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1
Schattgen, Stefan A. "Sensing of Endogenous Nucleic Acids by the Innate Immune System during Viral Infection: A Dissertation." eScholarship@UMMS, 2003. http://escholarship.umassmed.edu/gsbs_diss/764.
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Innate sensing of nucleic acids lies at the heart of antiviral host defense. However, aberrant activation of innate sensors by host nucleic acids can also lead to the development of autoimmune diseases. Such host nucleic acids can also be released from stressed, damaged or dying cells into the tissue microenvironment. It however remains unclear how the extracellular nucleic acids impacts the quality of the host immune responses against viral infections. Using a mouse model of influenza A virus (IAV) infection, we uncovered an important immune-regulatory pathway that tempers the intensity of the host-response to infection. We found that host-derived DNA from necrotic cells accumulates in the lung microenvironment during IAV infection, and is sensed by the DNA receptor Absent in Melanoma 2 (AIM2). AIM2-deficiency resulted in severe immune pathology highlighted by enhanced recruitments of immune cells, and excessive systemic inflammation after IAV challenge, which led to increased morbidity and lethality in IAV-infected mice. Interestingly, these effects of AIM2 were largely independent of its ability to mediate IL-1β maturation through inflammasome complexes. Finally, ablation of accumulated DNA in the lung by transgenic expression of DNaseI in vivo had similar effects. Collectively, our results identify a novel mechanism of cross talk between PRR pathways, where sensing of hostderived nucleic acids limits immune mediated damage to virus infected tissues.
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Schattgen, Stefan A. "Sensing of Endogenous Nucleic Acids by the Innate Immune System during Viral Infection: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/764.
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Innate sensing of nucleic acids lies at the heart of antiviral host defense. However, aberrant activation of innate sensors by host nucleic acids can also lead to the development of autoimmune diseases. Such host nucleic acids can also be released from stressed, damaged or dying cells into the tissue microenvironment. It however remains unclear how the extracellular nucleic acids impacts the quality of the host immune responses against viral infections. Using a mouse model of influenza A virus (IAV) infection, we uncovered an important immune-regulatory pathway that tempers the intensity of the host-response to infection. We found that host-derived DNA from necrotic cells accumulates in the lung microenvironment during IAV infection, and is sensed by the DNA receptor Absent in Melanoma 2 (AIM2). AIM2-deficiency resulted in severe immune pathology highlighted by enhanced recruitments of immune cells, and excessive systemic inflammation after IAV challenge, which led to increased morbidity and lethality in IAV-infected mice. Interestingly, these effects of AIM2 were largely independent of its ability to mediate IL-1β maturation through inflammasome complexes. Finally, ablation of accumulated DNA in the lung by transgenic expression of DNaseI in vivo had similar effects. Collectively, our results identify a novel mechanism of cross talk between PRR pathways, where sensing of hostderived nucleic acids limits immune mediated damage to virus infected tissues.
3
Adindu, Uzowuru Cosmas. "Inflammasome : Investigating the effect of NEK7 in the activation of the NLRP3 Inflammasome." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-18937.
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Inflammation is a biological defence mechanism applied by living organisms against foreign invaders. In the response to DAMPs and PAMPs, organisms use inflammatory multi-protein complexes to fight the attackers. The most studied inflammasome proteins are NLRP3, ASC and Caspase-1. This study is aimed at understanding the role of NEK7 protein in the NLRP3 inflammasome’s activation, using CRISPR/Cas9 system. To determine the effect of CRISPR/Cas9 and transfection, mRNA expression was analyzed. The results obtained suggest that neither the transfection nor the NEK7 protein knockout have sufficiently worked. This study could not experimentally establish that NEK7 triggers NLRP3 inflammasome activation because ELISA was not conducted to verify the levels of cytokines emitted, due to there being no statistical differences between the samples. Above all, the research question in this thesis project was not answered because the instability of the ACTB reference gene negatively influenced the results. However, previous related studies conclude that NEK7 plays a crucial role in the activation of the NLRP3 inflammasome.
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Janczy, John Roger. "Mechanisms for activation and inhibition of inflammasomes." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/1643.
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Activation of the cysteine protease caspase-1 and the subsequent processing and secretion of the pro-inflammatory cytokines IL-1Β and IL-18 is central to the inflammatory response as well as the induction of adaptive immune responses. Caspase-1 is activated as a part of a high-molecular weight multi-protein complex termed the inflammasome. The NLRP3 inflammasome is by far the best studied of these complexes, and it is the most promiscuous in terms of activating signals. The diversity of NLRP3 activating signals makes it likely that NLRP3 does not recognize each agonist directly, rather it detects a molecule that is generated, revealed, or altered by cellular stress. Recent studies have indicated that mitochondrial dysfunction is crucial for NLRP3 inflammasome activation, yet the activating ligand has not yet been identified. Appropriate and timely activation of this inflammatory pathway is required for host immunity to a variety of pathogens, however dysregulated activation leads to autoinflammation and potentially autoimmunity. Hence it is important to identify mechanisms for inflammasome activation and regulation. Therefore, this dissertation has focused on investigating the mechanisms for activation and regulation of the NLRP3 inflammasome, and the biological consequences of these changes. We show that the mitochondrial lipid cardiolipin is required for NLRP3 inflammasome activation. We have also identifying a novel mechanism by which inflammasome activation is regulated. Data presented in this dissertation shows that IgG immune complexes effectively suppress inflammasome activation and the subsequent processing and secretion of IL-1Α and IL-1Β. Furthermore we show that immunization with IgG immune complexes suppresses both Th2 and Th17 immune responses. Together these data provide novel insights into the activating and regulatory pathways of both the innate and adaptive immune systems.
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Srinivasan, N. "The role of inflammasomes in intestinal inflammation." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:04ad577c-a8dd-46eb-811a-79a3980ff806.
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Single Nucleotide Polymorphisms (SNPs) in the intracellular pattern recognition receptor gene NLRP3 are associated with susceptibility to Crohn’s disease, a form of inflammatory bowel disease (IBD). Following cell damage or infection, NLRP3 triggers the formation of inflammasomes, a multimolecular protein complex containing NLRP3, ASC and caspase-1, which mediate secretion of IL-1β and IL-18. NLRP3 inflammasome activation in macrophages has been implicated in protection against several pathogens, but whether NLRP3 activation in tissue cells contributes to protective immunity against bacterial pathogens is unknown. We show that upon infection with the attaching/effacing (A/E) intestinal pathogen Citrobacter rodentium, Nlrp3-/- and Asc-/- mice displayed higher bacterial colonization, more weight loss and exacerbated intestinal inflammation. We further show that Nlrp3 inflammasome activation in intestinal epithelial cells (IECs) acts rapidly after infection to limit bacterial replication and penetration, and inhibits the development of inflammatory pathology in the gut. We also show that epithelial Nlrp3-mediated protection is independent of the classical inflammasome cytokines IL-1β and IL-18. Thus an Nlrp3-Asc circuit in IECs regulates early defense against a mucosal pathogen and limits inflammation in the intestine. Nlrp3 inflammasome activation has also been implicated in protection in acute models of experimental colitis, but its role in chronic models of colitis is unknown. We found that Asc signaling is necessary for the development of innate chronic intestinal inflammation driven by Helicobacter hepaticus. Thus while deficient inflammasome signaling in tissue cells increases susceptibility towards enteric pathogens, excessive inflammasome activation can drive chronic intestinal inflammation.
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Kannan, Harsha. "The Inflammasome in Acute Myocarditis." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/3108.
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Acute myocarditis is an acute inflammatory syndrome characterized by myocardial damage and dysfunction often due to a viral infection followed by a variable development over time. There are currently no specific treatments and standard treatments for heart failure are generally applied. The inflammasome is a recently identified macromolecular structure that occupies a central role in the amplification of the inflammatory response and promotion of cell death during acute and chronic infections. We hypothesized the formation of the inflammasome in acute myocarditis. To investigate, samples of patients were collected from the Cardiomyopathy Registry in Trieste, with 12 cases of biopsy-proven myocarditis and 11 cases of autopsy-proven myocarditis stained for major components of the inflammasome through immunofluorescence; 10 of the 12 (83.3%) biopsy cases and 8 of the 11 (72.7%) autopsy cases presented formation of the inflammasome in a variety of cells including resident cells (i.e. cardiomyocytes, endothelial cells, fibroblasts) and infiltrating cells (i.e. leukocytes) while varying in intensity and distribution. Control samples of 5 subjects not presenting with any acute cardiac events showed no formation of the inflammasome. While further studies should look to elucidate the correlation of inflammasome-formation and progression of disease, this finding paves the way for further insight into the pathophysiology of acute myocarditis.
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Poli, Caroline. "Il-26 : une cytokine pro-inflammatoire stimulant les cellules immunitaires innées myéloïdes." Thesis, Angers, 2017. http://www.theses.fr/2017ANGE0068.
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Physiologiquement, l’ADN, séquestré dans les noyaux, n’est pas détecté par les récepteurs de danger du système immunitaire. En revanche, au cours d’inflammations chroniques, une rupture de la tolérance vis-à-vis de l’ADN du soi, consécutivement à sa libération dans le milieu extracellulaire par les cellules mortes, a été démontrée. L’accès de l’ADN extracellulaire aux récepteurs intracellulaires est médié par des molécules cargo capables de s’associer à l’ADN extracellulaire et d’induire sont internalisation.L’IL-26, qui est un membre de la famille de l’IL-10, a été décrite comme une cytokine pro-inflammatoire, dont les mécanismes moléculaires restent mal connus. Nous avons démontré que l’IL-26 se lie à l’ADN, permet son internalisation dans le cytosol des cellules myéloïdes et la sécrétion de cytokines pro inflammatoires via la voie STING et la voie des inflammasomes. La modélisation informatique de l’IL-26, basée sur la structure de l’IL-10,met en évidence des caractéristiques structurales similaires aux molécules cargo : un domaine de liaison à l’ADN, deux hélices amphipathiques et un motif d’ancrage à la membrane. De plus, des complexes circulants d’IL-26-ADN sont retrouvés dans les sérums de patients en poussée aigues de vascularite à ANCA,une pathologie inflammatoire chronique. L’IL-26 est détectée dans les lésions de glomérulonéphrite aigue nécrosante à croissants, ainsi qu’au niveau des cellules musculaires lisses artérielles de ces patients. Ces dernières sécrètent de l’IL-26 en présence de cytokinespro-inflammatoires. En conclusion, l’IL-26 établit d’une boucle d’autoamplification entre une mort cellulaire intense et l’inflammationIn physiological conditions, self-DNA released by dying cells is not detected by intracellular DNA sensors. Inchronic inflammatory disorders, unabated inflammation has been associated with a break in innate immune tolerance to self-DNA. However, to gain access to intracellular DNA sensors, extracellular DNA has to complex with DNA-binding molecules. IL-26 is a member of the IL-10 cytokine family, overexpressed in numerous chronic inflammatory diseases, which biological activity remains unclear. We demonstrate here that IL-26 binds to DNA and shuttles it in the cytosol of human myeloid cells. As a consequence, IL-26 allows extracellular DNA to trigger proinflammatory cytokine secretion by monocytes, in a STING- and inflammasome-dependent manner. Supporting these biological properties, IL-10-based modelling predicts two DNA-binding domains, two amphipathic helices, and an in-plane membrane anchor in IL-26, structural features of cationic amphipathic cell penetrating peptides. In line with these properties, patients with active autoantibody-associated vasculitis, a chronic relapsing autoimmune inflammatory disease associated with extensive cell death, exhibit high levels of both circulating IL-26 and IL-26-DNA complexes. Moreover, in patients with crescentic glomerulonephritis, IL-26 is expressed by renal arterial smooth muscle cells and deposits in necrotizing lesions. Accordingly, human primary smooth cells secrete IL-26 in response to proinflammatory cytokines. In conclusion, IL-26 expressed in lesions confers proinflammatory properties to DNA released by dying cells, setting up a positive amplification loop between extensive cell death and unabated inflammation
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de, Almeida Pereira Milton César. "Inflammasome signalling during Salmonella Typhimurium infection." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/283642.
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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.
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Palazón, Pablo. "Mechanisms and consequences of inflammasome activation." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/mechanisms-and-consequences-of-inflammasome-activation(995047bf-afce-496f-86be-efb0034ad490).html.
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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.
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Cirelli, Kimberly M. "Rodent inflammasome activation by Toxoplasma gondii." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/105635.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.Cataloged from PDF version of thesis.
Includes bibliographical references.
Toxoplasma gondii is an obligate intracellular pathogen capable of chronically infecting nearly all warm-blooded animals, including humans. The chronic stage is characterized by the presence of semi-dormant cysts in brain and muscle tissues. These cysts are crucial in the success of Toxoplasma as they are orally infectious and allow for the transmission of the parasite between hosts. As the host immune response drives cyst formation, the establishment of this chronic infection relies on the parasite's ability to find a balance between activation of a host immune response and evasion of parasiticidal mechanisms. This balance is achieved through the modulation of host cell processes by parasite proteins secreted from specialized secretory organelles known as rhoptries and dense granules. Here, we report that Toxoplasma activates the inflammasomes in mice and rats. The inflammasomes are a set of cytoplasmic pattern recognition receptors (PRRs). Activation of the inflammasomes results in caspase-1 activation and the cleavage and release of the pro-inflammatory cytokines, Interleukin (IL)-1[beta] and IL-18. IL-1p is an important mediator of local inflammation and neutrophil recruitment. IL- 18 induces Interferon (IFN)-[gamma], which is a critical cytokine in the control of Toxoplasma. A form of cell death, termed pyroptosis, can accompany inflammasome activation. The NLRP3 inflammasome is activated in mouse macrophages, leading to the secretion of IL-1[beta] in vitro. The NLRP1 and NLRP3 inflammasomes play a major role in mouse survival and control of parasite replication in vivo. The NLRPI inflammasome is activated in infected macrophages from rats that are able to completely clear infection. Toxoplasma infection leads to the secretion of active IL-I[beta] and IL-18. Activation of the NLRP1 inflammasome leads to pyroptosis, a programmed form of cell death. Pyroptosis prevents parasite replication within the host cell and likely promotes clearance by nearby immune cells. Using a chemical mutagenesis screen, we identified three Toxoplasma dense granule proteins (GRAs), GRA18, GRA27 and GRA28, essential for NLRP1 inflammasome activation and pyroptosis in rat macrophages. Our work has identified Toxoplasma gondii as a novel activator of the rodent inflammasomes and demonstrated host cell death as a mechanism to control parasite replication. We have also identified three novel parasite proteins required for this activation, providing insight into interactions between parasite and host, which may aid in the treatment of human infection.
by Kimberly M. Cirelli.
Ph. D.
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Jamilloux, Yvan. "Les inflammasomes : de la régulation aux maladies auto-inflammatoires." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1089/document.
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Les inflammasomes sont des complexes protéiques intracellulaires qui ont un rôle majeur dans l'immunité innée. Leur activation conduit à la mort de la cellule dans un contexte hyperinflammatoire. Compte-tenu des effets potentiellement délétères, tissulaires et systémiques, les inflammasomes sont strictement régulés. A l'heure actuelle, la compréhension des mécanismes conduisant à leur activation et leur régulation reste partielle. Dans une première partie de cette thèse, nous avons utilisé une technique de biotinylation proximale (BioID) pour identifier les protéines interagissant avec l'inflammasome. Nous avons identifié 111 protéines dont la relation étroite avec l'inflammasome était vraisemblable. Parmi ces 111 protéines, 25% avaient d'ailleurs déjà été décrites comme des protéines interagissant avec le complexe. L'identification d'un adaptateur majeur de l'autophagie, p62/sequestosome-1 (p62), nous a conduit à focaliser notre attention sur son rôle dans la régulation de l'inflammasome. Nous avons d'abord démontré que l'interaction entre p62 et l'inflammasome existait, sur le plan biochimique. Par la suite, nous avons prouvé que p62 était un substrat du complexe et que l'activation de ce dernier entrainait le clivage de p62 au niveau d'un résidu aspartique en position 329. Enfin, nous avons caractérisé les conséquences fonctionnelles de ce clivage, en montrant que les fragments protéiques générés entrainaient une régulation positive ou négative du complexe. Nous avons alors émis l'hypothèse que p62 pourrait réguler l'inflammasome de manière différente selon le signal activateur. Dans une seconde partie, translationnelle, nous nous sommes intéressés aux conséquences des mutations dans la séquence de gènes codant les constituants de l'inflammasome ou des protéines régulatrices du complexe. Celles-ci sont à l'origine des maladies auto-inflammatoires monogéniques. Ces maladies sont caractérisées par des épisodes récurrents de fièvre associés variablement à d'autres symptômes systémiques. La plus fréquente est la fièvre méditerranéenne familiale (FMF), avec une prévalence estimée entre 1 et 5 pour 10 000 habitants en France. Les mutations du gène MEFV, codant la pyrine, sont à l'origine de la FMF. La pyrine peut induire la formation d'un inflammasome spécifique. Récemment, le mécanisme d'activation de l'inflammasome pyrine a été mieux caractérisé : certaines toxines (comme la toxine B du Clostridium difficile, TcdB) induisent l'activation de l'inflammasome pyrine. Nous avons utilisé ces nouvelles connaissances afin d'explorer les conséquences de l'activation de l'inflammasome pyrine par la TcdB dans les monocytes des patients atteints de FMF, comparés aux monocytes de donneurs sains. Nos résultats indiquent que ces mutations induisent un abaissement du seuil d'activation de l'inflammasome pyrine. Par ailleurs, les corrélations génotype/phénotype indiquent qu'il existe un effet de dosage génétique, en lien avec le nombre d'allèles mutés. Ces résultats ouvrent de nouvelles perspectives pour les patients atteints de FMF, tant dans la compréhension de la physiopathologie que dans la possibilité de mise au point de tests fonctionnels pour le diagnostic de la maladieInflammasomes are intracellular multiprotein complexes that have a major role in innate immunity. Their activation leads to hyperinflammatory cell death. In view of potentially deleterious effects, the inflammasomes are strictly regulated. At present, the understanding of the mechanisms leading to their activation and regulation remains partial. In a first part of this thesis, we used a technique of proximity-dependent biotinylation (BioID) to identify the proteins interacting with the inflammasome. We identified 111 proteins with a close relationship to the inflammasome. Among these 111 proteins, 25% had already been described as proteins interacting with the complex. The identification of a major adaptor of autophagy, p62/sequestosome-1 (p62), led us to focus our attention on its role in the regulation of inflammasome. We first demonstrated that the interaction between p62 and inflammasome was real, at the biochemical level. Subsequently, we proved that p62 was a substrate of the complex and that the activation of the latter led to the cleavage of p62 at the aspartate 329. Finally, we characterized the functional consequences of this cleavage and showed that the protein fragments generated led to a positive or negative regulation of the complex. We thus hypothesized that p62 could regulate the inflammasome differently according to the activator signal. In a second translational part, we looked at the consequences of mutations in the sequence of genes coding components of the inflammasome or proteins regulating it. These are the cause of monogenic auto-inflammatory diseases. These diseases are characterized by recurrent episodes of fever associated with other systemic symptoms. The most frequent is Familial Mediterranean Fever (FMF), with prevalence estimated at between 1 and 5 per 10 000 inhabitants, in France. Mutations of the MEFV gene, encoding pyrin, cause FMF. Pyrine may trigger the formation of a specific inflammasome. Recently, the mechanism of activation of the pyrin inflammasome has been better characterized: toxins (such as toxin B of Clostridium difficile, TcdB) induce the activation of the pyrin inflammasome. We used this new knowledge to investigate the consequences of TcdB on pyrin inflammasome activation in monocytes from FMF patients compared to monocytes from healthy donors. Our results indicate that these mutations induce a decreased threshold of activation of the pyrin inflammasome. In addition, genotype / phenotype correlations indicate a gene-dosage effect, related to the number of mutated alleles. These results open new perspectives for patients with FMF, in understanding the pathophysiology of the diseass and in developing functional diagnostic tests
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Groslambert, Marine. "Etude de l'impact fonctionnel des modifications post-traductionnelles dans l'activation de l'inflammasome NLRP3." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSEN022.
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L'inflammation est un processus déclenché suite à la détection de pathogènes et de dommages tissulaires. Elle conduit à la sécrétion de cytokines pro-inflammatoires par les cellules immunitaires innées ainsi qu'au déclenchement de la pyroptose, mort cellulaire pro-inflammatoire. NLRP3 est une protéine senseur de stress cellulaire régulant le déclenchement de ces processus via la formation d'une plateforme multiprotéique appelée inflammasome. L'activation non contrôllée de NLRP3 conduit au développement d'une maladie auto-inflammatoire appelée CAPS (Cryopyrin associated periodic syndrome). De plus, l'inflammasome est impliqué dans le développement et la sévérité des symptômes de nombreuses maladies multifactorielles (diabète de type 2, athérosclérose, maladie de Parkinson et d'Alzheimer, sclérose en plaques, cancers...). Les mécanismes régulant l'activation de NLRP3 ne sont pas encore compris, mais les modifications post-traductionnelles de NLRP3 sont impliquées dans ce processus. Notre laboratoire a identifié différents sites d'ubiquitination et de phosphorylation sur NLRP3 par des approches biochimiques. Via la création de lignées cellulaires NLRP3 knock out reconstituées pour exprimer NLRP3 muté sur les résidus précédemment identifiés et de souris NLRP3 knock-in par la technique de CRISPR/Cas9, le travail de thèse a consisté en l'étude de l'impact fonctionnel de ces modifications. Ces résultats montrent que les substitutions de deux lysines identifiées comme étant ubiquitinées conduisent à une dérégulation de l’activation de l'inflammasome NLRP3 dans les cellules primaires. Un nouveau point de contrôle de l'activation de NLRP3 a ainsi pu être mis en lumièreInflammation is triggered after the sensing of pathogens or tissue damages. This process leads to the secretion of pro-inflammatory cytokines by innate immune cells and to the triggering of a pro-inflammatory form of cell death called pyroptosis. NLRP3 protein is a sensor of cellular stress and regulates the triggering of these events through the formation of a multiproteic platform called inflammasome. NLRP3 activation has to be tightly controlled as its deregulation leads to the development of an auto-inflammatory disease called CAPS (Cryopyrin associated periodic syndrome). Moreover, NLRP3 inflammasome is associated with the development and the severity of numerous multifactorial diseases (type 2 diabetes, atherosclerosis, Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, cancers…). The mechanisms involved in the regulation of NLRP3 activation are not fully understood. Recently, post-translational modifications of NLRP3 were shown to be important for the regulation of NLRP3 activation. Our lab has identified several phosphorylation and ubiquitination sites on this protein through biochemical studies. This phD work aims to identify the functional impact of these modifications. Thus, the generation of reconstituted cell lines expressing NLRP3 mutated on the previously identified residues and the generation of NLRP3 knock in mice via CRISPR/Cas9 technology were performed. The results show that substitution of two lysine residues previously identified as ubiquitinated leads to the deregulation of NLRP3 inflammasome activation in primary cells. This work highlights a new point of control in NLRP3 activation
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Kader, Shoxan. "Modulation of nlrp3 inflammasome by sp110 : Regulation and inhibition of NLRP3 inflammasome in Sp110 deficient THP-1 cells." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-12907.
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Chesnokov, Anton P. Mr. "Modulation of NALP3 Inflammasome Genes by Estrogen." Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/biology_theses/33.
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Immunity is known to be sexually dimorphic. This dimorphism may be attributed to the action of different hormones. Aluminum is a component of several vaccines and acts as an adjuvant of immunogenicity. The activation of the Nalp3 inflammasome plays a role in aluminum’s adjuvancy. Estrogen affects immune cells by regulating the expression of genes involved in immune-related mechanisms; such as the modulation of cytokine secretion. We hypothesized that estrogen modulates the aluminum-induced secretion of IL-1β and IL-18. Using an ex vivo mouse macrophage model this study examined: (i) the effects of estrogen on Nalp3 inflammasome genes expression and (ii) the estrogen receptor involved in the modulation of these genes. Our results indicate that estrogen up-regulates Nalp3 gene expression via ERα/β heterodimerization, and caspase-1 activity may be indirectly modulated due to the up-regulation of SPI-6 via ERβ.
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Tremel, Nadin. "Bedeutung des NLRP3-Inflammasoms bei der Pneumokokkenmeningitis." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-170983.
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He, Qiong. "Mechanism of linezolid-induced NLRP3 inflammasome activation." Thesis, University of Iowa, 2012. https://ir.uiowa.edu/etd/4855.
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Activation of the NLRP3 inflammasome has been shown in response to numerous activators; here we show that the oxazolidinone antibiotic linezolid results in both the NLRP3-dependent in vitro release of the proinflammatory cytokine IL-1 Α; and in vivo neutrophilic influx following its intraperitoneal administration. Clinical use of linezolid is commonly limited by hematologic side effects; herein we also show NLRP3-deficiency protected animals against linezolid-induced effects on the bone marrow. Importantly, all previously described activators of the NLRP3 inflammasome have required the generation of reactive oxygen species (ROS). Linezolid is however unique amongst NLRP3 agonists in that its ability to activate the NLRP3 inflammasome in a ROS-independent manner. The pathways for ROS-dependent and ROS-independent NLRP3 activation converge upon mitochondrial dysfunction and specifically the mitochondrial lipid cardiolipin. We demonstrated that interference with cardiolipin synthesis specifically inhibits NLRP3 inflammasome activation. These findings firstly suggests that ROS generation is not the canonical activator of NLRP3 but rather an intermediary step leading to the mitochondrial perturbation that is tied to NLRP3 inflammasome activation and also implicate the involvement of mitochondrial lipid cardiolipin in this process; secondarily, linezolid-induced NLRP3 activation may account for thetoxicity associated with prolonged usage of this antibiotic.
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Elliott, Eric Isaac. "Regulation of NLRP3 inflammasome activation by mitochondria." Diss., University of Iowa, 2018. https://ir.uiowa.edu/etd/6099.
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Pattern recognition receptors coordinate innate immune responses by sensing infection or injury. Nucleotide-binding, leucine rich repeat, and pyrin domain-containing protein 3 (NLRP3) is a cytosolic PRR which perceives diverse pathogenic and sterile insults. NLRP3 orchestrates inflammatory signaling responses by forming inflammasomes with the adaptor protein apoptosis-associated speck like protein with a caspase recruitment domain (ASC) and the cysteine protease caspase-1. Assembly of the intracellular macromolecular inflammasome complex culminates in proximity-induced autocatalysis of caspase-1. Caspase-1 activation promotes cell death by pyroptosis and activation and secretion of proinflammatory cytokines interleukin (IL)-1β and IL-18. While NLRP3-mediated inflammation protects against bacterial, fungal, viral, and parasitic infections, aberrant NLRP3 activation is implicated in numerous inflammatory diseases and heritable syndromes. Mechanistically, inflammasome activation requires a preliminary NF-κB-activating priming step (signal 1) and a subsequent NLRP3-specific stimulus (signal 2). While there is enormous molecular diversity among NLRP3-specific agonists, this second signal appears to engage a common pathway involving cation flux. Furthermore, NLRP3 associates with mitochondria and mitochondrial damage is implicated in NLRP3 activation, although the precise role for mitochondria in inflammasome assembly remains controversial. We previously demonstrated that the mitochondrial phospholipid cardiolipin binds to NLRP3 and is critical for NLRP3 inflammasome activation.
Here, we further investigated how mitochondria contribute to NLRP3 activation. We found that liposomes containing molar concentrations of cardiolipin that resemble mitochondrial cardiolipin levels can induce NLRP3-dependent caspase-1 autoactivation. Unexpectedly, we discovered that caspase-1 binds directly to cardiolipin, causing inflammasome-independent caspase-1 complex formation and autocatalysis at higher cardiolipin densities. Finding that caspase-1 and NLRP3 are independently capable of binding to cardiolipin, we more thoroughly examined the association of inflammasome components with mitochondria. Normally confined within mitochondrial inner membranes, cardiolipin relocates to outer membranes of stressed mitochondria. We found that reactive oxygen species (ROS) produced in response to signal 1 facilitate cardiolipin externalization to the outer membrane during priming. We also determined that this coincides with ROS-dependent recruitment of NLRP3 and caspase-1 to the outer membrane of mitochondria at priming. In contrast, we found that NLRP3 activation by the signal 2 agonist nigericin induces calcium-dependent recruitment of the adaptor ASC to mitochondria and caspase-1 activation. Finally, to determine what type of mitochondrial damage was necessary to promote NLRP3 inflammasome activation, we examined how different NLRP3 agonists affect mitochondria. We found substantial variability in the extent of mitochondrial damage induced among different NLRP3 agonists. Collectively, our findings illustrate that mitochondria serve as innate immune signaling platforms through multiple stages of NLRP3 inflammasome activation. Further, paralleling lipid A interactions with caspase-11, we have demonstrated that caspase-1 is capable of binding to the phospholipid cardiolipin.
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Riteau, Nicolas. "Immunité innée et inflammasome : rôle des signaux de dangers endogènes." Phd thesis, Université d'Orléans, 2011. http://tel.archives-ouvertes.fr/tel-00684046.
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La théorie du danger développée par Polly Matzinger stipule que l'attrait principal du système immunitaire ne réside pas dans la distinction entre le soi " à protéger " et le non-soi " à combattre ". Toute situation potentiellement délétère pour l'hôte, avec émission de signaux de dangers endogènes, est à ce titre capable de générer une réponse immunitaire afin de mobiliser les acteurs capables de permettre un retour à une situation basale. L'exposition des poumons de façon répétée à des agents toxiques environnementaux se traduit par une inflammation et une fibrose pulmonaire en condition stérile, c'est-à-dire sans intervention de micro-organisme. L'administration de Bléomycine dans les poumons de souris est un bon modèle pour étudier les molécules endogènes ou signaux de dangers engagés et les voies de signalisations associées. Nous avons identifié l'ATP extracellulaire et l'acide urique, provenant des cellules stressées ou endommagées, comme capables d'induire l'activation d'un complexe protéique cytoplasmique appelé inflammasome Nlrp3. Celui-ci conduit à la maturation de l'interleukine-1β, cytokine pro-inflammatoire. Dans une seconde partie, nous nous sommes intéressés aux mécanismes moléculaires d'activation de l'inflammasome Nlrp3 en réponse à des agents particulaires, responsables notamment de pathologies pulmonaires après inhalation. Nos résultats montrent que de l'ATP endogène est libéré activement par les cellules mises en contact avec des particules. Par un mécanisme autocrine/paracrine l'ATP va ensuite signaler sur plusieurs récepteurs purinergiques membranaires. Cette signalisation purinergique est importante dans la capacité des cellules à produire de l'interleukine-1β mature. Au final, les travaux présentés dans ce manuscrit attestent du rôle critique de molécules endogènes dans la mise en place d'une réponse immunitaire innée basée sur l'activité de l'inflammasome Nlrp3.
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McKeithen, Danielle N. "The Role of the Inflammasome During Chlamydia Infection." DigitalCommons@Robert W. Woodruff Library, Atlanta University Center, 2016. http://digitalcommons.auctr.edu/cauetds/151.
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Chlamydia trachomatis (C. trachomatis) is the most prevalent sexually transmitted bacteria with devastating reproductive consequences that lead to tubal factor infertility (TFI). Recent studies have implicated apoptosis – associated speck – like protein containing a caspase recruitment domain (ASC) as an adaptor of inflammasomes that stimulate IL – 1β and IL – 18 secretion, pro – inflammatory cytokines with critical functions in host defense against a variety of pathogens. Therefore, for the first time, we are reporting the use of ASC-/- mice in a mouse model of Chlamydia infection that might provide some information on the role of inflammasomes in the pathogenesis of Chlamydia infection. In this study, wild type (WT) and ASC-/- mice were infected with Chlamydia. Infectivity, pathology of the upper genital tract (UGT), and, fertility were evaluated. In addition, expression of ASC – dependent inflammasomes and the activation of immune cells within the genital tract (GT) were studied. Results showed that Chlamydia infectivity in ASC-/- mice was significantly higher (p-/- mice which, when compared to infected WT mice, was exhibited by decrease in average number of pups and percent pregnancy. There was also severe UGT damage in ASC-/- mice compared to WT mice, correlating with the higher number of hydrosalpinx observed on the UGT of Chlamydia infected ASC-/- mice. Furthermore, IL – 1β and IL – 18 production as well as immune cell activation were down regulated in the GT of Chlamydia infected ASC-/- mice. This finding indicates that in absence of ASC, host innate and adaptive immunity is impaired. Results imply that ASC plays a protective role in the mucosal immunity against GT Chlamydia infection.
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Lei, Andrea. "Inflammasome regulation and activation in the intestinal epithelium." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:6db34fe2-36ae-44f4-a4ac-2464a333c8f8.
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Microbiota colonisation of the intestinal tract makes it difficult for pattern recognition receptors (PRR) to discriminate between beneficial microbes and harmful pathogens. We aim to define the roles of cytosolic Nod-like receptors (NLR) in intestinal immunity and homeostasis. Upon activation, some NLR form inflammasomes that mediate the release of inflammatory cytokines and pyroptosis, an inflammatory form of cell death. NLR activation in the non-hematopoietic compartment was shown to be protective during acute intestinal infection. To identify the cell type responsible for this protection, we generated transgenic mice in which the key inflammasome adaptor molecule Asc is selectively ablated in intestinal epithelial cells (IEC) (AscΔVC) and observed that inflammasomes are important for controlling Citrobacter rodentium clearance in these mice. To further dissect the importance of pathogen clearance by IEC inflammasome, ex vivo cultures of primary IEC organoids were established. Thus far this system has revealed profound differences in inflammasome regulation between IEC organoids and bone marrow-derived macrophages (BMDM). This research will inform our understanding of cell type-specific regulation of inflammasomes.
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Deplano, Simona. "Role of P2X7-mediated inflammasome activation in glomerulonephritis." Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/17794.
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Glomerulonephritis is a major cause of kidney failure and current treatment is based on nonspecific immunosuppressive therapies. The purinergic P2X7 receptor (P2X7R) is usually not detectable in renal tissue. However, previous studies have demonstrated an increased glomerular P2X7R expression in animal models of glomerulonephritis. Furthermore, P2X7R knock-out mice have been shown to be significantly protected from antibody-mediated glomerulonephritis. P2X7R activation represents a fundamental step for the activation of the NLRP3 inflammasome which leads to the processing and release of IL-1β and IL-18. The role of the inflammasome activation in glomerulonephritis is not clear yet. The work presented in this thesis describes three aspects of P2X7R activation: cytokine production, signalling cascade following ATP stimulation and inflammasome activation. My data show that macrophages from wild type mice produce higher levels of IL-1β and IL-18 compared to macrophages from P2X7 deficient mice. ATP stimulation activates several signalling pathways in macrophages. Among them, the ribosomal pathway appears to be strictly regulated by P2X7R. To investigate the role of the inflammasome activation in glomerulonephritis I have compared macrophages from the susceptible rat strain Wistar-Kyoto with macrophages from the resistant strain Lewis. WKY macrophages express higher P2X7 mRNA and protein levels, release higher levels of IL-1β and IL-18 and exhibit a greater caspase-1 activity. Similarly, WKY nephritic glomeruli show higher P2X7, IL-1β, IL-18 and caspase-1 levels compared to Lewis glomeruli. Finally, in the attempt to identify genes responsible for the inflammasome regulation, I have examined macrophages and nephritic glomeruli from congenic rats. My data seem to indicate that the susceptibility locus Crgn2 contains one or more genes that control IL-1β and IL-18 release in macrophages. Further studies are certainly required to verify the relevance of these data. The results are important in understanding the pathogenesis of glomerulonephritis and identification of new potential therapeutic targets.
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Vladimer, Gregory I. "Inflammasomes and the Innate Immune Response Against Yersinia Pestis: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/649.
Full textAbstract:
Yersinia pestis, the causative agent of plague, is estimated to have claimed the lives of 30-50% of the European population in five years. Although it can now be controlled through antibiotics, there are still lurking dangers of outbreaks from biowarfare and bioterrorism; therefore, ongoing research to further our understanding of its strong virulence factors is necessary for development of new vaccines. Many Gram-negative bacteria, including Y. pseudotuberculosis, the evolutionary ancestor of Y. pestis, produce a hexa-acylated lipid A/LPS which can strongly trigger innate immune responses via activation of Toll-like receptor 4 (TLR4)-MD2. In contrast, Y. pestis grown at 37ºC generates a tetra-acylated lipid A/LPS that poorly induces TLR4-mediated immune activation. We have reported that expression of E. coli lpxL in Y. pestis, which lacks a homologue of this gene, forces the biosynthesis of a hexa-acylated LPS, and that this single modification dramatically reduces virulence in wild type mice, but not in mice lacking a functional TLR4. This emphasizes that avoiding activation of innate immunity is important for Y. pestis virulence. It also provides a model in which survival is strongly dependent on innate immune defenses, presenting a unique opportunity for evaluating the relative importance of innate immunity in protection against bacterial infection. TLR signaling is critical for the sensing of pathogens, and one implication of TLR4 engagement is the induction of the pro-forms of the potent inflammatory cytokines IL-1β and IL-18. Therefore Y. pestis is able to suppress production of these which are generated through caspase-1-activating nucleotide-binding domain and leucine-rich repeat (NLR)-containing inflammasomes. For my thesis, I sought to elucidate the role of NLRs and IL-18/IL-1β during bubonic and pneumonic plague infection. Mice lacking IL-18 signaling led to increased susceptibility to wild type Y. pestis, and an attenuated strain producing a Y. pseudotuberculosis-like hexa-acylated lipid A. I found that the NLRP12, NLRP3 and NLRC4 inflammasomes were important protein complexes in maturing IL-18 and IL-1β during Y. pestis infection, and mice deficient in each of these NLRs were more susceptible to bacterial challenge. NLRC4 and NLRP12 also directed interferongamma production via induction of IL-18 against plague, and minimizing inflammasome activation may have been a central factor in evolution of the high virulence of Y. pestis. This is also the first study that elucidated a pro-inflammatory role for NLRP12 during bacterial infection.
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Martin, Bradley N. "The Cellular and Molecular Mechanisms of ASC-dependent Inflammasomes in Neuroinflammation." Case Western Reserve University School of Graduate Studies / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1459420965.
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Abais, Justine M. "Redox Triggering of Podocyte NLRP3 Inflammasomes and Glomerular Injury in Hyperhomocysteinemia." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3357.
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Hyperhomocysteinemia (hHcys), an important pathogenic factor contributing to the progression of end-stage renal disease (ESRD), has been shown to activate NOD-like receptor protein 3 (NLRP3) inflammasomes and cause podocyte dysfunction and glomerular sclerosis. hHcys induces aggregation of the three inflammasome components – NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1 – and its activation is indicated by increased caspase-1 activity and secretion of interleukin-1β (IL-1β). The aims of the present study sought to elucidate the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated redox signaling in hHcys-induced NLRP3 inflammasome activation, to dissect the contribution of common endogenous reactive oxygen species (ROS) including superoxide (O2•−), hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hydroxyl radical (•OH), and to explore the molecular mechanisms by which the NLRP3 inflammasome senses changes in oxidative stress through thioredoxin-interacting protein (TXNIP). Specific inhibition of the gp91phox subunit of NADPH oxidase markedly reduced Hcys-induced caspase-1 activity and IL-1β production in cultured podocytes. Concurrently, gp91phox−/− or administration of a gp91ds-tat peptide also exhibited diminished glomerular inflammasome formation and activation in mice fed a folate-free (FF) diet to induce hyperhomocysteinemia and displayed glomerular protection as shown by prevention of hHcys-induced proteinuria, albuminuria and glomerular sclerosis. Interestingly, dismutation of O2•− by 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl and administration of H2O2 decomposer catalase either in cultured podocytes or hyperhomocysteinemic mice inhibited hHcys-induced NLRP3 inflammasome aggregation and activation. Hyperhomocysteinemic mice also demonstrated a significant increase in glomerular TXNIP binding to NLRP3, confirmed by confocal microscopy, size-exclusion chromatography, and co-immunoprecipitation studies. Blockade of TXNIP by genetic interference or by the calcium channel blocker verapamil prevented this hHcys-induced TXNIP-NLRP3 binding, NLRP3 inflammasome formation and activation, as well as protected hyperhomocysteinemic mice from glomerular dysfunction and damaged morphology. In conclusion, hHcys-induced NADPH oxidase activation is importantly involved in the switching on of NLRP3 inflammasomes in podocytes, where NADPH oxidase-derived O2•− and H2O2 primarily contribute to NLRP3 inflammasome activation. TXNIP binding to NLRP3 is a key signaling mechanism allowing NLRP3 inflammasome to sense these changes in oxidative stress. These findings greatly enhance our understanding of the early pathogenesis of hHcys-induced glomerular sclerosis, which may identify new therapeutic targets for prevention or treatment of ESRD.
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Thompson, Joyce K. "The Role of Inflammasomes in Asbestos-Induced Mesothelial to Fibroblastic Transition." ScholarWorks @ UVM, 2017. http://scholarworks.uvm.edu/graddis/691.
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Malignant Mesothelioma (MM) is a fatal disease with a low median survival between 8 to 12 months after diagnosis. MM has a long latency period (10-60 years), is causally related to asbestos exposure, and is refractory to all available modes of therapy. Despite the causal association between asbestos exposure and MM however, the mechanisms by which asbestos induces this deadly disease remain unclear. Chronic inflammation due to the presence of asbestos fibers is believed to play an important role in all aspects of MM pathogenesis, from development to progression and resistance. Chronic inflammation has been shown to promote dysregulated wound repair, fibrosis and epithelial to mesenchymal transition (EMT). One of the inflammatory pathways that asbestos activates is the inflammasome (a multiprotein scaffold that assembles in response to various stimuli to facilitate the activation of caspase-1), which has been implicated in several chronic inflammatory diseases and disorders. The nucleotide binding oligomerization domain (NOD) - like receptor containing a pyrin domain 3 (NLRP3) inflammasome, both as a whole or via its components [NLRP3, apoptosis related speck-like protein containing a CARD (caspase activating and recruitment domain) (ASC) and caspase-1] as well as its products, IL-1β and IL-18, has been implicated in the development of EMT during chronic inflammation.
Asbestos fibers, especially the amphiboles, are non-biodegradable and thus persist in tissues of the body for years after exposure. In mesothelial cells, the squamous epithelial-like cells that line the serosal cavities of the body, from which MM originates, asbestos chronically activates the NLRP3 inflammasome. Asbestos also activates the NLRP3 inflammasome in human macrophages that can lead to the establishment of a chronic inflammation environment. We therefore hypothesized that asbestos dependent regulation of the inflammasome played a role in mesothelial to fibroblastic transition to facilitate eventual neoplastic transformation of the mesothelial cells.
Using in vitro models, siRNA knockdown approaches as well as in vivo models of asbestos exposure utilizing inflammasome component knockout mice, we demonstrate that asbestos-induced reactive oxygen species generation modulates the redox state of the endogenous antioxidant, thioredoxin, causing its dissociation from thioredoxin interacting protein to promote activation of the inflammasome. We also show that the inflammasome plays a role in asbestos-induced mesothelial to fibroblastic transition (MFT) (a form of EMT occurring in the mesothelial cells) both in vitro and in vivo with a requirement for caspase-1 in vivo to promote thickening of the submesothelium. Through our studies, we have identified tissue factor pathway inhibitor 2 (TFPI2) and fibroblast growth factor 2 (FGF2) as molecules that are upregulated in response to asbestos exposure with potential roles in the progression of asbestos-induced MFT. There is a dearth of diagnostic biomarkers that enable early detection of MM, thus with further studies these two molecules could be explored as biomarkers of asbestos exposure/disease progression. TFPI2 levels were downregulated in response to blockage of IL-1β signaling and thus could be harnessed as a potential marker for therapy efficiency with further studies.
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Vladimer, Gregory I. "Inflammasomes and the Innate Immune Response Against Yersinia Pestis: A Dissertation." eScholarship@UMMS, 2001. http://escholarship.umassmed.edu/gsbs_diss/649.
Full textAbstract:
Yersinia pestis, the causative agent of plague, is estimated to have claimed the lives of 30-50% of the European population in five years. Although it can now be controlled through antibiotics, there are still lurking dangers of outbreaks from biowarfare and bioterrorism; therefore, ongoing research to further our understanding of its strong virulence factors is necessary for development of new vaccines. Many Gram-negative bacteria, including Y. pseudotuberculosis, the evolutionary ancestor of Y. pestis, produce a hexa-acylated lipid A/LPS which can strongly trigger innate immune responses via activation of Toll-like receptor 4 (TLR4)-MD2. In contrast, Y. pestis grown at 37ºC generates a tetra-acylated lipid A/LPS that poorly induces TLR4-mediated immune activation. We have reported that expression of E. coli lpxL in Y. pestis, which lacks a homologue of this gene, forces the biosynthesis of a hexa-acylated LPS, and that this single modification dramatically reduces virulence in wild type mice, but not in mice lacking a functional TLR4. This emphasizes that avoiding activation of innate immunity is important for Y. pestis virulence. It also provides a model in which survival is strongly dependent on innate immune defenses, presenting a unique opportunity for evaluating the relative importance of innate immunity in protection against bacterial infection. TLR signaling is critical for the sensing of pathogens, and one implication of TLR4 engagement is the induction of the pro-forms of the potent inflammatory cytokines IL-1β and IL-18. Therefore Y. pestis is able to suppress production of these which are generated through caspase-1-activating nucleotide-binding domain and leucine-rich repeat (NLR)-containing inflammasomes. For my thesis, I sought to elucidate the role of NLRs and IL-18/IL-1β during bubonic and pneumonic plague infection. Mice lacking IL-18 signaling led to increased susceptibility to wild type Y. pestis, and an attenuated strain producing a Y. pseudotuberculosis-like hexa-acylated lipid A. I found that the NLRP12, NLRP3 and NLRC4 inflammasomes were important protein complexes in maturing IL-18 and IL-1β during Y. pestis infection, and mice deficient in each of these NLRs were more susceptible to bacterial challenge. NLRC4 and NLRP12 also directed interferongamma production via induction of IL-18 against plague, and minimizing inflammasome activation may have been a central factor in evolution of the high virulence of Y. pestis. This is also the first study that elucidated a pro-inflammatory role for NLRP12 during bacterial infection.
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Guey, Baptiste. "Inflammasomes : des mécanismes d’activation de la caspase-1 à la progression tumorale." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1113/document.
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L'inflammasome est une plateforme moléculaire composée d'un récepteur de l'immunité innée tels que NLRP3 ou NLRP1b, de la protéine adaptatrice ASC et de la caspase-1. Il joue un rôle essentiel dans le déclenchement de la réponse inflammatoire via l'activation de la caspase-1 qui mène à la sécrétion de cytokines pro-inflammatoires telle que l'IL-1ß. Dans un premier axe de recherche, nous avons mis en évidence que les macrophages de souris déficientes pour ASC traités avec l'activateur de l'inflammasome NLRP1b, la toxine létale de bacillus anthracis, étaient capables de sécréter la forme mature de l'IL-1ß en absence de clivage de la caspase-1 pourtant décrit comme un événement indispensable à son activité. En reconstituant des macrophages caspase-1 KO avec une forme mutante non-clivable de la caspase-1, nous avons démontré que la forme entière de la caspase-1 est capable d'induire la sécrétion d'IL-1ß en réponse à la toxine létale alors qu'elle n'est pas fonctionnelle au sein de l'inflammasome NLRP3.Dans un second axe de recherche, mon travail de thèse s'est intéressé à comprendre le rôle de l'inflammasome au cours de la progression tumorale. En effet, l'IL-1ß est une cytokine fréquemment retrouvée dans le microenvironnement tumorale mammaire suggérant donc l'activation de l'inflammasome au sein des tumeurs. Au moyen d'un modèle de tumeurs in vivo, nous avons montré que l'absence de la caspase-1 et de ASC dans les cellules immunitaires chez la souris conduit à une réduction de la croissance tumorale. De plus, l'absence de ces deux protéines provoque également un plus fort recrutement et une meilleure activité des cellules NK au sein des tumeurs.En plus d'identifier un nouveau mécanisme d'activation de la caspase-1, mon travail de thèse a permis de mettre en évidence le rôle de l'inflammasome dans l'altération des cellules NK au cours de la progression tumorale mammaire. Ces données permettent d'envisager l'inflammasome comme une cible thérapeutique dans le cancerThe inflammasome is a molecular platform composed of an innate immune receptor such as NLRP3 or NLRP1b, of the adaptor protein ASC and of the caspase-1. It plays an essential role in triggering inflammatory response through the activation of caspase-1 that leads to the secretion of pro-inflammatory cytokines such as IL-1ß.In a first research axis, we showed that ASC deficient mice macrophages treated with an NLRP1b inflammasome activator, the lethal toxin of Bacillus anthracis, were able to secrete the mature form of IL-1ß in absence of any cleavage of caspase-1 previously described as an essential event for its function. By reconstituting caspase-1 KO macrophages with an uncleavable mutant form of caspase-1, we showed that the entire form of the protein is able to induce IL-1ß secretion upon lethal toxin treatment but is nonfunctional upon NLRP3 inflammasome activation.In a second research axis, my PhD work focused on underlying the inflammasome role during tumor progression. Indeed, IL-1ß is frequently found within breast tumor microenvironment suggesting that inflammasome is activated in tumors. Using in vivo tumor model, we showed that the absence of caspase-1 of ASC in immune cells lead to a delay tumor growth. In addition, the absence of these two proteins also causes a stronger recruitment and an enchanced activity of NK cells within mammary tumors
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Kaymakanov, Nikolay. "Aktivierung des AIM2 Inflammasoms in menschlichen Keratinozyten durch Bienengift." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-144874.
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Keller, Martin. "The inflammasome : a key regulator of unconventional protein secretion /." Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17640.
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Pache, Florence [Verfasser]. "Differentielle Aktivierung des NLRP3-Inflammasoms durch Pneumolysin / Florence Pache." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2014. http://d-nb.info/1062536908/34.
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Andersen, Kirstin. "Die Rolle des NLRP3-Inflammasoms bei Immunkomplex-vermittelten Glomerulonephritiden." Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-179270.
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Das proinflammatorische Zytokin IL–1beta ist maßgeblich an der renalen Schädigung im Rahmen der Immunkomplex-Glomerulonephritis beteiligt. Für die Spaltung von pro-IL–1beta in seine extrazelluläre sezernierbare Form sind Inflammasome zuständig. Für das NLRP3-Inflammasom, das auf eine Vielzahl endogener Gefahrensignale (DAMPs) reagiert, wurde eine funktionelle Rolle bereits in tubulointerstitiellen Nephropathien gezeigt, wohingegen die Funktion in glomerulären Erkrankungen bis dato nicht geklärt ist. Um zu untersuchen,ob NLRP3 und sein Adaptermolekül ASC am Geschehen der Immunkomplex-vermittelten glomerulären Inflammation beteiligt sind, wurde ein T-Zell-abhängiges autologes NTN Modell induziert. Dies erfolgte in Mäusen mit einer Defizienz der Inflammasomkomponenten NLRP3 (C57BL/6-Nlrp3tm1Tsc) und ASC (C57BL/6-Pycardtm1V md). Die renale Expression von NLRP3/ASC Inflammasom-Komponenten und pro–IL–1beta stiegen während der NTN an und waren insbesondere in dendritischen Zellen (DC) vermehrt nachweisbar. Diese Tatsache war assoziiert mit der renalen Produktion von aktivem IL–1beta, was auf die Aktivierung des Inflammasoms hindeutet. Eine Defizienz für Nlrp3 oder Asc verringerte den renalen Schaden signifikant, ebenso die Leukozyteninfiltration und die T–Zell Aktivierung. Übereinstimmend mit einem ASC-abhängigen Verlust der inflammasomvermittelten IL–1beta-Aktivierung war die renale und lienale Produktion von aktivem IL–1beta in Asc-defizienten Mäusen vermindert. Überraschenderweise blieb die IL–1beta-Bildung bei Nlrp3-defizienten Tieren unverändert, was auf nicht-kanonische proinflammatorische NLRP3–abhängige Effekte im Modell der NTN hinweist. Ein solcher möglicher Effekt könnte die NLRP3-vermittelte Freisetzung von proinflammatorischem HMGB–1 als ein neuer nicht-kanonischer Mechanismus von NLRP3 und ASC während der Immunkomplex-Glomerulonephritis sein. Auf Grund dessen wäre die Blockade sowohl von NLRP3, als auch von ASC eine möglicher nutzbringender therapeutischer Ansatz im Rahmen der Behandlung von Immunkomplex-vermittelten Glomerulonephritiden.The proinflammatory cytokine IL-1beta significantly contributes to renal injury in immune complex glomerulonephritis. Production of mature IL-1beta depends on activation of the inflammasome that cleaves pro-IL-1beta into its secretable form. A functional role of the NLRP3-containing inflammasome, which responds to a variety of endogenous danger signals, has been demonstrated in tubulointerstitial nephrophathies, but its function in glomerular disease remained unknown. To investigate whether NLRP3 and its adapter molecule ASC contributes to immune complex-mediated glomerular inflammation, we induced T-cell dependend autologous nephrotoxic serum nephritis (NTN) in Nlrp3- and Asc-deficient mice. Renal expression of NLRP3/ASC inflammasome components and pro-IL-1beta increased during NTN, and was abundant in renal DCs. This was associated with renal production of mature IL-1beta, indicating activation of the inflammasome. Nlrp3- and Asc-deficiency significantly attenuated glomerular injury, leukocyte infiltration and T-cell activation. Consistent with a loss of ASC-dependent inflammasome-mediated IL-1beta activation, renal and splenic production of mature IL-1beta was abrogated in Asc-deficient mice. Surprisingly, IL-1beta secretion remained intact in Nlrp3-deficient mice indicating noncanonical pro-inflammatory effects of NLRP3 in autologous nephrotoxic serum nephritis. This may include NLRP3-mediated glomerular release of pro-inflammatrory HMGB-1 as a non canonical function of NLRP3 and ASC in glomerulonephritis. Thus, therapeutic blockade of both NLRP3 and ASC may be beneficial in glomerulonephritis.
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Doull, Laura Elizabeth. "Inflammasome activation in ruminant cells infected with Chlamydia abortus." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/22082.
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Chlamydia abortus is the most common known infectious cause of ovine abortion worldwide but is rarely linked with bovine abortion. The reasons for this differential pathogenesis are unknown but may involve differences in innate immune recognition and immune responsiveness. This is supported by the observation that chlamydial abortion in sheep is associated with an inflammatory cytokine/chemokine cascade that is not commonly observed in cattle. Studies with other Chlamydia species have demonstrated that innate inflammatory pathways including inflammasome activation contribute to both pathogen clearance and pathology. Pattern recognition receptors (PRRs) activate these innate immune signalling pathways but are relatively poorly characterized in ruminants. We hypothesize that the ruminant hosts differ in their ability to innately sense C. abortus infection and activate the inflammasome. The main aims of this project were to: analyse PRR expression in innate immune cells; assess cytokine production from innate immune cells in response to C. abortus; investigate the role of PRRs in the induction of innate immune responses to C. abortus; and, conduct RNA-seq analysis on macrophages following infection with C. abortus to identify important immune signalling pathways. Ruminant oro-nasal turbinate cells, monocyte derived dendritic cells (MDDCs) and monocyte derived macrophages (MDMs) express the cell-surface PRRs TLR2 and TLR4 and also the intracellular PRRs NOD 1 and NLRP3. Oro-nasal turbinate cells produce CXCL8 late into the chlamydial developmental cycle independent of IL-1β. In contrast, ruminant MDMs and MDDCs secrete early IL-1β in response to C. abortus infection. In MDMs and MDDCs, live and UV-inactivated C. abortus induced TNF-α and CXCL8 but live infection was required for IL-1β secretion. Therefore, intracellular C. abortus multiplication is necessary to stimulate the IL-1β processing pathway within these cells. In order to determine PRR function, NOD1 and NLRP3 were knocked down in ruminant MDMs using siRNA. In both ovine and bovine MDMs, NOD1 was identified as a factor in C. abortus mediated IL-1β production. NLRP3 knockdown in bovine but not ovine MDMs also reduced IL-1β production, indicating species-specific differences in C. abortus recognition. The RNA-seq analysis of ruminant MDMs identified novel pathways of immune activation by C. abortus and potentially important species-specific differences. An improved understanding of the innate immune pathways activated in susceptible and resistant hosts following C. abortus infection will inform on disease pathogenesis and could contribute to novel chlamydial vaccine design.
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Bittante, Alessandra. "Molecular basis of NAIP/NLRC4 inflammasome activation by flagellin." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275741.
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The overall aim of this project was to determine the molecular mechanisms by which the flagellin gene from Salmonella enterica serovar Typhimurium (S. Typhimurium) activates the NAIP/NLRC4 inflammasome and its contribution to the host protective immune response against salmonellosis. Inflammasomes are multi-protein complexes formed in response to the activation of pattern recognition receptors (PRRs). The NOD-like receptor (NLR)-family of inflammasome complexes are formed from the cytosolic NLR receptors, ASC adaptor and caspase-1 in response to pathogen- associated molecules or danger-associated signals. The NAIP/NLRC4 inflammasome is activated by the S. enterica flagellar filament protein (FliC), the SPI-1 type III secretion system needle (PrgI) and inner rod proteins (PrgJ). Recognition of these bacterial ligands by the NAIP receptors allows oligomerisation with NLRC4 and subsequent recruitment of caspase-1. Caspase-1 mediates pyroptosis, while recruitment of ASC is also required for cleavage of pro-IL-1β and pro-IL-18 to their active forms by caspase-1. Differential recognition of the flagellar filament proteins (flagellin) by the NAIP/NLRC4 inflammasome forms an important part of my thesis. Here, I have looked at the molecular mechanisms and immunological consequences of the differential recognition of flagellin by the NAIP/NLRC4 inflammasome using S. Typhimurium SL1344 and the non-pathogenic E. coli strain K12-MG1655. An important part of my work was to try and determine which regions of fliC are required for NAIP/NLRC4 inflammasome activation and whether they can be mutated while preserving motility. To do this a panel of ten strains expressing chimeric fliC genes were created and characterised in macrophage infection experiments and bacterial motility assays. My results confirm the C-terminus of FliC is critical for both inflammasome activation and motility in agreement with published reports. To further investigate this differential recognition by the NAIP/NLRC4 inflammasome I modified S. Typhimurium strain of moderate virulence (M525P) to express flagellin from E. coli K12-MG1655. This strain (M525PΔfliC::fliCK12-MG1655CmR) retained motility and both in vitro and in vivo characterisation was carried out in macrophages and using a murine model of sublethal salmonellosis respectively. Activation of the NAIP/NLRC4 inflammasome was impaired in murine macrophages infected with M525PΔfliC::fliCK12-MG1655CmR when compared to those infected with M525P. Mice infected with M525PΔfliC::fliCK12-MG1655CmR had increased liver and spleen bacterial burdens compared to those infected with M525P, indicating that optimal NAIP/NLRC4 inflammasome activation is key for efficient control of microbial spread in vivo. The role of NAIP receptors in inflammasome formation was further investigated with the use of CRISPR/Cas9 to generate mutant murine macrophage cell lines. To investigate the consequence of gene deletions cell lines were designed to lack NAIP 1, 2, 5 and 6, while others were designed to express tagged NAIP proteins to elucidate the cellular localisation of the NAIP proteins during inflammasome formation by microscopy. Characterisation of these cell lines is ongoing, with extensive optimisation of the CRISPR/Cas9 technique undertaken during this study.
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Fulp, Jacob W. "THE DEVELOPMENT OF NOVEL INHIBITORS OF THE NLRP3 INFLAMMASOME." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5457.
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Inflammasomes are intracellular multimeric protein complexes that regulate inflammation by controlling the maturation of cytokines, interleukin-1β (IL-1β) and interleukin-18 (IL-18). Additionally, activation of these inflammasome complexes has been implicated in an inflammatory form of death known as proptosis. Of the known inflammasomes, the NOD-like receptor family pyrin domain containing 3 (NLPP3) inflammasome, is the most elucidated. Under physiological conditions, NLRP3-mediated inflammation promotes healing and the elimination of cellular debris and pathogens. However, the dysregulation of IL-1β, IL-18 and pyroptosis are instrumental in the development of multiple pathologies. Furthermore, studies suggest that the NLRP3 inflammasome mediates detrimental neuroinflammation and contributes significantly to the development of several neurodegenerative diseases. This includes Alzheimer’s disease, multiple sclerosis, Parkinson's disease, and amyotrophic lateral sclerosis. Novel NLRP3 inflammasome inhibitors (NLRP3Is) are needed as pharmacological tools to complement ongoing molecular and genetic studies to aid in defining the roles of NLRP3 in neurological diseases. Development of such inhibitors also has significant translational potential. In order to develop novel small molecule inhibitors of the NLRP3 inflammasome, we conducted an in-depth structure-activity-relationship study of a known NLRP3 inhibitor. Two new lead compounds, 54 and 64, were identified. These compounds were potent and selective NLRP3 inhibitors in both in BMDMs and J774A.1 cell-lines. Importantly, a study utilizing mice challenged with LPS demonstrated that both of these compounds have in vivo activity. Collectively, these results strongly encourage further development of new analogs based on this promising chemical scaffold.
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Gao, Jiangyuan. "NLRP3 inflammasome activity in RPE : role in AMD pathogenesis." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/62512.
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Purpose:
Age-related macular degeneration (AMD) is a devastating eye disease causing irreversible vision loss in the elderly. Retinal pigment epithelium (RPE), an important cell type afflicted in AMD, undergoes cell death in the late stages of the disease. Salient factors underlying AMD pathogenesis are aging, drusen components and NLRP3 inflammasome activity. The purpose of this dissertation is to elucidate the molecular interactions among these factors and how they contribute to RPE damage.
Methods:
The effects of aging on drusen components, in particular the membrane attack complex (MAC) and amyloid beta (Aβ) were examined in rats at different age. To determine the role of MAC in inflammasome activation in RPE, aurin tricarboxylic acid complex (ATAC), was administrated to naïve rats. To understand Aβ’s role in inflammasome activation, Aβ intravitreal injections were made into rat eyes in vivo and Vinpocetine was used to ameliorate the inflammatory responses. An in vitro RPE cell culture model was established to further investigate the relationship between inflammasome and X-chromosome linked inhibitor of apoptosis (XIAP). Statistical significance was set at p ≤ 0.05.
Results:
An age-dependent increase in MAC, Aβ, and NF-κB activation was observed in the RPE-choroid in vivo. Blocking MAC formation with ATAC led to a prominent reduction in inflammasome activity (caspase-1 cleavage and cytokine secretion), but not in NF-κB activity. Aβ intravitreal injections triggered inflammasome activation evidenced by enhanced caspase-1 cleavage and IL-1β/IL-18 release, which was suppressed by Vinpocetine mediated NF-κB inhibition. The robust inflammasome activity further led to gasdermin D-mediated activation of the pyroptotic pathway and a significant reduction in XIAP, which in turn enhanced IL-18 secretion.
Conclusion:
Aging is a strong risk factor for AMD, which increases the deposition of MAC and Aβ in the outer retina. The elevated levels of MAC and Aβ are triggers for inflammasome activation. By demonstrating a causal relationship between inflammasome activation and XIAP reduction, this dissertation suggests the precise regulation of XIAP, together with the suppression of MAC and NF-κB, may be crucial for controlling inflammasome activity and hence provides new avenues to prevent AMD.Medicine, Faculty of
Graduate
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DeSantis, David A. "The NLRC4 Inflammasome and its Regulation of Liver Disease Pathogenesis." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1433949804.
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Ellis, Amanda. "Investigating the contribution of the inflammasome to persistent pain states." Thesis, King's College London (University of London), 2013. https://kclpure.kcl.ac.uk/portal/en/theses/investigating-the-contribution-of-the-inflammasome-to-persistent-pain-states(e311c2ef-64d4-46c4-9798-7900282c6eb7).html.
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Neuropathic pain, a debilitating condition, occurs as a consequence of nerve injury with symptoms such as spontaneous pain, hyperalgesia, and allodynia commonly reported. In animal models of peripheral nerve injury, there is accumulation and proliferation of microglial cells in the injured spinal cord and macrophages within the injured nerve and dorsal root ganglion (DRG), contributing to the development of pain-like behaviours. The pro-inflammatory cytokine interleukin-1β (IL-1β) plays a key role in acute and chronic inflammation. It causes potent mechanical and thermal hyperalgesia when injected into peripheral tissues, and increased expression of IL-1β in the spinal cord, DRG, and injured nerve is seen in several animal models of inflammatory and neuropathic pain. A key step in the release of active IL-1β is the cleavage of pro-IL-1β by active caspase-1, generating mature IL-1β. Activation of this enzyme requires assembly of the inflammasome, a multi-protein complex. The active complex contains a central scaffold protein (eg nod-like receptor-1 (NLRP1), NLRP3), the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and pro-caspase-1. It is known to assemble in response to a variety of exogenous and endogenous danger signals (eg adenosine-5’-triphosphate (ATP), monosodium urate (MSU) crystals). Data presented in this thesis demonstrates that, following L5 Spinal Nerve Transection (SNT), an animal model of neuropathic pain, inflammasome components NLRP3, ASC, and caspase-1 are upregulated in the injured DRG and the ipsilateral lumbar dorsal horn. Immunohistochemical analysis reveals that ASC and caspase-1 are both highly co-localised with macrophage and microglial marker ionised calcium-binding adaptor molecule-1 (IBA1), but not with astrocytic marker glial fibrillary acid protein (GFAP) or neuronal marker NeuN. While daily intrathecal administration of Ac-YVAD-CMK, a cell-permeant caspase-1 inhibitor, significantly attenuated established behavioural hypersensitivity following L5 SNT, ASC-/- and NLRP3-/- mice developed behavioural hypersensitivity normally in the Partial Sciatic Nerve Ligation (PSNL) model of neuropathic pain, indicating an inflammasome-independent role of caspase-1 in neuropathic pain. In a model of central inflammatory pain, pharmacological inhibition of caspase-1 prevented the development of hindpaw mechanical hypersensitivity following intrathecal administration of lipopolysaccharide (LPS). ASC-/- mice had a significantly reduced mechanical hindpaw hypersensitivity following intrathecal administration of LPS. However NLRP3-/- mice developed hindpaw hypersensitivity normally following intrathecal administration of LPS, indicating a NLRP3-independent role for ASC and caspase-1 activation in this animal model of central inflammatory pain. Finally, although ASC appears to contribute to the development of hypersensitivity in a model of central inflammatory pain, ASC-/- mice developed behavioural hypersensitivity normally following intraplantar administration of complete Freund’s adjuvant (CFA) in an animal model of persistent peripheral inflammatory pain. Thus data presented in this thesis reveal a complex role for the inflammasome in animal models of pain, with differential contribution of inflammasome components to behavioural hypersensitivity in animal models of neuropathic and central and peripheral inflammatory pain.
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Hamade, Hussein. "Analyse des mécanismes moléculaires et cellulaires conduisant à une inflammation dans l'intestin et une progression tumorale induits par la perte de la sous-unité d'intégrine Alpha6 chez la souris." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ062/document.
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Le laboratoire a établi un modèle de souris α6ΔIEC qui développe une inflammation chronique intestinale associée à la formation d’adénocarcinomes colorectaux. Ce modèle correspond à une délétion ciblée à l’épithélium intestinal de l’intégrine α6β4. Mon projet de thèse a consisté à définir les mécanismes qui influencent la transformation de lésions inflammatoires en adénocarcinomes. La caractérisation du modèle α6ΔIEC a permis de mettre en évidence plusieurs altérations : détachement de l’épithélium, régénération du tissu, prolifération, augmentation de la perméabilité intestinale, hypersécrétion du mucus, ségrégation anormale des bactéries, inflammation chronique et formation de tumeurs.Pour étudier la séquence et la cinétique des mécanismes, j’ai développé un modèle de souris inductible (α6ΔIECTAM). Cette lignée présente, deux semaines après l’invalidation de l’intégrine α6,les mêmes signes d’inflammation que les souris α6ΔIEC. Mon approche a consisté à dissocier les processus impliqués dans chacune des étapes-clés de la pathologie afin de définir la contribution respective de l’infection par les bactéries et du stress mécaniqueWe generated a new mouse model, α6ΔIEC, in which the genetic ablation of α6 integrin from intestinal epithelial cells triggered the development of spontaneous colitis and colorectal cancer. My main goal was to define the mechanisms by which inflamed lesions degenerate into infiltrating adenocarcinomas. Loss of α6 integrin in this model resulted in epithelial barrier damage, enhanced permeability, altered mucus layers, abnormal bacterial segregation, chronic inflammation and tumor development.In order to define the sequence of events and the mechanisms involved at each stage of the disease, from inflamed to tumor lesions, I developed an inducible mouse model, α6ΔIECTAM, in which α6 integrin ablation was induced by tamoxifen treatment. This line recapitulates all aspects of inflammation observed in the α6ΔIEC model, as early as two weeks after tamoxifen treatment. In particular, I tried to define the respective contribution of infection by bacteria and mechanical stress during disease progression
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Paoletti, Audrey. "Étude des étapes précoces de l’infection par le VIH-1 : identification d’un nouveau point de contrôle immunitaire immunitaire impliquant le récepteur P2Y2 et la protéine NLRP3." Thesis, Université Paris-Saclay (ComUE), 2015. http://www.theses.fr/2015SACLS240.
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Plus de 34 millions de personnes dans le monde vivent avec le virus de l’immunodéficience humaine de type 1 (VIH-1). Cette pandémie est partiellement contrôlée par l’utilisation de combinaison d’agents antirétroviraux spécifiques. Cependant l’émergence de nouvelles souches virales multi-résistantes nécessite le développement de nouvelles stratégies antirétrovirales. Notre laboratoire porte une attention particulière à la compréhension des évènements cellulaires et viraux impliqués dans les étapes précoces du cycle réplicatif du VIH-1. Récemment, nous avons révélé l’existence d’une nouvelle voie de signalisation cellulaire, impliquant un canal membranaire (la Pannexine-1), un signal de danger (l’ATP extracellulaire) et un récepteur purinergique (P2Y2) participant à l’entrée du VIH-1 dans ses cellules cibles. Etant donnée que ces trois évènements cellulaires sont également des acteurs de la réponse immunologique, nous avons décidé de poursuivre l’étude du rôle des protéines de la réponse immunologique innée dans les étapes précoces d’infection par le VIH-1.Au cours de mes travaux de thèse, nous avons révélé une interaction entre le récepteur purinergique P2Y2 et la protéine de l’inflammasome NLRP3. Dans un premier temps, nous avons démontré que la migration cellulaire dépendante du récepteur purinergique P2Y2 est réprimée pendant l’activation de l’inflammasome NLRP3. A l’inverse, nous avons également observé que la polarisation des macrophages, la sécrétion de l’interleukine-1β et la pyroptose déclenchées par l’activation de NLRP3 sont sous le contrôle de l’autophagie induite par l’activation du récepteur purinergique P2Y2. Ces résultats suggèrent que l’interaction entre NLRP3 et P2Y2 constitue un nouveau point de contrôle immunologique qui régule les fonctions des macrophages. A la suite de ces travaux, nous avons analysé le rôle de ce point de contrôle immunologique lors de l’infection par le VIH-1 et avons démontré que l’activation de l’inflammasome NLRP3 empêche l’activation de la voie de signalisation purinergique qui implique l’ATP, la pannexine-1 et le récepteur P2Y2, et qui permet l’entrée du VIH-1 dans ses cellules cibles. Nos travaux de recherche mettent ainsi en lumière la capacité de l’inflammasome NLRP3 à représenter un nouveau facteur de restriction inductible du VIH-1. L’ensemble de mes travaux recherche souligne l’existence au niveau des macrophages d’un nouveau point de contrôle du système immunitaire impliquant la protéine NLRP3 et le récepteur P2Y2 et qui peut être moduler afin de développer de nouvelles approches thérapeutiques permettant de lutter contre l’émergence de virus résistants aux traitements rétroviraux classiquesIn 3 decades infection with the virus of the human immunodeficiency of type 1 (HIV-1) caused over than 34 million deaths and the surge of new multiresistant virus strains require the development of novel antiretroviral strategies.Our laboratories revealed a new signaling pathway involving in the early step of HIV-1 infection, involving a hemichannel (Pannexin-1), a common danger signal (extracellular ATP) and a purinergic receptor (P2Y2). These three cellular events are also players in the immune response; we decided to continue the study of proteins involved in the innate immune response during the early stages of infection by HIV -1.Here we demonstrated during this work a new interaction between the purinergic receptor P2Y2 and protein of the inflammasome NLRP3. We demonstrate that P2Y2-stimulated migration of macrophages is inhibited by NLRP3 inflammasome activation. Conversely, NLRP3-dependent macrophage polarization, interleukin-1 β secretion and pyroptosis are under the control of P2Y2-induced autophagy.Finally, the results suggest that the interaction between NLRP3 and P2Y2 is a new immunological checkpoint that regulates macrophage functions. Following this work, we analyzed the role of this immunological control during infection by HIV -1 and have demonstrated that activation of the inflammasome NLRP3 prevents the activation of the purinergic signaling channel involving ATP, pannexin -1 and the P2Y2 receptor, and which allows the entry of HIV -1 in its target cells. Our research and bring to light the capacity of the NLRP3 inflammasome to represent a new inducible restriction factor of HIV-1.All of this research work highlights the existence in macrophages of a new immune system checkpoint involving NLRP3 protein and P2Y2 receptor and can be modulated in order to develop new therapeutic approaches to fight against the emergence of viruses resistant to conventional retroviral treatments
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Verma, Deepti. "Genetic Variations in the NLRP3 Inflammasome : Susceptibility Factor for Chronic Inflammation." Doctoral thesis, Linköpings universitet, Cellbiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-70077.
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NLRP3 has been recognized as one of the key components of innate immunity. Upon activation, NLRP3 forms a multiprotein complex called as the ‘inflammasome’ which leads to the activation of pro-inflammatory caspase-1 which subsequently results in the formation of Interleukin (IL)-1β and IL-18. Mutations in the NLRP3 gene can lead to its constitutive activation resulting in an uncontrolled production of IL-1β. These mutations have been implicated in hereditary inflammatory diseases, often grouped under Cryopyrin associated periodic syndromes (CAPS, cryopyrin being an alternative name for NLRP3). Paper I in this thesis presents the case of a patient with a long history of arthritis and antibiotic resistant fever, but without the typical symptoms of CAPS. The patient was a heterozygous carrier of two common polymorphisms, Q705K in NLRP3 and C10X in CARD-8. Experimental studies indicated elevated activity of caspase-1 and IL-1β levels in the patient and a total clinical remission was achieved by IL-1β blockade. These two polymorphisms simultaneously occur in almost 4% of the control population, suggesting the possibility of a genetic predisposition for inflammation in these individuals. We, therefore, investigated a cohort of rheumatoid arthritis (RA) patients in paper II, and found that carrying the combined polymorphisms resulted in increased RA susceptibility and a more severe disease course. Hypothetically, this subgroup might benefit from IL-1β blockade. Paper III presents two patients: siblings, who did not fit into a typical CAPS phenotype. The inflammatory symptoms in both the patients appeared in adult life. A novel and functional M299V mutation in NLRP3 was detected in the siblings who neither had common symptoms nor the same disease severity. Consequent with inflammasome activation, abnormally elevated caspase-1 activity and IL-1β levels were seen. Patients in papers I and III highlight the risk of missing out such patients if attempting a very conventional diagnosis. Paper IV dissects the functional role of Q705K in NLRP3 using THP-1 cells in an in vitro model. Moderately elevated IL-1β and IL-18 levels could be observed in the THP-1 cells expressing Q705K, as compared to the wild type expressing cells, indicating a gain-of-function. Due to the presence of this alteration in healthy individuals it can be classified as a low-penetrance alteration. Additional studies are warranted to elucidate the mechanistic details of this polymorphism.
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Möcking, Jonas [Verfasser]. "Investigating the molecular basis of human NLRP1 inflammasome activation / Jonas Möcking." Bonn : Universitäts- und Landesbibliothek Bonn, 2020. http://d-nb.info/1207074691/34.
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Faure, Emmanuel. "Implications de la reconnaissance de Pseudomonas aeruginosa par le NLRC4-Inflammasome." Phd thesis, Université du Droit et de la Santé - Lille II, 2013. http://tel.archives-ouvertes.fr/tel-01060187.
Full textAbstract:
L'inflammasome est complexe protéique intracellulaire de l'immunité innée permettant la reconnaissance de pathogènes intracellulaires. NLRC4, un Nod-like récepteur permettant l'activation de l'inflammasome est impliqué dans la reconnaissance du flagelle ainsi que du système de sécrétion de type 3 (SST3) de Pseudomonas aeruginosa, une bactérie majoritairement extracellulaire. Nous avons donc déterminer l'impact de la reconnaissance de P. aeruginosa par le NLRC4-inflammasome in vivo dans un modèle murin de pneumonie aiguë. De façon surprenante, l'activation du NLRC4-inflammasome par le SST3 de P. aeruginosa contribue à diminuer la survie de l'hôte en diminuant la clairance bactérienne pulmonaire et en augmentant la lésion pulmonaire induite. En effet, la perte de l'activation de l'inflammasome chez les souris NLRC4/- permet d'une part, une réponse précoce méfiée par l'IL-17A. Cette réponse dépendant de l'IL-17A conduit à une expression majeure de peptides antimicrobiens par l'épithélium pulmonaire et diminue la lésion pulmonaire en diminuant le recrutement des cellules immunitaires inflammatoires. L'administration d'IL-18 recombinante murine ou l'inhibition de cette voie par un anticorps anti-IL-17A inhibe cette réponse IL-17A dépendante. Ces résultats mettent en évidence un nouveau rôle du SST3 de P. aeruginosa, qui en plus de son effet cytotoxique et de la translocation d'exotoxines, permet d'activer l'inflammasome pour échapper à la réponse immunitaire innée de l'hôte en inhibant une voie IL-17 dépendante.
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Ward, Christopher. "An investigation of the mechanisms underlying HIV-1-mediated inflammasome activation." Thesis, Cardiff University, 2017. http://orca.cf.ac.uk/104950/.
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Around 35 million people are living with HIV-1 infection globally. Untreated infection results in progression to AIDS and mortality. Current antiretroviral treatment is not curative and does not treat the underlying inflammatory processes caused by the presence of HIV-1, a cytotoxic and tissue toxic virus. HIV-1 triggers overwhelming and dysregulated pro-inflammatory cytokine response during infection, including deranged interleukin-1 beta and interleukin-18 levels. We investigate the mechanisms underlying how HIV-1 activates the inflammasome, a multimolecular complex involved in the production of potent inflammatory markers such as interleukin-1 beta and interleukin-18. The inflammasome is often activated in a two-signal process requiring initial sensing by Toll-like receptors to generate signal 1, priming inflammasome components; and signal 2 sensed by NOD-like receptors which recruit and activate the inflammasome. Human leukocytes were stimulated with live HIV-1 or its cognate envelope proteins gp120 or gp41, or transfected with plasmids encoding HIV-1 viroporin Vpu or gp41. NLRP3 knock-down attenuated inflammasome activation and interleukin-1beta/interleukin-18 secretion following HIV-1, gp120 or gp41 stimulation. gp120 induced inflammasome activation also required the presence of TLR2 and TLR4, suggesting a degree of cooperation in effecting signal 1. Dual NLRP3/NLRC4 knockdown reduced the inflammasome response to HIV-1 gp41 stimulation which was found to be dependent on chloride anion flux across integral lipid rafts in the trans-Golgi network. The results imply a critical role for TLR2 and TLR4 in recognising HIV-1 proteins providing signal 1 for inflammasome formation. Ion fluxes in the cell due to HIV-1 infection are the catalyst that triggers inflammasome activation via NLRP3 or NLRC4, and secretion of interleukin-1 beta and interleukin-18. The findings contribute to the general understanding of HIV-1 recognition by the human innate immune system and help to further clarify precisely how HIV-1 causes striking dysregulation in cytokine profiles in people living with HIV-1 infection.
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Ulland, Tyler Kent. "Mvin mediates Francisella Tularensis virulence through evasion of AIM2 inflammasome activation." Thesis, University of Iowa, 2010. https://ir.uiowa.edu/etd/751.
Full textAbstract:
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.
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Potter, Ryan. "NLRP INFLAMMASOME : CRISPR knockout of RIPK3 gene in THP-1 monocytes." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-18980.
Full textAbstract:
Inflammation is a biological response to harmful stimuli which assists the overall immune response in defending the organism against potential threats like bacteria and other pathogens. This response is largely regulated by multiprotein complexes known as inflammasomes, which use one or more multi-stage networks to create a coordinated response across cell groups. The NLRP3 inflammasome in particular is thought to have many interacting partners in a complex inflammation and cell-death management network. RIPK3 is one protein that has been implicated in regulation of inflammation and cell death pathways in connection with the NLRP3 inflammasome. In this study, we attempt to create a CRISPR/CAS9 construct to knockout the RIPK3 gene in THP-1-ASC-GFP monocytes with ViafectTM Transfection in order to examine the apparent effects at various stages of activation. Transfected cells were then quantitatively examined using qPCR. While the Guide-itTM CRISPR/CAS9 Systems kit produced high quantity, high quality DNA plasmids, this study found that the ViafectTM system resulted in reduced RNA isolation among cells differentiated with PMA as compared to non-differentiated counterparts. qPCR performed on cDNA generated from the RNA extractions resulted in highly erratic Cq values with standard deviations well above the acceptable limitations among technical replicates in both experimental and control gene samples. Additionally, this study found that the reference gene used, ACTB, did not maintain stability across samples. Due to mistakes and time constraints, the experiment failed to provide any substantial evidence of activity; however, an architecture is developed for optimization of future studies.
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Yang, Rui. "Interaction between caspases and their substrates in the inflammasome signaling pathway." Case Western Reserve University School of Graduate Studies / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1559917811566556.
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Bueter, Chelsea L. "Disparate Activation of the Inflammasome by Chitin and Chitosan: A Dissertation." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/687.
Full textAbstract:
Chitin is an abundant polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons. The immunological properties of both chitin and its deacetylated derivative chitosan are of relevance due to frequent natural exposure and their increasing use in translational applications. Depending on the preparation studied and the endpoint measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. Highly purified chitosan and chitin were prepared and the capacity of these glycans to stimulate the release of the inflammasomeassociated cytokine IL-1β was examined. Chitosan was shown to be a potent inflammasome activator in mouse bone marrow macrophages, macrophages polarized towards a M1 or M2 phenotype, dendritic cells, peritoneal cells, and human PBMCs. Acetylation of the chitosan to chitin resulted in a near total loss of IL-1β activity in all cell types tested. The size of the chitosan particles played an important role, with small particles eliciting the greatest activity. An inverse relationship between size and stimulatory activity was demonstrated using chitosan passed through size exclusion filters as well as with chitosan-coated beads of defined size. Partial digestion of chitosan with pepsin resulted in a larger fraction of small phagocytosable particles and more potent inflammasome activity. Inhibition of phagocytosis with cytochalasin D abolished the IL- 1β stimulatory activity of chitosan, offering an explanation for why the largest particles were nearly devoid of activity. Thus, the deacetylated polysaccharide chitosan potently activates the NLRP3 inflammasome in a phagocytosis-dependent manner. The reason for chitin’s inability to elicit IL-1β is unknown, but it does not appear to be due to active inhibition of the inflammasome and while chitin appears to be more readily digested by macrophage cell lysates, it does not occur at a rate which would likely impact inflammasome activation. There are three proposed mechanisms for NLRP3 inflammasome activation: K+ efflux, ROS, and lysosomal destabilization. The contributions of these mechanisms were tested and it was revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, the laminin receptor was evaluated as a potential chitin receptor. These studies provide insight into the activating properties of chitin and chitosan and highlight the importance of matching particle size and degree of acetylation to the level of activity desired for translational applications.
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Bueter, Chelsea L. "Disparate Activation of the Inflammasome by Chitin and Chitosan: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/687.
Full textAbstract:
Chitin is an abundant polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons. The immunological properties of both chitin and its deacetylated derivative chitosan are of relevance due to frequent natural exposure and their increasing use in translational applications. Depending on the preparation studied and the endpoint measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. Highly purified chitosan and chitin were prepared and the capacity of these glycans to stimulate the release of the inflammasomeassociated cytokine IL-1β was examined. Chitosan was shown to be a potent inflammasome activator in mouse bone marrow macrophages, macrophages polarized towards a M1 or M2 phenotype, dendritic cells, peritoneal cells, and human PBMCs. Acetylation of the chitosan to chitin resulted in a near total loss of IL-1β activity in all cell types tested. The size of the chitosan particles played an important role, with small particles eliciting the greatest activity. An inverse relationship between size and stimulatory activity was demonstrated using chitosan passed through size exclusion filters as well as with chitosan-coated beads of defined size. Partial digestion of chitosan with pepsin resulted in a larger fraction of small phagocytosable particles and more potent inflammasome activity. Inhibition of phagocytosis with cytochalasin D abolished the IL- 1β stimulatory activity of chitosan, offering an explanation for why the largest particles were nearly devoid of activity. Thus, the deacetylated polysaccharide chitosan potently activates the NLRP3 inflammasome in a phagocytosis-dependent manner. The reason for chitin’s inability to elicit IL-1β is unknown, but it does not appear to be due to active inhibition of the inflammasome and while chitin appears to be more readily digested by macrophage cell lysates, it does not occur at a rate which would likely impact inflammasome activation. There are three proposed mechanisms for NLRP3 inflammasome activation: K+ efflux, ROS, and lysosomal destabilization. The contributions of these mechanisms were tested and it was revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, the laminin receptor was evaluated as a potential chitin receptor. These studies provide insight into the activating properties of chitin and chitosan and highlight the importance of matching particle size and degree of acetylation to the level of activity desired for translational applications.
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Gonçalves, Amanda Costa. "Papel do inflamassoma NLRP3 em modelo de infecção sistêmica por Sporothrix schenckii /." Araraquara, 2016. http://hdl.handle.net/11449/146721.
Full textAbstract:
Orientador: Iracilda Zeppone CarlosBanca: Fernanda Freitas Anibal
Banca: Luciana Simon Pereira Crott
Banca: Angela Maria Victoriano de Campos Soares
Banca: Cleni Mara Marzocchi Machado
Resumo: A esporotricose é uma infecção fúngica causada pelas espécies do complexo Sporothrix, incluindo Sporothrix schenckii sensu stricto. Os receptores de reconhecimento padrão (PRRs), pertencentes às células da resposta imune inata, reconhecem os padrões moleculares associados a patógenos (PAMPs). Uma classe de PRRs que tem sido associada ao reconhecimento de fungos é a dos receptores Nod-like (NLR). Após o reconhecimento de PAMPs, os receptores NLR family pyrin domain-containing 3 (NLRP3), em conjunto com a molécula adaptadora Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) e com a caspase-1, formam o inflamassoma NLRP3. Quando ativado, este complexo protéico promove a maturação das citocinas pró-inflamatórias IL-1β e IL-18, que influenciam no desenvolvimento das respostas imune Th17 e Th1, respectivamente. O inflamassoma NLRP3 também é responsável pela piroptose, uma morte celular inflamatória regulada por caspase-1. Neste contexto, o objetivo deste estudo foi avaliar o papel do inflamassoma NLRP3 em modelo de infecção sistêmica por S. schenckii, utilizando um camundongo selvagem (WT) e três diferentes camundongos knockout (KO): NLRP3-/-, ASC-/- e Casp-1-/-. Todos os animais KO foram mais suscetíveis que os WT na infecção por S. schenckii. A ativação do inflamassoma NLRP3 foi essencial para a liberação ex vivo de NO, IL-1β, IL-17, IL-18, mas não para IFN-γ. Além disso, S. schenckii desencadeou maior expressão de caspase-1 ativa e maior porc... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Sporotrichosis is a mycosis caused by fungi from the Sporothrix schenckii species complex, whose prototypical member is Sporothrix schenckii sensu stricto. Pattern recognition receptors (PRRs) recognize and respond to pathogen-associated molecular patterns (PAMPs) and shape the following adaptive immune response. A family of PRRs most frequently associated to fungal recognition is the nucleotide-binding oligomerization domain-like receptor (NLR). After recognition of a PAMP, NLR family pyrin domain-containing 3 (NLRP3) binds to apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 to form the NLRP3 inflammasome. When activated, this complex promotes maturation of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 that influence the development of the Th17 and Th1 immune responses, respectively. In addition, the NLRP3 inflammassome is responsible for pyroptosis, a highly inflammatory cell death regulated by caspase-1. In this work, we aimed to evaluate the role of the NLRP3 inflammasome to the outcome of the S. schenckii infection using a wild-type mice (WT) and three different knockout mice (KO): NLRP3-/-, ASC-/-, and Casp-1 -/- . All the KO mice were more susceptible to the infection than the WT during the S. schenckii infection. Furthermore, the NLRP3 inflammasome seems to be critical to the ex vivo release of NO, IL-1β, IL-18, and IL-17, but not IFN-γ. The S. schenckii infection led to increased activation of caspase-1 and cell death by pyroptosis in WT mice. Also, a role for the inflammasome in shaping the adaptive immune response was suggested by lower frequencies of type 17 helper T (Th17) and Th1/Th17 cells in S. schenckii-infected KO mice. On the other hand, absence of any of the inflammasome components resulted in increased frequency of cytotoxic T cells in this infection... (Complete abstract click electronic access below)
Doutor
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Saxena, Pallavi. "Role of Inflammatory Cytokine Signaling in Control of Bacterial Infection." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/41076.
Full textAbstract:
The immune system rapidly mounts an innate immune response to invading pathogens that is accompanied by antigen-presentation, to promote the development of the adaptive immune response. These responses orchestrate through signal transduction by PRRs that recognize PAMPs, which results in the expression of various cytokines and mediators to promote pathogen control. Herein, we investigated the role of the type I interferon (IFN)- and the p38MAPK- pathways in response to infection with Salmonella Typhimurium (ST). We delved into the mechanisms through which IFNAR1-signaling results in host susceptibility against ST and show that while STAT2 and IRF9 promote susceptibility against ST, this is antagonized by STAT1. Our results indicate that IFNAR1-signaling induces IL-10 production through the ISGF3 complex, which indeed inhibits the production of IL-1β (via NLRP3 and caspase-1)
resulting in a state of resistance against ST. Furthermore, our work elucidates that MK2, which is a p38MAPK substrate promotes host resistance, which is contradictory to type I IFNs despite the fact that MK2 regulates cytokine expression in a similar pattern to IRF9. We demonstrate that MK2 inhibits inflammasome signaling via NLRP3, caspase-1 and caspase11. We also reveal a role for MK2 in regulating IL-1β production via distinct signaling pathways including inhibition of MSK1/2 besides activation of the autophagic machinery; which also contribute to the enhanced inflammasome activation seen in Mk2- deficient cells. Thus, our observations illuminate the fact that the type I IFN pathway and the p38MAPK pathway are only dependent on each other to a certain extent in modulating the innate immune
response to Salmonella infection, thereby bringing about varied outcomes in the infected host.