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.

Les virus influenza aviaires faiblement pathogènes (IAFP) ciblent principalement les épithéliums des voies respiratoires et intestinales chez les poulets (Gallus gallus) infectés. Cependant, les virus influenza aviaires hautement pathogènes (IAHP) mènent à une maladie systémique fatale avec une localisation particulière aux endothéliums. L’objectif de cette thèse a été d’explorer les relations entre la réplication des virus influenza aviaires (IA) et la réponse antivirale de l’hôte dans deux modèles cellulaires originaux obtenus chez le poulet : des cellules épithéliales pulmonaires (CLEC213) et des cellules endothéliales d’aortes (chAEC). Les résultats clés sont les suivants : (i) la réplication productive des virus IA dans les chAEC dépend du clivage de l’hémagglutinine et de l’échappement viral à la réponse immunitaire innée ; (ii) les CLEC213 sont très permissives aux virus IA et présentent une faible réponse antivirale médiée par la signalisation TLR3 et MDA5 ; (iii) les fonctions régulatrices de SOCS1 et SOCS3, sur le signal des interférons et des cytokines, sont conservées chez le poulet. Nous proposons que certains virus IA peuvent exploiter les fonctions pro-virales de SOCS1 et SOCS3 à leur avantage de manière spécifique au type cellulaire. / Low pathogenic avian influenza (LPAI) viruses essentially target the epithelia of the respiratory and intestinal tract in the infected chicken host (Gallus gallus). However, highly pathogenic avian influenza (HPAI) viruses induce a peracute fatal systemic disease and exhibit a striking endothelial cell tropism. The objective of the present thesis was to explore the interdependencies of AI virus replication and the antiviral host response in two novel avian cell culture models: chicken lung epithelial cells (CLEC213) and chicken aortic endothelial cells (chAEC). The salient findings from this study are that (i) productive AI virus replication in chAEC is dependent on hemagglutinin cleavability and appears to be related to innate immune escape; (ii) CLEC213 are highly permissive to AI virus infection, due to a cell type-specific diminished TLR3- and/or MDA5-mediated antiviral signaling response; (iii) the interferon and cytokine regulatory functions of SOCS1 and SOCS3 are conserved in the chicken. Based on our data, we propose a model that predicts that certain AI viruses may exploit the proviral functions of SOCS1 and SOCS3 in a cell type-specific manner.

Le syndrome d'ischémie-reperfusion (IR), inhérent à la transplantation rénale, est caractérisé par un infiltrat leucocytaire important et des lésions tissulaires graves dont les signaux initiateurs restent à ce jour peu décrits. Postulant que la libération d'alarmines par les cellules en nécrose est décisive dans ce processus, l'objectif principal du présent travail a été d'étudier la contribution de l'alarmine IL-33 dans la genèse des lésions tissulaires dans un modèle murin d'IR rénale. Nos résultats montrent que l'IL-33 est rapidement libérée du rein après IR comme protéine circulante, dès une heure de reperfusion. Les souris IL-33gt/gt, déficientes en IL-33, sont moins sensibles aux lésions induites par l’IR, comme l'attestent le maintien de la fonction rénale et des lésions histologiques atténuées avec un recrutement de polynucléaires neutrophiles (PNN) diminué par rapport aux souris contrôles. Ceci est associé à la perte du recrutement de cellules iNKT productrices d'IFN-γ/IL-17A. Parallèlement, les souris Jα18KO, déficientes en cellules iNKT et protégées contre les lésions d'IR, possèdent également des niveaux élevés d'IL-33 circulante. Nous proposons donc que l'IL-33 endogène contribue aux lésions d'IR en favorisant le recrutement de cellules iNKT, conduisant ainsi à un recrutement amplifié de PNN au niveau du rein lésé. Notre étude, en identifiant l'alarmine IL-33 comme un médiateur précoce de la réponse immunitaire innée induite par l'IR rénale, mettant en jeu les cellules iNKT, contribue à la compréhension des mécanismes impliqués dans la genèse des lésions associées à la greffe rénale et permet de proposer de nouvelles stratégies thérapeutiques. / Ischemia-reperfusion (IR) injury in renal transplantation is characterized by leukocyte infiltration and tissue damage. However, the signals that initiate these events remain poorly understood. Assuming that alarmin release by necrotic cells during IRI is critical, the main objective of the present study was to investigate the role of alarmin IL-33 in kidney injury using a mouse model of renal IR. We observed release of IL-33 shortly after kidney IR concomitantly with an increase in plasma levels of IL-33 within one hour of reperfusion. IL-33 deficient mice (IL-33gt/gt) exhibited reduced renal IR-induced injury, as attested by function preservation, reduced histological change and attenuation of neutrophil recruitment compared to control mice. This was associated with the loss of IFN-γ/IL-17A-producing iNKT cell recruitment. In the meantime, iNKT cell-deficient (Jα18KO) mice, also protected against IRI, have increased levels of circulating IL-33.These findings lead us to propose that endogenous IL-33 contributes to kidney IRI by promoting iNKT cell recruitment and cytokine production, thereby promoting amplified neutrophil recruitment to the injured kidney. The present study identifies the nuclear alarmin interleukin (IL)-33 as an important and early mediator of innate immune response, involving iNKT cells, following experimental kidney ischemia-reperfusion in mice. Our findings contribute to a better understanding of IR-induced injury and may lead to new therapeutic insights into renal-induced injury associated with renal transplantation.

Universidade Federal de Uberlândia / CHAPTER II: Infection in insects stimulates a complex defensive response. Recognition of pathogens may be accomplished by plasma or hemocyte proteins that bind specifically to bacterial or fungal polysaccharides. Several morphologically distinct hemocyte cell types cooperate in the immune response. Hemocytes attach to invading organisms and then isolate them by phagocytosis, by trapping them in hemocyte aggregates called nodules, or by forming an organized multicellular capsule around large parasites. In the current investigation the cellular population in the hemolymph third instar larvae of M. scutellaris has been characterized by means of light microscopy analysis and phagocytosis assays were performed in vivo by injection of 0,5μm fluorescence beads in order to identify the hemocyte types involved in phagocytosis. Four morphotypes of circulating hemocytes were found in 3rd instar larvae: prohemocytes, plasmatocytes, granulocytes and oenocytoids. The results presented plasmatocytes and granulocytes involved in phagocytic response of foreign particles in 3rd instar larvae of M. scutellaris. CHAPTER III: Insects are continuously exposed to potentially pathogenic microorganisms and eukaryotic parasites, but only a few encounters result in infection. Insects possess a complex and efficient system of biological defense against pathogens and parasites. This system involves the following: the integument and gut as physical barriers to infection, coordinated responses of several subpopulations of hemocytes when these barriers are breached, and the induced synthesis of antimicrobial peptides and proteins, primarily by the fat body. The purpose in the present study was to verify a Toll receptor (MsToll) expression in Melipona scutellaris. By semiquantitative RT-PCR we evaluate the MsToll levels at different development stages and in different M. scutellaris workers tissues. The MsToll expression in the immune response was evaluated by real time RT-PCR in workers infected with Escherichia coli (gram-negative). Our data showed lower MsToll expression in the larval stage compared with other development stages. The specific tissue analysis showed that its expression in intestine was significantly higher compared with other tissues analyzed. Furthermore, the MsToll levels in innate immune response of M. scutellaris showed four folds enhanced in bees infected with E. coli compared with control. / CAPITULO II: Infecção em insetos estimula uma resposta defensiva complexa. O reconhecimento de patógenos pode ser realizado pelos hemócitos ou proteínas que se ligam especificamente em microorganismos com padrões moleculares específicos, os chamados (PAMPs). Diferentes células da hemolinfa cooperam na resposta imune. Os hemócitos reconhecem os patógenos e os isolam por fagocitose, formando nódulos ou, cápsula multicelular em torno do parasita. Nesse trabalho foram identificadas as células da hemolinfa da abelha sem ferrão Melipona scutellaris e caracterizados os hemócitos envolvidos no processo de fagocitose utilizando beads de 0,5μm de diâmetro, em média, com fluorescência vermelha. Na hemolinfa do 3° instar larval de M. scutellaris foram distinguidos quatro tipos de hemócitos: prohemócitos, plasmatócitos, granulócitos e oenocitóides. No ensaio de fagocitose foram identificados plasmatócitos e granulócitos, com beads fluorescentes fagocitados no citoplasma. CAPITULO III: Insetos são continuamente expostos a microrganismos potencialmente patogênicos, mas apenas alguns contatos resultam em infecção. Insetos possuem um complexo e eficiente sistema de defesa contra patógenos e parasitas, que envolve o tegumento e intestino como barreiras físicas para infecção; respostas coordenadas de vários tipos de hemócitos quando estas barreiras são violadas e a síntese de peptídeos antimicrobianos e proteínas, principalmente pelo corpo gorduroso. Nosso objetivo foi clonar e sequenciar parcialmente um gene do sistema imune inato MsToll da abelha Melipona scutellaris. Por análises de RT-PCR semiquantitativo avaliou-se os níveis de expressão de MsToll em diferentes estágios do desenvolvimento e em diferentes tecidos de operárias de M. scutellaris. A expressão de MsToll na resposta imune foi avaliada por RT-PCR tempo real em operarias infectadas com Escherichia coli (gram-negativa). Os resultados mostraram menor expressão do gene MsToll nos estágios larvais quando comparados com os demais estágios do desenvolvimento. A análise tecido específico de MsToll mostrou que em intestino sua expressão foi significativamente maior quando relacionado com os demais tecidos analisados. Com relação aos níveis de MsToll na resposta imune observou-se o aumentou de quatro vezes dos níveis desse transcrito em abelhas infectadas com E. coli comparadas com o controle. / Mestre em Genética e Bioquímica

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-02-24T13:29:14Z No. of bitstreams: 1 micaelpetersxavier.pdf: 17481766 bytes, checksum: fb76bff140085a73dc148ca7493df8b3 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-02-24T15:36:12Z (GMT) No. of bitstreams: 1 micaelpetersxavier.pdf: 17481766 bytes, checksum: fb76bff140085a73dc148ca7493df8b3 (MD5) / Made available in DSpace on 2017-02-24T15:36:12Z (GMT). No. of bitstreams: 1 micaelpetersxavier.pdf: 17481766 bytes, checksum: fb76bff140085a73dc148ca7493df8b3 (MD5) Previous issue date: 2013-08-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O desenvolvimento de sistemas computacionais que simulam o funcionamento de tecidos ou mesmo de órgãos completos é uma tarefa extremamente complexa. Um dos muitos obstáculos relacionados ao desenvolvimento de tais sistemas é o enorme poder computacional necessário para a execução das simulações. Por essa razão, o uso de estratégias e métodos que empregam computação paralela são essenciais. Este trabalho foca na simulação temporal e espacial, em uma seção tridimensional de tecido, do comportamento de algumas das células e moléculas que constituem o sistema imunológico humano (SIH) inato. Com o objetivo de reduzir o tempo necessário para realizar a simulação, foram utilizadas múltiplas unidades de processamento gráfico (Graphics Processing Unit, GPUs) em um ambiente de agregados computacionais. Apesar do alto custo de comunicação imposto pelo uso de múltiplas GPUs, as abordagens e técnicas utilizadas neste trabalho para implementar as versões paralelas do simulador mostraram-se efetivas para alcançar o objetivo de redução do tempo de simulação. / The development of computer systems that simulate the behavior of tissues or even whole organs is an extremely complex task. One of the many obstacles related to the development of such systems is the huge computational resources needed to execute the simulations. For this reason, the use of strategies and methods that employ parallel computing are essential. This work focuses on the spatial-temporal simulation of some human innate immune system (HIS) cells and molecules in a three-dimensional section of tissue. Aiming to reduce the time required to perform the simulation, multiple graphics processing units (GPUs) were used in a cluster environment. Despite of high communication cost imposed by the use of multiple GPUs, the approaches and techniques used in this work to implement parallel versions of the simulator proved to be very effective in their purpose of reducing the simulation time.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-03-02T17:47:54Z No. of bitstreams: 1 pedroaugustoferreirarocha.pdf: 4715587 bytes, checksum: dfef00badf9cc3d7c79c1b4c62d3abfd (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-03-06T19:58:07Z (GMT) No. of bitstreams: 1 pedroaugustoferreirarocha.pdf: 4715587 bytes, checksum: dfef00badf9cc3d7c79c1b4c62d3abfd (MD5) / Made available in DSpace on 2017-03-06T19:58:07Z (GMT). No. of bitstreams: 1 pedroaugustoferreirarocha.pdf: 4715587 bytes, checksum: dfef00badf9cc3d7c79c1b4c62d3abfd (MD5) Previous issue date: 2012-08-27 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Dois mecanismos são utilizados pelo Sistema Imunológico Humano (SIH) para defender o organismo contra doenças causadas pelos mais distintos agentes patogênicos: o sistema inato e o sistema adaptativo. O primeiro é composto por células e substâncias químicas que utilizam um mecanismo genérico de defesa para prevenir ou limitar infecções ocasionadas pela maioria dos patógenos. Já o segundo mecanismo é ativado pelo primeiro, baseando-se na habilidade de reconhecer e de recordar agentes patogênicos específicos, colaborando para a montagem de um ataque mais potente a cada vez que o mesmo patógeno é encontrado. Apesar de ser muito estudado, muitas questões sobre o funcionamento do SIH ainda estão em aberto em virtude de sua complexidade e do grande número de interações, nos mais diversos níveis, entre seus distintos componentes. Neste sentido, ferramentas computacionais podem se constituir em um poderoso ferramental para auxiliar nas pesquisas sobre o tema. O presente trabalho está inserido neste escopo, dividindo-se em duas partes. Na primeira parte, o trabalho apresenta os resultados de uma análise de sensibilidade em um modelo matemático-computacional que simula a resposta imunológica inata ao lipopolissacarídeo (LPS), com o objetivo de encontrar os parâmetros mais sensíveis deste modelo. Além disto, a segunda parte do trabalho propõe uma adaptação do modelo original para um modelo tridimensional. As simulações realizadas nas duas partes do trabalho mostraram-se computacionalmente caras, demandando longos períodos de tempo para serem concluídas. Assim, GPGPUs (General Purpose Graphics Processing Units) foram utilizadas para reduzir os tempos de execução. O uso de GPGPUs permitiu que acelerações de 276 vezes para a análise de sensibilidade massiva e de 87 vezes para a computação do modelo em três dimensões fossem obtidas. / Two mechanisms are used by the Humman Immune System (HIS) to protect the body against diseases caused by distinct pathogens: the innate and the adaptive immune system. The first one is composed of cells and chemicals that use a generic mechanism of defense to prevent or limit infections caused by most pathogens. The second mechanism is activated by the first one. It has the ability to recognize and remember specific pathogens, contributing to the assembly of a more powerful attack each time the same pathogen is encountered again. Despite being widely studied, many questions about the functioning of the HIS are still open because of its complexity and the large number of interactions of its components on distinct levels. In this sense, computational tools are a powerful instrument to assist researchers on this field of study. This work is inserted in this scope and it is split into two parts. In the first part, this work presents the results of a sensitivity analysis on a mathematical-computational model that simulates the innate immune response to lipopolysaccharide (LPS). The main objective of the sensitivity analysis was to find the most sensitive parameters of the mathematical model. The second part of this work proposes the extension of the original model to a three-dimensional one. The simulations in the two parts of the work proved to be computationally expensive, requiring long periods of time to complete. Thus, GPGPUs (General Purpose Graphics Processing Units) were used to reduce execution times. The use of GPGPUs allowed speedups of 276 times for sensitivity analysis, when compared to the sequential one, and of 87 times for computations using the three dimensions model.

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.

Human immunodeficiency virus type 1 (HIV-1) is a lentivirus that causes acquired immunodeficiency syndrome (AIDS). Since the first cases of AIDS were described in 1981, HIV-1 has become one of the most serious public health threats in the world. There are approximately 38 million people worldwide currently living with HIV-1. 28 million of these people have access to antiretroviral therapy (ART) that is highly effective in reducing viral load to undetectable levels, thereby curbing the risk of viral transmission and preventing progression to AIDS. Despite their effectiveness in suppressing HIV-1 viremia, systemic inflammation remains as a hallmark of HIV-1 infection in vivo. This persistent immune activation is often associated with non-AIDS related complications, including elevated risk of neurocognitive and cardiovascular disorders. Several different mechanisms may contribute to this chronic immune activation and inflammation in people living with HIV-1 on ART. One of the contributing factors might be HIV-1 RNA expressed from the provirus. Even though ART potently suppresses HIV-1 replication, it fails to eradicate proviruses established prior to initiation of ART. Ongoing activation of CD4+ T cells and macrophages by HIV-1 proviral transcripts might contribute to the persistent inflammation that remains even after HIV-1 suppression by ART. Previously, our laboratory has shown that induction of innate immune signaling after HIV-1 challenge of primary human dendritic cells (DCs), macrophages, or CD4+ T cells requires integration, transcription from the nascent provirus, and nuclear export of intron-containing HIV-1 RNA through the Rev-CRM1 pathway. However, these studies failed to identify the innate immune sensor of intron-containing HIV-1 RNA. Here we conducted a targeted loss-of-function screen, using shRNA-expressing lentivectors in human DCs to identify this innate immune receptor. Of the twenty-one candidate genes targeted for knockdown by shRNA, the innate immune response to HIV-1 was inhibited only by knockdown of IFIH1, MAVS, and XPO1. The effect of IFIH1 and MAVS knockdowns on HIV-1-induced immune activation was confirmed in macrophages, and rescue of the knockdown with non-targetable coding sequence showed that IFIH1 protein was required. IFIH1 mutants that are defective for interaction with MAVS blocked activation, demonstrating that MAVS acts downstream of IFIH1 in this system. Since both IFIH1 and DDX58 signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation; the IFIH1-specific inhibitor Nipah virus V protein blocked the activation by HIV-1. RNA-Seq showed that IFIH1-knockdown in DCs globally disrupted the induction of IFN-1-stimulated genes. Altogether, results presented in this thesis reveal that IFIH1 is required for innate immune activation by intron-containing RNA from the HIV-1 provirus, and potentially contributes to chronic inflammation in people living with HIV-1.

The principle of trained immunity represents innate immune memory due to sustained, mainly epigenetic, changes triggered by endogenous or exogenous stimuli in bone marrow (BM) progenitors (central trained immunity) and their innate immune cell progeny, thereby triggering elevated responsiveness against secondary stimuli. BM progenitors can respond to microbial and sterile signals, thereby possibly acquiring trained immunity-mediated long-lasting alterations that may shape the fate and function of their progeny, for example, neutrophils. Neutrophils, the most abundant innate immune cell population, are produced in the BM from committed progenitor cells in a process designated granulopoiesis. Neutrophils are the first responders against infectious or inflammatory challenges and have versatile functions in immunity. Together with other innate immune cells, neutrophils are effectors of peripheral trained immunity. However, given the short lifetime of neutrophils, their ability to acquire immunological memory may lie in the central training of their BM progenitors resulting in generation of reprogrammed, that is, “trained”, neutrophils. Although trained immunity may have beneficial effects in infection or cancer, it may also mediate detrimental outcomes in chronic inflammation. Here, we review the emerging research area of trained immunity with a particular emphasis on the role of neutrophils and granulopoiesis.

No description available.