O reconhecimento da flagelina é compartilhado pelo receptor transmembrânico TLR5 e citosólico NAIP5/NLRC4. Entretanto, pouco se sabe sobre os mecanismos efetores individuais induzidos a partir do reconhecimento extra e intracelular da flagelina. Aqui, nós demonstramos que macrófagos estimulados com a flagelina citosólica (FLA-BSDot) induziu a expressão de iNOS, enzima responsável pela produção do óxido nítrico (NO). A expressão de iNOS foi dependente do eixo NAIP5/NLRC4/caspase-1 e independente de IL-1β, IL-18 e MyD88, descartando a via de ativação dos TLRs. Ainda, esta via não requer a ativação do fator de transcrição IRF-1, mas envolve a ativação do NF-kB, assim como a clivagem da enzima PARP-1 (poly(ADP-ribose)polymerase-1). Por fim, avaliamos a relevância biológica desta via no controle das infecções por L. pneumophila e S. Typhimurium, dados que definem um mecanismo efetor adicional no controle de patógenos. / Recognition of flagellin is shared by transmembranic TLR5 and cytosolic NAIP5/NLRC4. However, little is known about the individual effector mechanisms induced by extra and intracellular flagellin. Here, we have demonstrated that cytosolic flagellin-stimulated macrophages (FLA-BSDot) induced iNOS expression, an enzyme responsible for the production of nitric oxide (NO). iNOS expression was dependent of the NAIP5/NLRC4/caspase-1 axis and independent of IL-1β, IL-18 and MyD88, discarding TLRs signaling pathway. Still, this pathway do not require the activation of IRF-1 transcriptional factor, but involves NF-kB activation as well as the cleavage of the enzyme, PARP-1 (poly(ADP-ribose)polymerase-1). Finally, we have evaluated the biological relevance of this pathway in the control of the infections by L. pneumophila e S. Typhimurium, which define an additional effector mechanism to the control of pathogens.

Orientador: Alexandrina Sartori / Resumo: A esclerose múltipla (EM) é uma doença inflamatória crônica e desmielinizante do sistema nervoso central (SNC). A imunopatogênese da EM envolve linfócitos Th1, Th17 e Tc autorreativos e ativação de macrófagos e microglia, os quais liberam vários mediadores pró-inflamatórios e radicais livres. Não há cura para a EM e as terapias disponíveis desencadeiam efeitos colaterais. Neste contexto, testamos o potencial terapêutico da cisteína selenizada (CS), um produto em desenvolvimento pela empresa Biorigin, no controle da encefalomielite autoimune experimental (EAE). O potencial profilático/terapêutico foi testado por administração de CS durante 30 dias (14 dias antes seguido de 16 dias pós-indução da EAE) e o potencial terapêutico por administração da CS a partir de 1 ou 7 dias após a indução da doença. Os 3 procedimentos reduziram escore clínico e incidência da EAE. A estratégia profilática/terapêutica reduziu discretamente a expressão de componentes do inflamassoma NLRP3 e o percentual de macrófagos e microglia ativada no SNC. As duas estratégias terapêuticas determinaram redução significativa no escore clínico, no percentual de macrófagos e microglia ativada e na expressão de MHCII por estas células. A terapia com CS também reduziu a ativação do inflamassoma NLRP3 e a expressão de mRNA para iNOS e CX3CR1.A redução na percentagem de células dendríticas ativadas (DCs) (CD11b+MHCII+) e o aumento no percentual de DCs tolerogênicas (CD11b-CD103+) sugere o estabelecimento deum perfil ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Its immunopathogenesis is complex and involves the interplay of distinct autoreactive T lymphocyte subsets and innate immune cells. The persistent inflammation observed in MS patients is related to oxidative stress and inflammasome activation which have been recently implicated in both, demyelination and axonal damage processes. Since the development of new therapeutic procedures remains necessary, we evaluated the efficacy of an antioxidant product to control experimental autoimmune encephalomyelitis (EAE) development. The selenized cysteine (SC) is an organic selenium, developed by a Brazilian company (Biorigin). For prophylactictherapeutic strategy, oral treatment with SC (45µg of selenium/dose) started 14 days before EAE induction and was daily extended until the acute phase of the disease. The precocious and delayed therapies have begun 1 or 7 days after MOG administration. The three therapeutic approaches reduced EAE incidence and clinical manifestations. Prophylactic-therapeutic strategy attenuated NLRP3 inflammasome activation and reduced the number of macrophages and activated microglia in the CNS. Precocious and delayed therapies were efficient in controlling EAE symptoms, reducing the percentage of macrophages and activated microglia and the MHCII expression. This protection was concomitant by less NLRP3 inflammasome activation and lower expression of mRNA for ... (Complete abstract click electronic access below) / Mestre

Inflammation is the response of the body to injury or threats. Immune cells such as macrophages have a crucial role in controlling and regulating this process. The potent pro-inflammatory cytokines interleukin (IL)-1beta and IL-18 are synthesized by macrophages as inactive precursors which activation follows a unique mechanism involving the activation of caspase-1 by assembly of a macromolecular complex called the inflammasome. However, the assembly of the inflammasome is a double-edged sword. Although inflammasome activation is necessary for a normal inflammatory response, its malfunction can trigger and contribute to inflammatory disorders such as gout, arthritis or cryopirin-associated periodic syndromes (CAPS). The fine regulation of this mechanism and the cell death associated with it is key for the outcome of the inflammatory process. In this thesis we tackle three aspects of the mechanisms and consequences of inflammasome activation. First we studied the role of the deubiquitinases USP7 and USP47 in inflammasome activation. We showed how USP7 and USP47 activity is increased upon danger signals and how that is necessary for the assembly of the inflammasome. We also pointed how their inhibition dampens the deubiquitination of ASC using a BRET2 assay. Second we examined how the activity of IL-18 is controlled by the release of IL-18BP during inflammasome activation. We showed how IL-18BP release increased upon membrane permeabilization and pyroptosis. This release happens in other types of lytic cell (necrosis and necroptosis) death but not in apoptosis. Finally, we showed that this IL-18BP acute release dampens IL-18 signalling and IFN gamma production by PBMCs. These results demonstrate a novel mechanism by which lytic cell death could dampen IL-18-driven inflammation and highlights a key role for IL-18BP in inflammasome related diseases. Finally we studied the role of inflammasome in lung epithelial cells as a model to investigate lung infections. We found that lung epithelial cells lack NLRP3 inflammasome activity and components, but express caspase-4 and caspase-8 which could have a role in the release of IL-1 family of cytokines. To conclude we showed how lung epithelial release IL-18 upon Aspergillus fumigatus infection. Overall, this thesis enhances our understanding of the mechanisms that control IL-1beta and IL-18 activity by regulating inflammasome activation and by understanding the consequences of its activation.

Legionella pneumophila, o agente etiológico da doença dos Legionários, é conhecida por desencadear a formação de poro em membranas de macrófagos derivados de medula óssea (BMMs) por mecanismos dependentes do sistema de secreção do tipo IV conhecido como Dot/Icm. Neste trabalho, foram utillizados vários mutantes de L. pneumophila em combinação com camundongos nocautes para investigar os fatores bacterianos e do hospedeiro envolvidos na formação de poro em BMMs. Observamos que apesar da atividade do Dot/Icm, a formação de poro não ocorre em BMMs deficientes para caspase-1 e Nlrc4. A formação de poro foi temporalmente associada com a secreção de IL-1b e precedeu a lise celular e a piroptose. A formação de poro foi dependente do Dot/Icm, mas independente de várias proteínas efetoras, da multiplicação bacteriana e da síntese de novo de proteínas. A flagelina, a qual é conhecida em ativar o inflamassoma de Nlrc4, foi necessária para a formação de poro; a bactéria mutante flaA falhou em induzir a permeabilização celular. Consequentemente, a transfecção da flagelina purificada foi suficiente para desencadear a formação de poro independente da infecção. Utilizando 11 diferentes espécies de Legionella, nós observamos alta formação de poro em resposta à L. micdadei, L. bozemanii, L. gratiana, L. jordanis e L. rubrilucens, e essa resposta estava correlacionada com a expressão de flagelina por essas espécies. Além disso, verificamos que as proteínas Asc e Caspase-11 apresentam fenótipo intermediário na formação de poro, sugerindo que outras vias podem estar envolvidas no processo. Observamos também que a formação de poro desencadeada por L. pneumophila difere daquela induzida pelo ATP. Em conjunto, nossos resultados sugerem que a formação de poro não é uma resposta específica de L. pneumophila nem o resultado de dano da membrana induzido pelo Dot/Icm. Ao invés disso, a formação de poro é uma resposta do hospedeiro altamente coordenada, dependente dos componentes do inflamassoma Nlrc4 e caspase-1 e é desencadeada em resposta a bactérias que expressam o sistema de secreção do tipo IV e flagelina. / Legionella pneumophila, the etiological agent of Legionnaires disease, is known to trigger pore formation in bone marrow-derived macrophages (BMMs) by mechanisms dependent on the type IVB secretion system known as Dot/Icm. Here, we used several mutants of L. pneumophila in combination with knockout mice to assess the host and bacterial factors involved in pore formation in BMMs. We found that regardless of Dot/Icm activity, pore formation does not occur in BMMs deficient in caspase-1 and Nlrc4/Ipaf. Pore formation was temporally associated with IL-1b secretion and preceded host cell lysis and pyroptosis. Pore-forming ability was dependent on bacterial Dot/Icm but independent of several effector proteins, multiplication and de novo protein synthesis. Flagellin, which is known to trigger the Nlrc4 inflammasome, was required for pore formation as flaA mutant bacteria failed to induce cell permeabilization. Accordingly, transfection of purified flagellin was sufficient to trigger pore formation independent of infection. By using 11 different Legionella species, we found robust pore formation in response to L. micdadei, L. bozemanii, L. gratiana, L. jordanis and L. rubrilucens, and this trait correlated with flagellin expression by these species. Furthermore, we found that Asc and Caspase-11 showed an intermediate phenotype in pore formation, suggesting that other pathways may be involved in this process. We also observed that the pore formation triggered by L. pneumophila differs from the pore induced by ATP. Together, the results suggest that pore formation is neither L. pneumophilaspecific nor the result of membrane damage induced by Dot/Icm activity; instead, it is a highly coordinated host cell response dependent on host Nlrc4 and caspase-1 and on bacterial flagellin and type IV secretion system.

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No. of bitstreams: 1 nunes_pr_me_bot.pdf: 4789833 bytes, checksum: 772d289b789606f7e85653d2c289a43a (MD5) Previous issue date: 2017-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introdução: A gestação é uma condição fisiológica que pode apresentar maior suscetibilidade ao desequilíbrio entre fatores pró e antioxidativos, evoluindo assim com dano celular e resposta inflamatória. A autofagia é um processo que elimina organelas e proteínas danificadas do citoplasma, com potente mecanismo anti-inflamatório responsável pela manutenção da homeostase celular. A autofagia pode controlar a inflamação por meio da inibição da ativação do inflamassoma, complexo essencial para a liberação de citocinas pró-inflamatórias. Assim, alterações nesses processos podem relacionar-se com disfunções celulares e doenças sistêmicas. Objetivos: Este projeto teve como objetivo avaliar se a exposição de explantes placentários a diferentes concentrações de peróxido de hidrogênio (H2O2) é capaz de induzir autofagia e levar a ativação do inflamassoma NLRP3. Métodos: Explantes placentários de gestantes normais obtidos após o parto foram cultivados em diferentes concentrações de H2O2 por 4 e 24 h após a avaliação da viabilidade dos mesmos nesses períodos. As enzimas superóxido dismutase (SOD) e catalase foram avaliadas nos sobrenadantes das culturas após 4 h. As expressões gênicas de marcadores de autofagia (LC3-II, beclin-1 e p62), do inflamassoma (NLRP3 e caspase-1) e das citocinas IL-1β, IL-10 e TNF-α foram avaliadas por RT-qPCR. Os níveis de gonadotrofina coriônica (hCG), proteína de choque térmico 70 (Hsp70) e citocinas IL-1β, IL-10 e TNF-α foram determinados por ensaio imunoenzimático (ELISA) após 24 h de cultura. Resultados: Os níveis de LDH foram crescentes conforme o tempo de cultura, sendo que as culturas de 24 h apresentaram-se com viabilidade celular adequada para o estudo. Os níveis proteicos de catalase e Hsp70, bem como a expressão gênica de LC3-II, beclin-1 e p62 apresentaram níveis crescentes e relacionados às maiores concentrações de H2O2. As concentrações proteicas de SOD, hCG e TNF-α foram maiores nas culturas com 100 µM de H2O2. A expressão gênica de TNF-α, IL-1β, NLRP3 e caspase-1 foram elevadas em 1000 µM de H2O2. Além disso, a expressão proteica de IL-1β também foi maior nessa concentração. As concentrações gênicas e proteicas de IL-10 decresceram de acordo com o aumento da concentração de H2O2. Conclusões: Os resultados obtidos demonstraram que o H2O2 é capaz de induzir o estado de estresse oxidativo placentário, induzir autofagia, ativar o inflamassoma e assim aumentar a produção de citocinas inflamatórias. / Introduction: Pregnancy is a physiological condition characterized by increased susceptibility to oxidative stress, which can lead to cell damage and inflammatory response. Autophagy is a process that removes damaged organelles and proteins from the cytoplasm. It works as potent anti-inflammatory mechanism, responsible for maintaining cellular homeostasis. Autophagy can control inflammatory responses by regulating the activation of inflammasome, an essential complex for pro-inflammatory cytokine release. Objectives: The aim of this study was to evaluate whether placental explants exposure to hydrogen peroxide (H2O2) is able to induce autophagy and inflammasome activation. Methods: Placental explants achieved from normal pregnant women after delivery were cultured in different concentrations of H2O2 for 4 and 24 h after viability evaluation for these periods. Superoxide dismutase (SOD) and catalase were evaluated in culture supernatants after 4 h. Gene expressions of autophagy markers (LC3-II, beclin-1 and p62), inflammasome (NLRP3 and caspase-1) and IL-1β, IL-10 and TNF-α were assessed by RT-qPCR. Levels of chorionic gonadotrophin (hCG), heat shock protein (Hsp70) and IL-1β, IL-10 and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA) after 24 h of culture. Results: LDH levels were increased according to culture time, and the 24 h cultures presented adequate cell viability for the study. The protein levels of catalase and Hsp70, as well as the gene expression of LC3-II, beclin-1 and p62 presented increasing levels and related to the higher concentrations of H2O2. Protein concentrations of SOD, hCG and TNF-α were higher in cultures with 100 μM of H2O2. Gene expression of TNF-α, IL-1β, NLRP3 and caspase-1 were raised in 1000 μM of H2O2. In addition, IL-1β protein expression was also higher at this concentration. Gene and protein concentrations of IL-10 decreased as the H2O2 concentration increased. Conclusions: Our results demonstrate that H2O2 is able to induce a state of placental oxidative stress, induce autophagy, activate the inflammasome and then increase the production of inflammatory cytokines. / FAPESP: 2014/25611-5

The innate immune system is the first line of defence against infection. It is comprised of physicochemical barriers and a variety of cell types including macrophages and dendritic cells. Pathogens express specific pathogen associated molecular patterns (PAMP) which are recognised by pattern recognition receptors (PRR) on macrophages to initiate an innate immune response. Gram-negative bacteria such as Salmonella enterica serovar Typhimurium express a range of bacterial PAMPs recognised by Toll-like receptors (TLRs) including lipopolysaccharides (LPS) recognised by TLR-4 and lipoproteins by TLR-2. The activation of TLRs results in activation of nuclear factor κB (NF-κB) to drive transcription of mRNA coding for pro-inflammatory proteins such as tumor necrosis factor α (TNF-α) and pro-interleukin (IL) 1β. Myeloid cells also possess intracellular PRRs including the nucleotide-binding domain and leucine-rich repeat (NLR) family. NLR family CARD domain- containing protein 4 (NLRC4) and NLR family pyrin domain-containing protein 3 (NLRP3) are the main NLRs engaged in recognising S. Typhimurium infection, leading to formation of the inflammasome. The inflammasome is a macromolecular complex assembled in the cytoplasm, and usually contains a NLR, the structural protein apoptosis-associated speck-like protein containing a CARD (ASC) and effector enzymes such as cysteine-dependent aspartate-directed protease (caspase) -1 and caspase-8. This structure is responsible for processing the cytokines pro- IL-1β and pro-IL-18 to their mature form and is involved in triggering a pro-inflammatory process of cell death termed pyroptosis. The formation of the inflammasome therefore results in cell death and secretion of proinflammatory cytokines which play important roles in controlling infections. Inflammasome activity must be tightly coordinated, as its dysregulation is associated with a variety of auto-inflammatory and auto-immune diseases. The signalling events leading to inflammasome assembly are poorly understood and the molecules involved in fine-tuning its activity are only beginning to be discovered. The aim of this thesis was to discover new molecules involved in inflammasome activation and/or in keeping its activity in check. To achieve this goal, I performed S. Typhimurium infection assays in primary bone marrow derived macrophages (BMDM) derived from C57BL/6 mice wild type (WT) and compared the resulting cellular viability, intracellular bacteria counts and IL-1β production to that of BMDMs derived from C57BL/6 mice lacking proteins involved with, or suspected to be involved with, innate immune activity. Amongst the proteins I studied, caspase recruitment domain 9 (CARD9) inhibited inflammasome-mediated IL-1β production. Multiple independent genome-wide association studies link this protein to inflammatory pathologies such as Crohn's disease, but its role in canonical inflammasomes was largely unexplored. To investigate how CARD9 inhibits inflammasome-mediated IL-1β production I have conducted assays in WT and Card9-/- BMDMs, including stimulation of specific NLRs with their purified ligands, infection with bacterial strains deficient in NLRC4 activation, and infection assays in presence of pharmacological inhibitors. By employing these approaches, I observed that CARD9 has a negative role on NLRP3-dependent IL-1β production. Specifically, in response to activation of the NLRP3 by Salmonella infection, CARD9 negatively regulates pro-IL-1β transcription, and decreases IL-1β processing by inhibiting spleen tyrosine kinase (SYK)-mediated NLRP3 activation and represses caspase-8 activity in the inflammasome. CARD9 expression is suppressed in the course of S. Typhimurium infection which may act as a mechanism to increase IL-1β production during the infection. In conclusion, I have established a connection between CARD9 and IL-1β production by the canonical NLRP3 inflammasome and elucidated some of the mechanisms involved in this process. I have also found evidence that other proteins are likely to be involved in inflammasome regulation and the elucidation of their roles will be addressed in future studies.

A morte celular necrótica é conhecida pelo seu caráter inflamatório, conferido pela grande quantidade de sinais de dano liberados. Durante a tuberculose primária progressiva é observada intensa lesão necrótica nos pulmões e disseminação do bacilo para outros órgãos. Neste estudo, nós hipotetizamos que a amplificação da necrose pulmonar, via reconhecimento de sinais de dano tecidual pelo receptor purinérgico P2X7 (P2X7R), poderia favorecer a progressão da doença, bem como a potencialização da resposta inflamatória. Vimos que o reconhecimento de extracelular ATP (eATP) via o receptor P2X7 é de suma importância para o desenvolvimento da doença grave, por favorecer a indução de necrose dos macrófagos infectados, o que facilitou o escape do bacilo. A adenosina, resultante da hidrólise do eATP, impactou na ativação da resposta imune adquirida. Nosso estudo provê uma nova perspectiva para o desenvolvimento de protocolos terapêuticos baseados na inibição do P2X7R e dos receptores de adenosina para evitar a indução de formas graves da doença. / Necrosis cell death is known as an inflammatory process due to the large amount of damage signals released. During the progressive primary tuberculosis, extensive necrotic lesions in the lung and intensive bacterial dissemination are observed. In this study, we hypothesized that the amplification of pulmonary necrosis through damage signals recognition by a purinergic receptor called P2X7 (P2X7R) could favor the progression of disease, as well as the augmentation of the inflammatory response. We found that the recognition of extracellular ATP (eATP) through P2X7R is crucial to the outcome of fatal tuberculosis by favoring the induction of necrosis of infected macrophages, which facilitated the bacterial escape. The adenosine, resulted from eATP hydrolyzation, impacted to the acquired immune response activation. Our study provides a new perspective to the development of therapeutic protocols based on the inhibition of P2X7R and adenosine receptor to avoid the induction of aggressive forms of tuberculosis.

The innate immune system are a cooperation of many components – receptors being one of them. Both membrane-bound and cytosolic receptors play a large role in the defence system against pathogens and danger. NLRP3 is a receptor which assembles a protein complex called inflammasome in response to cytosolic stress and is responsible for many autoimmune diseases if it malfunctions. The activation of the NLRP3 inflammasome leads to secretion of inflammatory cytokines and in many cases to programmed cell death. The structure, function and activation of the NLRP3 inflammasome is still not fully understood and the urge to understand the mechanisms behind are important for future medical improvements. The aim was to anchor the NLRP3 inflammasome by the cell membrane - By Overlap PCR, the NLRP3 cDNA was fused extracellular and trans-membrane parts of the TLR4 cDNA to anchor the NLRP3 to the membrane and in turn analyse the inflammasome with LPI™ technology. Multiple primers and a TLR4 nucleotide were designed and the NLRP3 was amplified with specific overhangs by PCR. The fusion protein was successfully linked together by Overlap PCR but not confirmed by sequencing. The gene fusion demands high quality primers for amplification and further evaluation must be made to the details of the laboratory. To anchor the protein complex to the cell membrane, continue to be of full importance and can be an asset in many structural studies and biopharmaceuticals trials.

Autofagia e ativação dos inflamassomas são dois processos celulares autônomos, que podem interagir entre si. Estes processos participam ativamente do controle de infecções ocasionadas por diversos patógenos intracelulares. Anteriormente, descrevemos o inflamassoma NLRP3 no controle do T. cruzi, agente causador da Doença de Chagas. Entretanto, o papel da autofagia nesse controle não era conhecido. Neste trabalho, foi demonstrado que o T. cruzi induz aumento da expressão de LC3-II, formação de autofagossomas e autolisossomos em macrófagos peritoneais (MPs) de camundongos C57BL/6 selvagens. Ainda, a manipulação farmacológica da autofagia interferiu com a capacidade dos MPs em controlar a infecção pelo T. cruzi, apontando esse processo como um mecanismo efetor envolvido no controle do protozoário por macrófagos. Nesse contexto, NLRP3 parece funcionar como um modulador do processo autofágico. Na ausência de NLRP3, a manipulação farmacológica da autofagia não interferiu no controle do T. cruzi por MPs. Isso se correlacionou ao fato do fluxo autofágico se encontrar interrompido em MPs de camundongos deficientes para NLRP3 em resposta à infecção, mas não em resposta à rapamicina e starvation. A razão do bloqueio no fluxo autofágico parece ser a incapacidade de MPs deficientes em NLRP3 em formar autolisossomos, fato visualizado em microscopia confocal e eletrônica. Interessante, NLRP3 parece agir independente de caspase-1/11 na regulação da autofagia. Por outro lado, a análise da expressão de genes autofágicos por PCR-array revelou que MPs de animais deficientes em NLRP3 apresentam alta expressão basal de genes relacionados com a formação e maturação de autofagossomas e autolisossomas. Em contrapartida, a infecção pelo T. cruzi inibe a expressão desses genes na ausência de NLRP3, ao contrário da indução observada em MPs selvagens. Juntos, esses dados mostram que NLRP3 induz autofagia funcional em resposta ao T. cruzi, sendo sua presença fundamental para impedir o escape do parasita pela inibição de genes autofágicos. / Autophagy and inflammasome activation are two cell-autonomous and cross-regulated processes involved in host resistance against infections. Our group previously described that NLRP3 inflammasome is required for the control of T. cruzi, causative agent of Chagas disease. However, the involvement of autophagy in this process was largely unknown. Here, we demonstrated that T. cruzi is able to induce an increase in LC3II expression, formation of autophagosome and autolysosomes in peritoneal macrophages (PMs) from C57BL/6 mice. Moreover, the pharmacological modulation of autophagic machinery influenced the trypanocidal ability of PMs, pointing out autophagy as an effector mechanism to control T. cruzi infection. In this sense, NLRP3 seems to be involved in the modulation of the autophagic process. In the absence of NLRP3, the pharmacological modulation of autophagy did not interfere in the control of T. cruzi by PMs. Furthermore, autophagic flux is blocked in these cells in response to infection, but not in response to rapamycin and starvation. In fact, whereas T. cruzi induces the formation of large autolysosomes (LC3&#43 and Lysotracker&#43)-containing amastigotes in WT macrophages, only small and single positive vesicles are found in the absence of NLRP3. Interesting, NLRP3 appears to act independently of caspase-1/11 on the regulation of autophagy. On the other hand, the PCR-array analysis of autophagic genes demonstrated that NLRP3-/- PMs have higher basal expression of genes related to the formation and maturation of autophagosomes and autolysosomes in comparison to WT cells. In contrast, T. cruzi inhibited the expression of these genes in the absence of NLRP3, unlike the induction observed in WT PMs. Together, these data show that NLRP3 induces functional autophagy in response to T. cruzi being its presence required to overcome the escape of the parasite by preventing its inhibition of autophagic genes.

Cytosolic double stranded DNA (dsDNA) is sensed as a “danger signal” by host cells. Detection of viral and bacterial nucleic acid is emerging as a major route for cells to identify an infection by a pathogen. Recognition of cytoplasmic DNA causes death of some cells and interferon (IFN) and cytokine induction, which are appropriate anti-viral responses. Responses to cytoplasmic DNA may not only be relevant to certain retrovirus, DNA virus and bacterial infections, but could also be generated by reverse transcription of endogenous retro-elements. Introduction of DNA into the cytoplasm of bone marrow derived macrophages (BMM) causes upregulation of MHC Class I, induction of IFNβ and other cytokines and cell death. Both cytokine induction and cell death were independent of recognition of “CpG motifs” through TLR9. In order to determine whether a single receptor was likely to mediate these responses, the types of DNA eliciting these responses was compared. Both cellular activation to produce cytokines and IFNβ, as well as cell death were seen only with dsDNA but not single stranded DNA (ssDNA). Both responses increased with increasing DNA length, with little detectable effect of a double stranded 22bp oligonucleotide (ODN). The sequences of DNA leading to optimal induction of IFNβ and death were different. Although all dsDNA induced death of primary macrophages, poly(dA):(dT) was a particularly potent and rapid pro-death stimulus. In contrast, poly(dA):(dT) was a relatively poor stimulus for IFNβ, even at doses which were minimally toxic, or in cells which are resistant to DNA induced cell death. The alternating co-polymer poly(dA-dT) was the most potent inducer of IFNβ. This data suggests that separate DNA receptors mediate cell death and IFNβ induction in response to dsDNA Transfected dsDNA also rapidly activated caspase 3, a classical pro-apoptotic caspase, in BMM as early as 2½ minutes post-transfection with DNA. Caspase 3 is an effector caspase which is activated by an upstream initiator caspase. Although the apical caspase in the DNA detection system has not been defined, use of Bcl2 overexpressing BMM and caspase 2-/- BMM showed that DNA-dependent caspase 3 activation did not occur via the mitochondrial damage or the caspase 2 activation pathways. The inflammatory caspase, caspase 1 was also activated in response to DNA transfection, although whether caspase 1 is responsible for cleavage of caspase 3 has not been established. Caspase 1 activation suggests the involvement of the inflammasome, which is important for processing pro-inflammatory cytokines such as IL-1β into their biologically active forms. Furthermore, there is recent evidence suggesting that DNA-transfected cells die by a caspase 1-dependent cell death called pyroptosis. Other work in our lab identified the HIN-200 family member and candidate lupus susceptibility factor p202 as a candidate receptor for cytoplasmic dsDNA; p202 bound stably and rapidly to transfected DNA. Here, knockdown studies revealed p202 to be a regulatory protein limiting DNA-induced caspase 1 and 3 activation. Conversely, the related pyrin domain-containing HIN-200 factor AIM2 (p210), a candidate tumour suppressor, was required for caspase 1 and 3 activation by cytoplasmic dsDNA. Recently published work suggests that AIM2 multimerises along the length of the DNA leading to the formation of an inflammasome complex. The pyrin domain of AIM2 recruits the adaptor protein ASC through homotypic pyrin domain interactions. ASC subsequently recruits caspase 1, which results in its auto-activation. The inhibitory effect of p202 on caspase activation is likely to be due to its lack of a pyrin signalling domain. p202 rapidly binds to cytoplasmic DNA, and may reduce the clustering of AIM2 pyrin domains which results in caspase activation. Consistent with this proposal, DNA-dependent caspase activation correlated inversely with p202 expresssion in 3 mouse strains. This work defines HIN-200 proteins as a new class of pattern recognition receptors mediating responses to dsDNA. Work in this thesis aimed to understand the biological role and mechanism of responses to cytoplasmic DNA. Responses to cytoplasmic DNA are likely to be relevant not only to infectious disease but also to autoimmune diseases such as systemic lupus erythmatosus (SLE), where DNA appears to act as an adjuvant, and even tumour progression where there is evidence for a role for active endogenous retro-elements. In addition, responses to DNA may limit transfection efficiency and the efficacy of non-viral gene therapy.