed hexameric ring formation. Investigation of the crystal structure of PNT1 revealed presumed active site which was found from other 3´-5´ exonuclease homologues. Through mutagenesis of four residues in the presumed active site, Glu52 was identified as a key residue for its 3´-5´ exonuclease activity.

Leishmania amazonensis é um parasito endêmico no Brasil, que ao infectar humanos, pode causar a leishmaniose cutânea. Cerca de 12 milhões de pessoas estão infectadas com parasitos do gênero Leishmania em 98 países, e anualmente, 350 milhões de indivíduos estão em risco de contrair esta doença. Enquanto o papel da imunidade adaptativa já foi extensivamente investigado, em modelos de infecção com Leishmania spp., os mecanismos de alguns receptores da imunidade inata, e principalmente dos citosólicos, como é o caso do AIM2, permanecem obscuros. Recentemente, o nosso grupo descreveu a importância do inflamassoma de NLRP3 no controle da infecção por L. amazonensis, o qual é mediado pela produção de óxido nítrico dependente da sinalização de IL-1R. Neste trabalho, foi demonstrado que a deficiência para IL-1R confere uma maior susceptibilidade a este parasito do que a deficiência para NLRP3. Sugerindo que outros inflamassomas, que operam via IL- 1R, possam estar envolvidos na resposta contra este patógeno. Dentre estes, o AIM2 destacase como sendo um receptor de DNA citosólico que mediante a sua ativação promove a secreção de citocinas pro-inflamatórias e piroptose. Neste contexto, nós demonstramos que o AIM2 promove o controle da infecção in vitro por L. amazonensis em macrófagos, e que estas células requerem este sensor de DNA para a secreção de IL-1?, expressão da enzima NOS2 e produção de óxido nítrico. Adicionalmente, também observamos que AIM2 promove resistência à infecção por L. amazonensis in vivo, contribuindo para o desenvolvimento de uma resposta Th1. Por fim, também demonstramos que o inflamassoma de AIM2 atua na produção de IL-1? e IL-1?, por células dendríticas, assim como na expressão de CD86 e CD40 e na secreção de IL-12 por estas células. Desta forma, foi demonstrado neste trabalho que o AIM2, ao ativar células da imunidade inata e adaptativa, promove o controle direto ou indireto da infecção por L. amazonensis. / Leishmania amazonensis is an endemic parasite in Brazil that can cause the cutaneous leishmaniasis in humans. Annually, around 12 million people are infected with Leishmania spp. parasites, in 98 countries. In addition, 350 million of individuals are on risk to develop this disease. While the role of the adaptive immune response was already extensively evaluated, on models of infection with Leishmania spp., the mechanisms of some innate immune receptors, and mainly the cytosolic ones, such as AIM2, remain unclear. Recently, our group described the importance of the NLRP3 inflammasome to the control of L. amazonensis mediated by NO, which is dependent of the IL-1R signaling. In this work, it was also reported that the Il-1r deficiency displayed a higher susceptibility to L. amazonensis infection in comparison to Nlrp3-deficient mice. Suggesting that other inflammasomes, which may operate through IL-1R, might be involved in the response to this pathogen. Among these, the AIM2 highlights as a cytosolic DNA sensor, which, after its activation, promotes the secretion of proinflammatory cytokines and pyroptosis. On this context, we demonstrated that AIM2 promotes the in vitro control of L. amazonensis in macrophages, and that these cells require this DNA sensor for the IL-1? secretion, NOS2 expression and NO production. In addition, we also observed that AIM2 promotes the in vivo resistance to L. amazonensis infection, contributing for the development of a Th1 response. Finally, we also demonstrated that AIM2 acts on the production of IL-1? and IL-1?, by dendritic cells, as well as on the CD86 and CD40 expression and IL-12 secretion by these cells. Therefore, in this work, we showed that the AIM2, leading to the activation of innate and adaptive immune cells, promotes the direct or indirect control of L. amazonensis infection.

Leishmania amazonensis é um parasito endêmico no Brasil, que ao infectar humanos, pode causar a leishmaniose cutânea. Cerca de 12 milhões de pessoas estão infectadas com parasitos do gênero Leishmania em 98 países, e anualmente, 350 milhões de indivíduos estão em risco de contrair esta doença. Enquanto o papel da imunidade adaptativa já foi extensivamente investigado, em modelos de infecção com Leishmania spp., os mecanismos de alguns receptores da imunidade inata, e principalmente dos citosólicos, como é o caso do AIM2, permanecem obscuros. Recentemente, o nosso grupo descreveu a importância do inflamassoma de NLRP3 no controle da infecção por L. amazonensis, o qual é mediado pela produção de óxido nítrico dependente da sinalização de IL-1R. Neste trabalho, foi demonstrado que a deficiência para IL-1R confere uma maior susceptibilidade a este parasito do que a deficiência para NLRP3. Sugerindo que outros inflamassomas, que operam via IL- 1R, possam estar envolvidos na resposta contra este patógeno. Dentre estes, o AIM2 destacase como sendo um receptor de DNA citosólico que mediante a sua ativação promove a secreção de citocinas pro-inflamatórias e piroptose. Neste contexto, nós demonstramos que o AIM2 promove o controle da infecção in vitro por L. amazonensis em macrófagos, e que estas células requerem este sensor de DNA para a secreção de IL-1?, expressão da enzima NOS2 e produção de óxido nítrico. Adicionalmente, também observamos que AIM2 promove resistência à infecção por L. amazonensis in vivo, contribuindo para o desenvolvimento de uma resposta Th1. Por fim, também demonstramos que o inflamassoma de AIM2 atua na produção de IL-1? e IL-1?, por células dendríticas, assim como na expressão de CD86 e CD40 e na secreção de IL-12 por estas células. Desta forma, foi demonstrado neste trabalho que o AIM2, ao ativar células da imunidade inata e adaptativa, promove o controle direto ou indireto da infecção por L. amazonensis. / Leishmania amazonensis is an endemic parasite in Brazil that can cause the cutaneous leishmaniasis in humans. Annually, around 12 million people are infected with Leishmania spp. parasites, in 98 countries. In addition, 350 million of individuals are on risk to develop this disease. While the role of the adaptive immune response was already extensively evaluated, on models of infection with Leishmania spp., the mechanisms of some innate immune receptors, and mainly the cytosolic ones, such as AIM2, remain unclear. Recently, our group described the importance of the NLRP3 inflammasome to the control of L. amazonensis mediated by NO, which is dependent of the IL-1R signaling. In this work, it was also reported that the Il-1r deficiency displayed a higher susceptibility to L. amazonensis infection in comparison to Nlrp3-deficient mice. Suggesting that other inflammasomes, which may operate through IL-1R, might be involved in the response to this pathogen. Among these, the AIM2 highlights as a cytosolic DNA sensor, which, after its activation, promotes the secretion of proinflammatory cytokines and pyroptosis. On this context, we demonstrated that AIM2 promotes the in vitro control of L. amazonensis in macrophages, and that these cells require this DNA sensor for the IL-1? secretion, NOS2 expression and NO production. In addition, we also observed that AIM2 promotes the in vivo resistance to L. amazonensis infection, contributing for the development of a Th1 response. Finally, we also demonstrated that AIM2 acts on the production of IL-1? and IL-1?, by dendritic cells, as well as on the CD86 and CD40 expression and IL-12 secretion by these cells. Therefore, in this work, we showed that the AIM2, leading to the activation of innate and adaptive immune cells, promotes the direct or indirect control of L. amazonensis infection.

Inflammasome sind große intrazelluläre Multiproteinkomplexe und stellen einen wichtigen Bestandteil des angeborenen Immunsystems dar. Sie werden durch eine Vielzahl mikrobieller Moleküle, Gefahrensignale und kristalliner Substanzen aktiviert und führen zur Produktion von reifem IL-1β. In dieser Arbeit wurde der Fokus auf zwei Vertreter dieser Inflammasome gelegt, dem AIM2 und NLRP3 Inflammasom. Ersteres wird über intrazytoplasmatische DNA aktiviert und Defekte in seiner Regulation sind beispielsweise pathogenetisch relevant bei der chronischen Entzündung im Rahmen einer Psoriasis (Dombrowski et al. 2011) oder bei der Entstehung von Kolonkarzinomen mit Mikrosatelliteninstabilität (Woerner et al. 2007). Das NLRP3 Inflammasom kann durch unterschiedlichste Substanzen, wie z.B. Cholesterolkristalle, ATP, SDS oder Uratkristalle aktiviert werden. Pathogenetisch von Bedeutung ist eine Fehlregulation u.a. bei der Entstehung von CEDs. Ziel dieser Arbeit war es, Kolonadenom und –karzinomzelllinen auf die Induzierbarkeit des AIM2 und NLRP3 Inflammasoms zu untersuchen. Zu diesem Zweck wurden die Zelllinien entsprechend der Inflammasom-typischen Signalwege stimuliert, bzw. mit dsDNA transfiziert, und anschließend mittels RT-qPCR, ELISA und Western Blot die AIM2- und/oder IL-1β Genexpression, sowie die IL-1β Proteinsekretion bestimmt. In den untersuchten Darmzelllinien konnte unter den gewählten Versuchsmodalitäten weder eine funktionelle AIM2, noch eine funktionelle NLRP3 Inflammasomaktivierung nachgewiesen werden. Ein möglicher Grund hierfür könnte das Fehlen von für die Signalkaskade wichtigen Proteinen in den Kolonzelllinien sein. Dieses könnte erklärt werden durch die Überlegung, dass sich die verwendeten Kolonadenom- und karzinomzelllinien im Vergleich zu normalen Kolonzellen in einem zu stark entdifferenzierten Zustand befanden und somit zur Inflammasomaktivierung nicht mehr in der Lage waren. Vielleicht bedarf es auch anderer Zytokinstimulations- bzw. Transfektionszeiten, um eine IL-1β Sekretion in den Kolonzelllinien zu induzieren. / Inflammasomes are intracellular multiprotein complexes that play an important role in the response of the innate immune system. Aim of this work was to investigate how colon adenoma cell lines and colon carcinoma cell lines react to stimulation with danger associated molecular patterns and transfection of double-stranded DNA and whether oligomerisation and activation of the AIM2 and NLRP3 Infalmmasome with subsequent IL-1 beta release is performed.

The innate immune response to pathogens by the host is dependent upon the interplay of both pathogen and host intrinsic factors. Nucleotide-binding domain leucine-rich repeat containing (NLR) and pyrin and HIN200 domain containing (PYHIN) proteins are intracellular sensors of damage-associated and pathogen-associated molecular patterns. The studies presented here focus on the PYHIN molecule, AIM2, and the NLR, NLRP12, and the importance of bacteria- and host-associated proteins in the coordination of the innate immune response to the Gram-negative pathogen Fracisella tularensis. We have found that several genes expressed by F. tularensis encode for proteins that, when disrupted, cause the bacteria to trigger hyper- or hypoactivation of the AIM2 inflammasome. Bacteria with a mutation in FTL_0724, which hyperactivates the AIM2 inflammasome, are highly attenuated in a mouse model of infection, and induce robust caspase-1 processing and secretion of IL-1β by bone marrow derived macrophages (BMDMs). In contrast the hypoactivating mutant of F. tularensis, FTL0699, reduces IL-1β secretion by BMDMs and remains virulent in in vivo models of infection. We have also investigated the role of host-expressed NLRs in F. tularensis infection models. We have found that NLRP12 is important in the coordination of the innate immune response to F. tularensis through the modulation of CXCL1 production. We hypothesized that decreased CXCL1 production by Nlrp12-deficient mice was in turn responsible for the diminished recruitment of neutrophils in to the lungs of Nlrp12-deficient mice following intranasal challenge with F. tularensis. Nlrp12-deficient mice were found to be highly susceptible to infection with F. tularensis, and succumbed to infection at a much higher rate than wild-type mice. Taken together these data demonstrate that a number of pathogen and host factors can play critical roles in the outcome F. tularensis infections.

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.

L'inflammasome est une voie de signalisation du système immunitaire inné impliquée dans la lutte contre les pathogènes et notamment dans la réponse aux infections bactérienne. L'activation de l'inflammasome entraine la sécrétion de cytokines pro-inflammatoires et une mort cellulaire caspase-1 dépendante. Des dérégulations de l'inflammasome conduisent aussi à des syndromes auto-inflammatoires graves ; il est donc essentiel de mieux comprendre sa régulation. Francisella tularensis est une bactérie intracellulaire facultative responsable de la tularémie. Son pouvoir pathogène est lié à sa capacité à s'échapper rapidement de son phagosome. Le système de surveillance du macrophage détecte la présence de F. tularensis via l'inflammasome AIM2. La détection de l'ADN bactérien induit la formation d'un large complexe composé de AIM2, le récepteur, d'ASC, l'adaptateur et de caspase-1, l'effecteur ; ce complexe forme un speck visible dans la cellule. Nous avons utilisé l'infection par F. tularensis de macrophages primaires murins pour étudier la régulation de l'inflammasome AIM2 dans un contexte physiologique. Nous avons ainsi identifié une boucle de rétrocontrôle, médiée par la caspase-1, qui régule négativement la formation/stabilité des specks AIM2. Nous avons étudié le rôle de facteurs vacuolaires et des espèces réactives de l'oxygène et de l'azote dans l'activation de l'inflammasome AIM2 lors de l'infection par Francisella. Nous avons ainsi mis en évidence le rôle clef des péroxynitrites dans cette activation. Nos résultats suggèrent que des décomposeurs catalytiques des péroxynitrites pourraient avoir un rôle thérapeutique dans les maladies liées à l'inflammasome / The inflammasome is an innate immune signaling pathway involved in the fight against pathogens. This pathway can also be activated by danger signals. Inflammasome activation induces the release of the pro-inflammatory cytokines IL-1b and IL-18 and cell death in a caspase-1 dependent manner. The inflammasome pathway is a key antibacterial pathway. Deregulation of the inflammasome pathway can lead to serious auto-inflammatory syndromes ; it is therefore critical to better understand inflammasome regulation. Francisella tularensis is a facultative intracellular bacterium responsible for tularemia. Its ability to cause disease is linked to its ability to rapidly escape from the phagosome into the host cytosol where it replicates. The macrophage surveillance system can detect F. tularensis presence in the cytosol through the AIM2 inflammasome. Recognition of DNA induces the formation of a large complex consisting of AIM2, the receptor; ASC, the adaptor and caspase-1, the effector; this complex is visible as a speck within the cell. We used F. tularensis infection of bone marrow derived macrophages to study the activation of the AIM2 inflammasome in a physiological context. We have identified a feedback loop, dependent on caspase-1, negatively regulating speck formation/stability. Then, we studied the role of vacuolar factors and reactive oxygen and nitrogen species in the AIM2 inflammasome activation during Francisella infection. We also described a key role for peroxynitrite in this activation. Our results suggest that catalytic decomposer of peroxynitrite may have a therapeutic potential in diseases linked to inflammasome

O diabetes tipo 1 (DM1) é uma doença autoimune caracterizada pela destruição das células ? presentes nas ilhotas pancreáticas por linfócitos T autorreativos, especialmente Th1 e Th17, levando o indivíduo a um estado de hiperglicemia. Embora existam diversos estudos que abordam a resposta imune adaptativa no contexto do DM1, poucos trabalhos tentaram elucidar o papel da resposta imune inata no desenvolvimento da doença. Neste contexto, observamos que camundongos WT pré-diabéticos possuem um aumento significativo na expressão gênica e proteica do receptor AIM2 e de moléculas relacionadas à sua via de ativação e sinalização (Caspase-1, IL-1? e IL-18) nos linfonodos pancreáticos (LNPs) e no íleo. Posteriormente, foi verificado que camundongos deficientes do receptor AIM2 tornaram-se mais suscetíveis ao DM1, comprovado por elevados níveis de glicose sanguínea e menor produção de insulina em relação aos animais selvagens (WT) após a administração com estreptozotocina (STZ). Tal suscetibilidade está relacionada a um processo de disbiose e aumento da translocação de bactérias da microbiota intestinal para os LNPs de camundongos AIM2-/-. De maneira interessante, o inflamassoma AIM2 foi ativado apenas na presença de DNA fecal de animais diabéticos, que possui uma microbiota em disbiose, uma vez que resultou na produção significativa da citocina IL-1?. Também foi constatado que a ativação do receptor AIM2 na mucosa intestinal regulou a expressão gênica e proteica de proteínas de junção celular, peptídeos antimicrobianos e mucinas, como forma de minimizar a translocação de bactérias da microbiota para os LNPs. Adicionalmente, foi visto que a ativação do receptor AIM2 contribui para a indução de células Th17 intestinais, para a migração de neutrófilos no intestino, assim como para a expressão das citocinas IL-23, IL-17 e IL-22 no íleo. Por fim, mostramos que o receptor AIM2 modulou negativamente a ativação de células dendríticas expressando TLR4 e TLR9, que correlacionou com o aumento de células Tc1 patogênicas nos LNPs. De forma geral, nossos resultados demonstram que a ativação do receptor AIM2 na mucosa intestinal desempenha um importante papel em controlar a homeostase da microbiota intestinal, manter a integridade da barreira intestinal, e consequentemente. / Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of ? cells present in the pancreatic islets by autoreactive T lymphocytes, especially Th1 and Th17, leading to a state of hyperglycemia. There are many studies that address the role of adaptive immune response, so only some studies have attempted to elucidate the role of the innate immune response in the context of T1D. In this regard, we observed that pre-diabetic WT mice have a significant increase in the gene and protein expression of the AIM2 receptor and in molecules related to its activation and signaling pathways (Caspase-1, IL- 1? and IL-18) in the pancreatic lymph nodes (PLNs) and in the ileum. Subsequently, it was verified that AIM2 receptor deficient mice became more susceptible to T1D, as proved by blood glucose levels and lower insulin production compared to wild-type mice (WT) after administration of streptozotocin (STZ). This susceptibility was related to a process of dysbiosis and increased translocation of bacteria from gut microbiota to PLNs in AIM2-/- mice. Interestingly, the AIM2 inflammasome was activated in the presence of fecal DNA from diabetic mice, which has a gut microbiota in dysbiosis, since resulted in significant production of IL-1?. It was found that activation of the AIM2 receptor in the intestinal mucosa regulated the gene and protein expression of tightjunction proteins, antimicrobial peptides and mucins in order to minimizing a bacterial translocation of the microbiota to the PLNs. In addition, it was seen that activation of the AIM2 receptor contributes to induction of intestinal Th17 cells, to neutrophil migration in the intestine, as well as for expression of IL-23, IL-17 and IL-22 cytokines in the ileum. Finally, we show that the AIM2 receptor negatively modulated the activation of dendritic cells expressing TLR4 and TLR9, which correlated with the increase of pathogenic Tc1 cells in the PLNs. In general, the results demonstrate that activation of the AIM2 receptor in the intestinal mucosa plays an important role in controlling the composition of gut microbiota homeostasis, maintaining the intestinal barrier function, and consequently reducing the bacterial translocation to the PLNs, conferring a protective effect to the immunopathogeny against to DM1.

O diabetes tipo 1 (DM1) é uma doença autoimune caracterizada pela destruição das células ? presentes nas ilhotas pancreáticas por linfócitos T autorreativos, especialmente Th1 e Th17, levando o indivíduo a um estado de hiperglicemia. Embora existam diversos estudos que abordam a resposta imune adaptativa no contexto do DM1, poucos trabalhos tentaram elucidar o papel da resposta imune inata no desenvolvimento da doença. Neste contexto, observamos que camundongos WT pré-diabéticos possuem um aumento significativo na expressão gênica e proteica do receptor AIM2 e de moléculas relacionadas à sua via de ativação e sinalização (Caspase-1, IL-1? e IL-18) nos linfonodos pancreáticos (LNPs) e no íleo. Posteriormente, foi verificado que camundongos deficientes do receptor AIM2 tornaram-se mais suscetíveis ao DM1, comprovado por elevados níveis de glicose sanguínea e menor produção de insulina em relação aos animais selvagens (WT) após a administração com estreptozotocina (STZ). Tal suscetibilidade está relacionada a um processo de disbiose e aumento da translocação de bactérias da microbiota intestinal para os LNPs de camundongos AIM2-/-. De maneira interessante, o inflamassoma AIM2 foi ativado apenas na presença de DNA fecal de animais diabéticos, que possui uma microbiota em disbiose, uma vez que resultou na produção significativa da citocina IL-1?. Também foi constatado que a ativação do receptor AIM2 na mucosa intestinal regulou a expressão gênica e proteica de proteínas de junção celular, peptídeos antimicrobianos e mucinas, como forma de minimizar a translocação de bactérias da microbiota para os LNPs. Adicionalmente, foi visto que a ativação do receptor AIM2 contribui para a indução de células Th17 intestinais, para a migração de neutrófilos no intestino, assim como para a expressão das citocinas IL-23, IL-17 e IL-22 no íleo. Por fim, mostramos que o receptor AIM2 modulou negativamente a ativação de células dendríticas expressando TLR4 e TLR9, que correlacionou com o aumento de células Tc1 patogênicas nos LNPs. De forma geral, nossos resultados demonstram que a ativação do receptor AIM2 na mucosa intestinal desempenha um importante papel em controlar a homeostase da microbiota intestinal, manter a integridade da barreira intestinal, e consequentemente. / Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of ? cells present in the pancreatic islets by autoreactive T lymphocytes, especially Th1 and Th17, leading to a state of hyperglycemia. There are many studies that address the role of adaptive immune response, so only some studies have attempted to elucidate the role of the innate immune response in the context of T1D. In this regard, we observed that pre-diabetic WT mice have a significant increase in the gene and protein expression of the AIM2 receptor and in molecules related to its activation and signaling pathways (Caspase-1, IL- 1? and IL-18) in the pancreatic lymph nodes (PLNs) and in the ileum. Subsequently, it was verified that AIM2 receptor deficient mice became more susceptible to T1D, as proved by blood glucose levels and lower insulin production compared to wild-type mice (WT) after administration of streptozotocin (STZ). This susceptibility was related to a process of dysbiosis and increased translocation of bacteria from gut microbiota to PLNs in AIM2-/- mice. Interestingly, the AIM2 inflammasome was activated in the presence of fecal DNA from diabetic mice, which has a gut microbiota in dysbiosis, since resulted in significant production of IL-1?. It was found that activation of the AIM2 receptor in the intestinal mucosa regulated the gene and protein expression of tightjunction proteins, antimicrobial peptides and mucins in order to minimizing a bacterial translocation of the microbiota to the PLNs. In addition, it was seen that activation of the AIM2 receptor contributes to induction of intestinal Th17 cells, to neutrophil migration in the intestine, as well as for expression of IL-23, IL-17 and IL-22 cytokines in the ileum. Finally, we show that the AIM2 receptor negatively modulated the activation of dendritic cells expressing TLR4 and TLR9, which correlated with the increase of pathogenic Tc1 cells in the PLNs. In general, the results demonstrate that activation of the AIM2 receptor in the intestinal mucosa plays an important role in controlling the composition of gut microbiota homeostasis, maintaining the intestinal barrier function, and consequently reducing the bacterial translocation to the PLNs, conferring a protective effect to the immunopathogeny against to DM1.

La inflamación hepática crónica es un fenómeno crucial en la patogénesis del carcinoma hepatocelular (HCC). En los últimos años, el proceso inflamatorio desencadenado por la activación del complejo del inflamasoma se ha asociado con diferentes enfermedades hepáticas. Sin embargo, su papel en el desarrollo de HCC está prácticamente sin explorar. En esta tesis, se estudia y analiza el impacto de diferentes componentes del inflamasoma, incluyendo la proteína caspasa 1 y los receptores AIM2 (del inglés absent in melanoma 2) y NLRP3 (del inglés NOD-like receptor family pyrin domain containing 3), en el desarrollo de HCC inducido por dietilnitrosamina (DEN) en ratones. La inactivación genética de AIM2, pero no de NLRP3, reduce el daño hepático y el desarrollo de HCC en este modelo. La deficiencia de AIM2 está asociada a una disminución en la activación del inflamasoma, la inflamación hepática y la respuesta proliferativa durante la fase de iniciación del HCC. También se identificó que AIM2 está altamente expresado en células de Kupffer, y que la producción de interleucina (IL)-1β, mediada por AIM2, en estas células se ve aumentada tras el daño hepático inducido por DEN. Además, la inhibición de IL-1β resultó en menor daño hepático, inflamación y proliferación durante la fase inicial de hepatocarcinogénesis; sin embargo, esta inhibición no fue suficiente para frenar la progresión de tumores hepáticos ya establecidos. Así, los datos de esta tesis indican que AIM2 promueve el proceso inflamatorio durante el daño hepático carcinogénico, contribuyendo al desarrollo genotóxico de HCC en ratones y, por tanto, reconociendo AIM2 como una posible diana terapéutica en esta enfermedad.