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. / The Danger Theory developed by Polly Matzinger specifies that what really matters for the immune system is not to distinguish between self « to protect » and non-self « to fight ». Any potentially detrimental situation for the host would be able to generate endogenous danger signal that are able to mount an immune response to eventually return to baseline. Lung repeated expositions to toxic environnemental pollutants lead to inflammation and then fibrosis in sterile condition, meaning without a role of micro-organism. Lung administration of Bleomycin in mice is a good model to define what are the endogenous molecules or danger signals and associated pathways. We have identified extracellular ATP and uric acid as danger signals released by stressed or damaged cells and able to produce pro-inflammatory interleukin-1β through activation of a cytoplasmic complex called the Nlrp3 inflammasome. We then became interested in the molecular mechanism of particle-induced Nlrp3 inflammasome activation in vitro. We demonstrate that particles such as silica or alum hydroxide lead to endogenous ATP release and further purinergic signaling via an autocrine/paracrine manner. We identified purinergic signaling as an essential triggering of interleukin-1β maturation in response to particles. As a whole, the work presented here highlights the critical role of two endogenous molecules, ATP and uric acid, in the Nlrp3-mediated immune response.

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 classiques. / In 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.

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 lumière. / Inflammation 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.

Flagellin is a highly immunogenic, bacterial protein considered to be abundant in the intestinal lumen. It has been reported to be an immunodominant antigen in patients with inflammatory bowel disease (IBD). In the work presented in this thesis several complementary murine models of IBD were employed to elucidate the role of innate immune sensing of flagellin in the development of intestinal inflammation. Pattern recognition receptors (PRRs) enable mammals to discriminate self from non-self through the recognition of microbial signatures, such as bacterial flagellin. Flagellin is detected by at least two distinct PRRs, NOD-like receptor 4 (NLRC4) and Toll-like receptor 5 (TLR5). Our experiments revealed that NLRC4 promoted protective effects during acute intestinal inflammation mediated by infection with Citrobacter rodentium, a close relative of enteropathogenic E. coli. Following infection with C. rodentium, Nlrc4^-/- mice developed more severe weight loss, increased bacterial colonisation levels and exacerbated intestinal inflammation compared to WT counterparts. Bone marrow chimera experiments revealed that NLRC4 expression in non-hematopoietic cells provided protection and intestinal epithelial cells expressed high NLRC4 mRNA levels. These results suggest that NLRC4 inflammasome activation in the intestinal epithelium provides potent, protective effects during infection with a mucosal pathogen. In contrast, TLR5 was shown to promote protective effects during chronic, T-cell mediated intestinal inflammation driven by adoptive transfer of naïve CD4+ T-cells into lymphopenic Rag^-/- hosts. The absence of TLR5 in Rag^-/- recipients resulted in accelerated and exacerbated IBD. Furthermore, chronic T-cell dependent colitis driven by Helicobacter hepaticus, a flagellated, enteric bacterium, was more severe in mice deficient in TLR5. Finally, construction of a H.hepaticus Type 6 secretion system deletion mutant revealed delayed pathogenicity in an innate model of intestinal inflammation, most likely due to reduced initial colonisation of mutant H.hepaticus.

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.

Leishmania species are vector-borne protozoan parasites that cause a spectrum of human diseases, with an estimated 12 million people infected in 88 countries. Inflammation plays distinct roles in the different clinical syndromes. Visceral leishmaniasis, in which parasites migrate from the site of infection and proliferate in liver and spleen, is accompanied by systemic immune suppression. Cutaneous leishmaniasis, where parasites remain at the site of inoculation and create a long-term ulcer, is associated with vigorous systemic immunity to the parasite. The innate immune sensing pathways responding to Leishmania spp. parasites are not fully described. NLR proteins are a class of structurally related cytosolic proteins. The most well described NLRs form inflammasome complexes that generate strong inflammatory responses to “danger” signals. Other NLRs do not form inflammasomes and have anti-inflammatory functions. While NLR proteins are known to be important in the immune response to many pathogens, the roles NLR proteins in leishmaniasis have only begun to be investigated. We hypothesized that NLR proteins affect the pathogenesis of leishmaniasis through their ability to modulate inflammatory responses. We hypothesized that inflammasome activation in cutaneous leishmaniasis would be detrimental, leading to greater disease pathology, and that the potential anti-inflammatory functions of the non-inflammasome NLRs, NLRP6, NLRP10, and NLRP12, would be protective, reducing tissue damage. In contrast, we hypothesized that in visceral leishmaniasis greater inflammation due to activation of the inflammasome would be protective and control parasite replication, while the anti-inflammatory NLRs would be permissive to parasite replication in the liver and spleen by contributing to the immunosuppressive strategy of the parasite. We used knockout mouse strains lacking the inflammasome adaptor protein ASC, and several non-inflammasome forming NLRs, to investigate NLR proteins in murine models of visceral or cutaneous leishmaniasis. Our data showed that NLR proteins have important functions in visceral leishmaniasis, where they are essential for appropriate parasite homing and replication in the liver and spleen. In cutaneous leishmaniasis, we found that NLRP10 is essential for controlling inflammation in the skin, limiting lesion development and tissue damage at the site of infection. Taken together our findings show important functions for NLR proteins in leishmaniasis, influencing localized tissue specific inflammation, the adaptive immune responses, and clearance or long term residence of the parasite in the infected organs. This research underscores the importance of localized inflammation at the infection site to the pathogenesis and the course of leishmaniasis.

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.

Inflammation plays an important role in responding to injury and combating infections. In this thesis, I examine how inflammation is regulated by cytokines responsible for driving initial immune responses to combat infections. Toll-Like receptor (TLR)-mediated activation of monocytes, macrophages and dendritic cells can lead to the co-expression of proinflammatory cytokines including IL-1β, IL-23, and IL-27. IL-23 and IL-27 belong to the IL-12 cytokine family yet have distinct functions; IL-23, along with IL-1β, regulates TH17 cell differentiation, while IL-27 supports TH1 proliferation and inhibits TH17 differentiation. Our lab has previously demonstrated that IL-27 can modulate inflammasome activation, the multi-protein regulatory complex that produces bioactive IL-1β; however, the mechanism behind this is poorly understood. Similarly, the effect of IL-27 on IL-23 expression has not been well described. Using the CD14+ THP-1 monocytic cell line as a model system, I investigated the role of IL-27 on LPS-mediated inflammasome activation and IL-23 expression. To induce inflammasome activation, CD14+ THP-1 cells were treated with LPS and/or IL-27, followed by treatment with ATP. I demonstrated that IL-27-enhanced inflammasome activation, which is associated with increased surface expression of LPS and ATP receptors: TLR4 and P2X7 respectively. Furthermore, costimulation resulted in increased secretion of ATP from CD14+ THP-1 cells. Inhibition of ATP signaling and inflammasome activation significantly decreased secreted IL-1β, suggesting that an ATP autocrine feedback loop is driving IL-1β secretion. Moreover, LPS and IL-27 costimulation increased IL-23 expression concurrent with that of IL-1β and ATP secretion. Furthermore I showed that IL-23 secretion is dependent on inflammasome activation and IL-1β, and ATP signaling following IL-27 and LPS priming. My data point to a novel mechanism of IL-27 enhanced LPS-induced IL-1β and IL-23 secretion from CD14+ THP-1 cells through an ATP autocrine feedback loop. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2014-06-26 15:18:20.124

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.

The pro-inflammatory cytokine interleukin 1 (IL-1) is involved in numerous physiological and pathological processes. It contributes to thermoregulation, sleep, feeding behaviour and notably to the exacerbation of non-communicable disorders such as cancer, heart disease, stroke and epilepsy, which are the greatest cause of mortality worldwide. Given this important role, IL-1 is tightly regulated, with regulation mechanisms present at the level of its synthesis, activation and receptor engagement. However, when studying IL-1 in vitro, little notice is taken of the disease microenvironment in which it acts. Acidosis is a hallmark of disease, often due to poor perfusion resulting in a shift to anaerobic respiration, a build-up of lactic acid and poor clearance of CO2. Additionally, highly active infiltrating immune cells favour anaerobic respiration and can contribute to this local acidosis. This thesis utilised primary cell cultures, cell lines and reporter cells to explore the mechanisms of IL-1 signalling under disease-relevant acidic conditions. Subsequently, a murine seizure model was developed to further explore IL-1 signalling in disease conditions in vivo. This work demonstrated that acidic pH itself did not induce IL-1β release, however, it did promote release of minimally active 20 kDa IL-1β in response to damage associated molecular patterns (DAMPs) such as ATP, monosodium urate crystals or calcium pyrophosphate dihydrate crystals. The cleavage of pro-IL-1β into 20 kDa IL-1β was mediated by cathepsin D and was also induced on addition of lactic acid to the culture media. This 20 kDa IL-1β was not further cleaved to the active mature 17 kDa IL-1β thus its production limits the spread of inflammation. The intranasal administration of kainic acid induces seizures in C57Bl/6J mice, however, IL-1β was not observed acutely in this model thus the presence of 20 kDa IL-1β in vivo was not confirmed. In recent years, the contribution of IL-1 to disease has become well established. However, despite successes in the development of novel therapeutics targeted at blocking IL-1 activity, such as anakinra, canakinumab or rilonacept to treat cryopyrin associated periodic syndromes, a number of studies have demonstrated poor efficacy and only minor improvements in patients when targeting IL-1. Thus further knowledge of the mechanisms of IL-1 signalling in disease is required to understand this system and develop improved novel therapeutics.