A lesão granulomatosa é caracterizada como um agregado compacto de fagócitos maduros formado em resposta à um estímulo persistente. Os mediadores pró-inflamatórios da família da IL-1, ao promoverem a ativação da imunidade inata e o remodelamento tecidual descontrolados, geram a fisiopatologia da paracoccidioidomicose, doença pulmonar granulomatosa causada pelo fungo P. brasiliensis. A principal via inflamatória envolvida na secreção de IL-1?, IL-18 e IL-1? é a ativação dos inflammasomas, complexos protéicos conhecidos pela sua capacidade de ativar proteoliticamente a enzima caspase-1. Neste estudo abordamos os mecanismos subjacentes às vias canônica e não canônica do inflamassoma, avaliando a importância funcional das caspases 1, 11 e 8 na resistência do hospedeiro durante a infecção por P. brasiliensis. Demonstramos que a resposta imunológica protetora mediada pelo IFN-?, após a liberação de IL-18 pela via da caspase-1, é o principal mecanismo responsável pelo controle fúngico quando o dano celular desencadeado pelo fungo é reconhecido pelo inflamassoma de NLRP3. No entanto, apesar do papel importante da caspase-1 na maturação da IL-1?, a falta de caspase-1 apenas reduz parcialmente os níveis de IL-1? durante a infecção por P. brasiliensis. Ao contrário do esperado, a deficiência de caspase-11 não prejudicou a produção de IL-1?, mas em vez disso, preveniu, na célula, a morte por piroptose e a secreção IL-1?, citocina importante para restringir o crescimento fúngico através da síntese de IL-17. Por fim, observamos que a ativação de caspase-8 pela sinalização de dectina-1 / Syk, além de mediar a maturação da IL-1?, que acontece de maneira independente de caspase-1 e 11, é necessária também para o funcionamento eficiente da via canônica de caspase-1, demonstrando uma rede interligada entre as vias canônica de caspse-1 e não canônica de caspase-8 para coordenar o processamento da IL-1?. Em conjunto, nossos resultados mostram contribuições distintas das vias canônica e não canônica do inflamassoma na produção de citocinas da família da IL-1, enfatizam a versatilidade desta plataforma em recrutar várias proteínas efetoras para adequar a resposta imunológica antifúngica e evidenciam a complexidade envolvida nas interações patógeno-hospedeiro. / Granuloma is a lesion characterized by a compact aggregate of mature phagocytes arising in response to a persistent stimulus. By driving uncontrolled innate immunity and tissue remodelling, IL-1 family pro-inflammatory mediators govern the pathophysiology of paracoccidioidomycosis, a granulomatous lung disorders caused by Paracocccidioides brasiliensis. A major inflammatory pathway involved in IL-1?, IL-18 e IL-1? secretion is the activation of inflammasomes, large multimolecular complexes best known for their ability to control activation of the proteolytic enzyme caspase-1. In this study we addressed the mechanisms that underlie canonical and non-canonical inflammasome pathways, assessing the functional importance of caspase-1, caspase-11 and caspase-8 in the regulation of inflammasome-mediated host resistance during P. brasiliensis infection. We found that IFN-?-mediated protective immune response following by caspase-1- dependent IL-18 release after is the key mechanism responsible for the fungal control after P. brasiliensis-induced cell damage recognition by NLRP3 canonical inflammasome pathway. Nonetheless, despite the important role of caspase-1 in the IL-1? maturation, the lack of caspase-1 only partially reduced IL-1? levels during P. brasiliensis infection. Unlike caspase-1, caspase-11 deficiency did not impair IL-1? production, cytokine strictly secreted by canonical caspase-1 inflammasome pathway. Instead, P. brasiliensis-triggered caspase-11 activation in an ill-defined manner leads to a rapid pore-mediated cell lysis and is required for IL-1? production during P. brasiliensis infection. IL-1?, in turn, is important for promoting the restriction of fungal growth trough IL-17-based inflammation. Finally, the caspase-8 induction by dectin-1/Syk signaling besides playing a role in mediating the caspase-1/11-independent IL-1? maturation is also required to efficient canonical caspase-1 inflammasome pathway, demonstrating a connected network between non-canonical caspase-8 and canonical caspase-1 inflammasome pathways to coordinate IL-1?. Taken together, our results revealed distincts contributions of both canonical and non-canonical inflammasome pathways in IL-1 family cytokine production and emphasizes the versatility of this platform to recruit several effector proteins to tailor the antifungal immune response, sheding new light on the complexity of this hostpathogen interaction.

A esclerose múltipla (EM), doença neurodegenerativa do sistema nervoso central (SNC) com característica auto-imune e inflamatória, com eventos iniciais, bem como a evolução da EM. É uma doença heterogênea (três principais formas clínicas) e multifatoriais. A imunidade inata demonstrou recentemente ser um fator importante na EM e as variantes genéticas dos componentes do inflamassoma têm sido associadas a doenças autoimunes e neurodegenerativas, com isso hipotetizamos que o inflamassoma e suas citocinas IL-1Beta e IL-18, podem representar importantes contribuintes na patogênese da EM e eventualmente explicar, pelo menos em parte, a heterogeneidade observada em pacientes com EM. Fizemos uma análise multivariada que foi realizada com base na forma clínica (recorrente remitente/RR, primária progressivo /PP ou secundário progressiva /SP, índice de gravidade (EDSS) e índice de progressão (IP). Os monócitos do sangue periférico (PBMC) dos pacientes foram examinados para ativação do inflamassoma (Produção de IL-1Beta e IL-18, clivagem de caspase-1). Com os objetivos de avaliar a contribuição do inflamassoma na EM, em termos de (a) efeito genético sobre o desenvolvimento, gravidade e / ou prognóstico, e (b) ativação complexa de células de sangue periférico como uma forma de avaliar a inflamação sistêmica. Para isso, utilizamos variantes genéticas funcionais em componentes do inflamassoma, que foram analisadas em uma coorte de pacientes com EM, pelo uso de ensaios específicos de alelos e qPCR. A analise multivariada resultou em associação com a variante -511C / T IL1B ganho de função, sendo essa mais frequente em formas progressivas (especialmente SP) do que em RR. A variante de ganho de função NLRP3 Q705K resultou mais frequente em pacientes com EDSS > 3 do que em pacientes com EDSS < 3 e, consequentemente, esse SNP está associado a um IP mais elevado. A análise de PBMC mostrou que as células de indivíduos EM, são mais propensas a responder a um estímulo NLRP3 clássico (isto é, LPS) do que as dos doadores saudáveis. Em conjunto, esses achados indicaram que os pacientes com EM apresentam uma desregulação no inflamassoma NLRP3, podendo ser avaliada no sangue periférico facilitando um prognóstico, e que esse perfil pode ser secundário a um mecanismo genético pró-inflamassoma / The multiple sclerosis (MS), neurodegenerative disease of the central nervous system (CNS) with autoimmune and inflammatory characteristics, with initial events, as well as the evolution of MS, are heterogeneous (three main clinical forms) and multifactorial. Innate immunity has recently been shown to be an important factor in MS and the genetic variants of the components of inflammassoma have been associated with autoimmune and neurodegenerative diseases, thereby hypothesizing that the inflammassoma and its IL-1Beta and IL-18 cytokines may represent important contributors in the pathogenesis of MS and possibly explain, at least in part, the heterogeneity observed in MS patients. We performed a multivariate analysis that was performed based on clinical form (recurrent recurrent / RR, progressive primary / PP or progressive secondary / SP, EDSS and progression index.) Peripheral blood mononuclear cells (PBMC) of patients were examined for inflammatory activation (IL-1Beta and IL-18 production, caspase-1 cleavage). With the objectives of evaluating the contribution of inflammassoma in MS in terms of (a) genetic effect on development, severity and / or prognosis, and (b) complex activation of peripheral blood cells as a way of assessing systemic inflammation. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. Multivariate analysis resulted in association with the -511C / T IL1B function gain, which is more frequent in progressive forms (especially SP) than in RR. The gain variant of NLRP3 Q705K function was more frequent in patients with EDSS > 3 than in patients with EDSS < 3 and, consequently, this SNP is associated with a higher PI. PBMC analysis showed that cells from MS individuals are more likely to respond to a classical NLRP3 (ie LPS) stimulus than healthy donors. Taken together, these findings indicated that patients with MS have a dysregulation in the NLRP3 inflammassoma and can be evaluated in the peripheral blood facilitating a prognosis and that this profile may be secondary to a pro-inflammatory genetic mechanism

Nlrp1b is a NOD-like receptor of the innate immune system that upon sensing of anthrax lethal toxin oliogmerizes and forms a protein scaffold that binds to and activates pro-caspase-1; this complex is called an inflammasome. Nlrp1b is highly polymorphic and different alleles display an all or none ability to sense lethal toxin. Here I show that Nlrp1b is cleaved in the FIIND domain, and that the cleaved fragments remain associated even after activation by lethal toxin. The inflammasome activity of an inactive allele was restored by three mutations, one of which also restored cleavage. A heterologous cleavage site was inserted into an uncleaved mutant of Nlrp1b; induced proteolysis of the cleavage site rescued inflammasome activity. An uncleaved mutant of Nlrp1b showed no deficiency in FIIND self-association, but did have reduced recruitment of pro-caspase-1. These data provide evidence that cleavage of Nlrp1b is required for proper recruitment and activation of caspase-1.

Nlrp1b is a NOD-like receptor of the innate immune system that upon sensing of anthrax lethal toxin oliogmerizes and forms a protein scaffold that binds to and activates pro-caspase-1; this complex is called an inflammasome. Nlrp1b is highly polymorphic and different alleles display an all or none ability to sense lethal toxin. Here I show that Nlrp1b is cleaved in the FIIND domain, and that the cleaved fragments remain associated even after activation by lethal toxin. The inflammasome activity of an inactive allele was restored by three mutations, one of which also restored cleavage. A heterologous cleavage site was inserted into an uncleaved mutant of Nlrp1b; induced proteolysis of the cleavage site rescued inflammasome activity. An uncleaved mutant of Nlrp1b showed no deficiency in FIIND self-association, but did have reduced recruitment of pro-caspase-1. These data provide evidence that cleavage of Nlrp1b is required for proper recruitment and activation of caspase-1.

A lesão granulomatosa é caracterizada como um agregado compacto de fagócitos maduros formado em resposta à um estímulo persistente. Os mediadores pró-inflamatórios da família da IL-1, ao promoverem a ativação da imunidade inata e o remodelamento tecidual descontrolados, geram a fisiopatologia da paracoccidioidomicose, doença pulmonar granulomatosa causada pelo fungo P. brasiliensis. A principal via inflamatória envolvida na secreção de IL-1?, IL-18 e IL-1? é a ativação dos inflammasomas, complexos protéicos conhecidos pela sua capacidade de ativar proteoliticamente a enzima caspase-1. Neste estudo abordamos os mecanismos subjacentes às vias canônica e não canônica do inflamassoma, avaliando a importância funcional das caspases 1, 11 e 8 na resistência do hospedeiro durante a infecção por P. brasiliensis. Demonstramos que a resposta imunológica protetora mediada pelo IFN-?, após a liberação de IL-18 pela via da caspase-1, é o principal mecanismo responsável pelo controle fúngico quando o dano celular desencadeado pelo fungo é reconhecido pelo inflamassoma de NLRP3. No entanto, apesar do papel importante da caspase-1 na maturação da IL-1?, a falta de caspase-1 apenas reduz parcialmente os níveis de IL-1? durante a infecção por P. brasiliensis. Ao contrário do esperado, a deficiência de caspase-11 não prejudicou a produção de IL-1?, mas em vez disso, preveniu, na célula, a morte por piroptose e a secreção IL-1?, citocina importante para restringir o crescimento fúngico através da síntese de IL-17. Por fim, observamos que a ativação de caspase-8 pela sinalização de dectina-1 / Syk, além de mediar a maturação da IL-1?, que acontece de maneira independente de caspase-1 e 11, é necessária também para o funcionamento eficiente da via canônica de caspase-1, demonstrando uma rede interligada entre as vias canônica de caspse-1 e não canônica de caspase-8 para coordenar o processamento da IL-1?. Em conjunto, nossos resultados mostram contribuições distintas das vias canônica e não canônica do inflamassoma na produção de citocinas da família da IL-1, enfatizam a versatilidade desta plataforma em recrutar várias proteínas efetoras para adequar a resposta imunológica antifúngica e evidenciam a complexidade envolvida nas interações patógeno-hospedeiro. / Granuloma is a lesion characterized by a compact aggregate of mature phagocytes arising in response to a persistent stimulus. By driving uncontrolled innate immunity and tissue remodelling, IL-1 family pro-inflammatory mediators govern the pathophysiology of paracoccidioidomycosis, a granulomatous lung disorders caused by Paracocccidioides brasiliensis. A major inflammatory pathway involved in IL-1?, IL-18 e IL-1? secretion is the activation of inflammasomes, large multimolecular complexes best known for their ability to control activation of the proteolytic enzyme caspase-1. In this study we addressed the mechanisms that underlie canonical and non-canonical inflammasome pathways, assessing the functional importance of caspase-1, caspase-11 and caspase-8 in the regulation of inflammasome-mediated host resistance during P. brasiliensis infection. We found that IFN-?-mediated protective immune response following by caspase-1- dependent IL-18 release after is the key mechanism responsible for the fungal control after P. brasiliensis-induced cell damage recognition by NLRP3 canonical inflammasome pathway. Nonetheless, despite the important role of caspase-1 in the IL-1? maturation, the lack of caspase-1 only partially reduced IL-1? levels during P. brasiliensis infection. Unlike caspase-1, caspase-11 deficiency did not impair IL-1? production, cytokine strictly secreted by canonical caspase-1 inflammasome pathway. Instead, P. brasiliensis-triggered caspase-11 activation in an ill-defined manner leads to a rapid pore-mediated cell lysis and is required for IL-1? production during P. brasiliensis infection. IL-1?, in turn, is important for promoting the restriction of fungal growth trough IL-17-based inflammation. Finally, the caspase-8 induction by dectin-1/Syk signaling besides playing a role in mediating the caspase-1/11-independent IL-1? maturation is also required to efficient canonical caspase-1 inflammasome pathway, demonstrating a connected network between non-canonical caspase-8 and canonical caspase-1 inflammasome pathways to coordinate IL-1?. Taken together, our results revealed distincts contributions of both canonical and non-canonical inflammasome pathways in IL-1 family cytokine production and emphasizes the versatility of this platform to recruit several effector proteins to tailor the antifungal immune response, sheding new light on the complexity of this hostpathogen interaction.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-07T12:49:00Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Mayara final [08-09-2016].pdf: 6000027 bytes, checksum: 8aba9ff907d1176a530d19ec2bb6fad1 (MD5) / Made available in DSpace on 2017-04-07T12:49:00Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Mayara final [08-09-2016].pdf: 6000027 bytes, checksum: 8aba9ff907d1176a530d19ec2bb6fad1 (MD5) Previous issue date: 2015-09-02 / CAPES / Doença inflamatória intestinal descreve um grupo heterogêneo de doenças inflamatórias crônicas do trato gastrointestinal. Os dois principais tipos de DII são retocolite ulcerativa idiopática e doença de Crohn. A patogênese dessas doenças é caracterizada pela inflamação persistente no intestino, envolvendo uma interação entre fatores genéticos, ambientais e imunológicos. Foram investigados aspectos clínico-epidemiológicos e analisados os polimorfismos dos genes da reposta imune em pacientes brasileiros com doença inflamatória intestinal em diferentes formas anátomo-clínicas. Um total de 101 pacientes foram analisados (43 - retocolite ulcerativa idiopática e 58 - doença de Crohn) para os polimorfismos dos genes do fator de necrose tumoral alfa (TNF-α -308 G/A; rs1800629), interleucina-10 (IL-10 -1082 G/A; rs1800896), domínio do recrutamento e ativação da caspase 15/receptor tipo NOD2 (CARD15/NOD2; rs2066844 e rs2066845), receptor tipo NOD contendo domínio pirina – NLRP1 (rs12150220), NLRP3 (rs35829419) e interleucina -1beta (IL-1β -511T/C; rs16944). A forma anatómica-clínica de DC predominante foi a fistulizante (29,31%), seguida por inflamatória (27,58%) e estenosante (27,58%). O grupo controle foi composto por 91 indivíduos saudáveis. Os genes do receptor tipo NOD contendo domínio pirina 1 e 3 e do domínio do recrutamento e ativação da caspase 15/receptor tipo NOD2 variantes R702W e G908R não foram associados à susceptibilidade a doença inflamatória intestinal. Em relação ao polimorfismo da interleucina 10, nenhuma diferença estatística foi encontrada entre os genótipos e alelos para a doença inflamatória intestinal comparado aos controles. Fator de necrose tumoral alfa mostrou uma associação estatisticamente significativa entre pacientes e controles de retocolite ulcerativa idiopática que sugere que a presença do alelo A predispõe o aparecimento de retocolite ulcerativa idiopática, mas não doença de Crohn. Verificou-se ainda que o genótipo AG da interleucina 1 foi associado com o desenvolvimento de retocolite ulcerativa idiopática. Os resultados sugerem que os polimorfismos de única base do fator de necrose tumoral alfa e da interleucina 1 estão envolvidos com a retocolite ulcerativa idiopática e podem contribuir para a patogênese na população brasileira estudada. / Inflammatory bowel disease describes a heterogeneous group of chronic inflammatory diseases of the gastrointestinal tract. The two main types of inflammatory bowel disease are ulcerative colitis and Crohn disease. The pathogenesis of the disease is characterized by unpredictable attacks of inflammation of the intestine, besides involving an interaction between genetic, environmental and immunological factors. Clinical and epidemiological aspects were investigated and the polymorphisms of genes of the immune response in Brazilian patients with inflammatory bowel disease in different anatomic-clinical forms were analyzed. A total of 101 patients were analyzed (43 - ulcerative colitis and 58 - Crohn disease) for the tumour necrosis factor alpha (TNF-α -308 G/A; rs1800629), interleukin-10 (IL-10 -1082 G/A; rs1800896), caspase activation and recruitment domains 15/ NOD like receptor 2 (CARD15/NOD2; rs2066844 and rs2066845), NOD like receptor pyrin domain containing – NLRP1 (rs12150220), NLRP3 (rs35829419) and interleukin-1beta (IL-1β 511T/C; rs16944) genes polymorphisms. The anatomic-clinical form of Crohn disease predominant was the fistulizing (29.31%), followed by inflammatory (27.58%) and stricturing (27.58%). A control group was composed by 91 healthy subjects group. NOD like receptor pyrin domain containing 1 and 3 and caspase activation and recruitment domains 15/ NOD like receptor 2 genes R702W and G908R variants were not associated to inflammatory bowel disease susceptibility. With respect to the polymorphism of interleukin-10, no statistical difference was found between the genotypes and alleles for inflammatory bowel disease compared to controls. Tumour necrosis factor alpha showed a statistically significant association between ulcerative colitis patients and controls which suggests that the presence of A allele predisposes the onset of ulcerative colitis but not Crohn disease. It was found yet that AG genotype of interleukin-1beta was associated with the development of ulcerative colitis. The results suggest that the tumour necrosis factor alpha and interleukin-1beta single nucleotide polymorphisms are involved with ulcerative colitis and may be contributing to pathogenesis in Brazilian population.

Background &amp; Aims: Celiac disease (CD) is a chronic inflammatory small-bowel enteropathy caused by permanent intolerance to gliadin in wheat gluten, and related proteins in ray and barley. It is disputed whether CD patients tolerate oats. The only treatment of CD is lifelong gluten-free diet (GFD). Only individuals that carry the HLA-DQ2 and/or DQ8 alleles, and eat gluten can develop CD. Dysbiosis in the gut microbiota is a suggested risk factor for CD. T cells in small intestinal mucosa, including intraepithelial lymphocytes (IELs), are known to be important in the pathogenesis of CD. In contrast, the role of intestinal epithelial cells (IECs) is poorly understood. In this thesis we investigated the role of IECs in the immune pathology of CD from duodenal mucosa of children with CD, clinical controls and treated CD. We also investigated the role of CD associated bacteria and oats supplemented GFD on the mucosal immune system. Results: A new CD-associated bacterium, Prevotella jejuni, was isolated and characterized. It is a saccharolytic and proteolytic anaerobe. More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were upregulated in IECs in active CD. In two in vitro models for intestinal epithelium, small intestine enteroids and T84 polarized tight monolayers, we showed that 70% of these genes were upregulated by interferon (IFN)-γ via the IRF1 pathway. IRF1 was also upregulated by the CD-associated bacteria P. jejuni and Actinomyces gravenitzii. IECs expressed the NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin (IL)-18, which induces IFN-γ in IELs. P. jejuni bound the intestinal epithelial cell lines T84, Caco2, HT29, and INT407, while Lachnoanaerobaculum umeaense preferentially bound Caco2. P. jejuni caused decreased transepithelial resistance over tight monolayers, while L. umeaense caused an increase. P. jejuni upregulated mRNAs for the detoxification molecules CYP1A1, CYP1A2, CYP1B1, and TIPARP, the chemokines CX3CL1, CXCL1, and CXCL10, the sialyltranserase ST3GAL4, and the inflammation promoting protein S100A3 in tight monolayers. L. umeaense upregulated the chemokines CCL20 and CXCL10, and down-regulated TLR2. In a randomized, double-blinded intervention trial comparing two study-groups, standard GFD and oat-containing GFD, we found that mRNAs for several immune effector molecules and tight junction proteins were only reduced in patients receiving GFD, but not in a substantial fraction of patients on GFD with oats. The down-regulatory cytokines IL-10 and TGF-β1, the cytotoxicity-activating NK-receptors NKG2C and NKG2E, and the tight junction protein claudin-4 remained elevated in the study group on GFD with oats. Conclusions: IECs are far from inactive in CD. A key factor in the epithelial reaction in CD appears to be over-expression of IRF1 in IECs. Dual activation of IRF1 and IRF1-regulated genes, both directly by P. jejuni and indirectly by IFN-γ via the IL-18-inflammasome, would drastically enhance the inflammatory response and lead to the pathological situation seen in active CD. P. jejuni harms the intestinal epithelium, i.e., it is a likely risk factor for CD, while L. umeaense strengthen barrier function and local immunity, possibly acting as a protective. A fraction of CD patients should avoid oats in the diet. / Doctoral thesis

Short ORF-encoded peptides (SEPs) are increasingly being identified as functional elements in various cellular processes. The current computational methods and experimental molecular biochemistry allow us to discover putative SEPs or micropeptides from proteogenomic datasets and experimentally validate them. Here, we identified a micropeptide produced from a putative long noncoding RNA (lncRNA) 1810058I24Rik which is downregulated in both human and murine myeloid cells exposed to lipopolysaccharide (LPS), as well as other TLR ligands and inflammatory cytokines. Analysis of lncRNA 1810058I24Rik subcellular localization revealed this transcript is localized in the cytosol, prompting us to evaluate its coding potential. In vitro translation with 35S-labeled methionine resulted in translation of a 47 amino acid micropeptide. Microscopy and subcellular fractionation studies in macrophages demonstrated endogenous expression of this peptide on the mitochondrion. We thus named this gene ‘Mitochondrial micropeptide-47 (Mm47)’. Functional studies using siRNA and Cripsr-cas9-mediated deletion in primary cells, showed that the transcriptional response downstream of TLR4 was not affected by Mm47 loss of function. In contrast, both the Crispr-cas9- and siRNA-targeted BMDM cells were compromised for Nlrp3 inflammasome responses. However, the primary macrophages derived from the Mm47 knockout mice do not require Mm47 for Nlrp3 activation, likely due to basal downregulation of a negative regulator microRNA of Nlrp3 called Mir-223. Notably, the Mm47-deficient mice are susceptible to influenza virus infection and succumb despite comparable antiviral and inflammatory response to wildtype mice. We hypothesize that the Mm47 deficiency may affect the antiviral resilience of mice due to secondary mitochondria dependent immunometabolic defect or failure of recovery from immune pathology, which warrants further investigation. This study therefore identifies a novel mitochondrial micropeptide Mm47 that is required for activation of the Nlrp3 inflammasome in cells and resistance to influenza virus infection. Broadly, this work highlights the presence of translatable ORFs is annotated noncoding RNA transcripts and underscores their importance in innate immunity and virus infection.

Central driving forces in the pathogenesis of liver disease are hepatocyte death and immune cell-driven inflammation. The interplay between outcomes, stemming from these two major cell types, is present from the earliest ethanol exposure, and are both determinants in advanced stages of liver disease particularly in alcoholic liver disease (ALD). The complexities associated with advanced ALD are many and therapies are limited. Due to the liver’s role in ethanol metabolism and filtering gut-derived products, it is becoming increasingly clear that innate immunity plays a central role in triggering activation of cell death and inflammatory pathways in ALD. We identified interferon regulatory factor 3 (IRF3) activation as a mediator of hepatocyte death as the first event after ethanol exposure, and the inflammasome as a protein complex responsible for the subsequent inflammatory cascade, driven by the NLRP3 inflammasome. Our novel findings in murine samples and human patients with alcoholic hepatitis demonstrate that ethanol-induced inflammasome activity results in Caspase-1-mediated pyroptosis and extracellular ASC aggregates in the liver and circulation. Pyroptosis can be abrogated by therapeutic inhibition of inflammasome components, NLRP3 or Caspase-1. Taken together, the event leading to mtDNA release into the cytoplasm is the inception of the pathogenesis of ALD, triggering hepatocyte death, culminating in a pro-inflammatory cascade driven by the NLRP3 inflammasome and pyroptotic release of ASC.

Interferon-inducible PYHIN protein family includes the DNA-binding proteins, AIM2 and IFI16, which form ASC-caspase 1 dependent inflammasomes, important in immunity against cytosolic bacteria, DNA viruses and HIV. The role of other members of this family in the recognition of DNA and/or regulation of immune responses is unclear. We identified an immune regulatory function of p205, another member of the PYHIN family, in the transcriptional control of immune genes. Knockdown of p205 in macrophages revealed that inflammasome activation due to dsDNA and ligands that engage the NLRP3 inflammasome were severely compromised. Detailed mechanistic analysis showed that loss of p205 was associated with a decrease in Asc mRNA and protein levels. p205 knockdown resulted in reduced RNA Polymerase II-mediated endogenous Asc gene transcription and mRNA processing, suggesting a co-transcriptional control of Asc gene expression. Ectopically expressed p205 induced expression of an Asc gene-luciferase reporter and collaborated with other transcription factors, such as c/EBPβ, p65/RelA, to further enhance expression. p205 knockdown also affected the expression of the immune genes Cd86, Cox2, Cxcl2, Il1α, Il10, Il12α, Il6 and Ifnα in LPS-stimulated macrophages. Together these findings suggest that p205 regulates inflammation through control of Asc gene expression, and other immune genes. Fungal infections activate both caspase 1-dependent and -independent inflammasomes. In an independent study, we show that Paracoccidioides brasiliensis fungal infection also induces caspase 8-dependent non-canonical inflammasome. Caspase 8-dependent IL-1β processing required dectin-1, Syk and Asc. Rip3-/- Casp8-/- mice infected with P. brasiliensis displayed increased fungal load and showed worse disease progression compared to wild type and Rip3-/- mice. These results revealed the importance of caspase 8 in activating and regulating inflammasome responses during fungal infection in vivo.