Le virus de l'hépatite B (HBV) infecte chroniquement plus de 250 millions de personnes. Des traitements existent permettant de contrôler la production de particules infectieuses. Cependant, aucun des traitements actuels ne permet d'éradiquer complètement l'infection. Il est donc nécessaire de développer de nouvelles stratégies thérapeutiques, incluant des approches immunothérapeutiques pour permettre un meilleur contrôle immunologique des infections HBV. Dans une étude récente menée au sein du laboratoire, il a été montré que l'IL-1ß est la cytokine ayant le plus fort effet antiviral contre la réplication d'HBV dans les hépatocytes. Dans le foie, la cytokine IL-1ß est principalement produite par les macrophages résidents (les cellules de Kupffer) ou infiltrant (monocytes inflammatoires différenciés en macrophages). De nombreuses études récentes ont montrés qu'HBV était capable de bloquer partiellement l'induction des réponses immunitaires innées. Il est donc important de déterminer si HBV est capable d'empêcher la production d'IL-1ß par les différents types de macrophages. L'objectif de cette thèse était d'étudier l'effet du virus sur le phénotype des macrophages et les implications de ces modifications phénotypiques sur l'établissement de l'infection dans les hépatocytes. Des monocytes du sang ou des macrophages du foie ont été purifiés, respectivement, du sang périphérique ou de résections hépatiques, et ont été exposés au virus pendant leur différentiation et/ou leur activation pour les monocytes, ou seulement pendant leur activation pour les macrophages hépatiques déjà différenciés. Il a été démontré que le virus de l'hépatite B est capable d'inhiber la sécrétion d'IL-6 et d'IL-1ß par les macrophages pro-inflammatoires. De plus, HBV est capable d'inhiber la sécrétion d'IL-1ß par les macrophages hépatiques stimulés par différents ligands. Finalement, les surnageants de macrophages pro-inflammatoires sont capables de bloquer l'établissement de l'infection, ce qui n'est pas le cas quand les macrophages ont été exposés au virus. Il apparait donc qu'HBV est capable de modifier le phénotype des macrophages pour favoriser l'établissement et la persistance de l'infection. La compréhension des mécanismes de subversion du phénotype des macrophages par le virus de l'hépatite B serait un premier pas vers le développement de nouvelles stratégies thérapeutiques / Hepatitis B virus (HBV) chronically infects over 250 million people worldwide. Several treatments can be used to prevent the formation of de novo particles. However, they do not allow the total eradication of the infection. Therefore, it is necessary to develop new therapeutic strategies, including immune-therapeutic ones, which would be more likely to lead to an immunological control of HBV infections. We have recently shown that IL-1ß is the most effective antiviral cytokine against the replication of HBV in vitro. In the liver, IL-1ß is mainly produced by resident macrophages (also called Kupffer cells) or infiltrating cells (inflammatory monocytes differentiated into macrophages). Recent studies have shown that HBV is able to partially inhibit the induction of innate immune responses. Hence, it was necessary to determine if HBV was also able to block the production of IL-1ß by the different types of macrophages.The objective of this thesis was to study the effect of HBV on macrophage phenotypes and the impact of those modifications on the establishment of HBV infection in hepatocytes.Blood monocytes and liver macrophages were purified, respectively, from peripheral blood or hepatic resections, and were exposed to HBV during their differentiation and/or activation for monocytes, or only during activation for liver macrophages which are already in a differentiated state. HBV was able to partially inhibit the secretion of IL-6 and IL-1ß by pro-inflammatory macrophages. Moreover, HBV was able to inhibit IL-1ß secretion by liver macrophages stimulated by different ligands and, conditioned medium of pro-inflammatory macrophages could inhibit the establishment of infection in hepatocytes. This effect was reverted when macrophages were exposed to HBV, concomitantly with a lower production of IL-6 and IL-1ß.In summary, HBV is able to modify macrophage phenotypes to favour the establishment and persistence of HBV infection. The full understanding of the mechanistic basis of how HBV phenotypically modifies macrophages will be a first step towards the development of new therapeutic strategies

Cytomegaloviruses (CMV), the prototypical β-herpesviruses, have co-evolved with their hosts and thus acquired multiple strategies for modulation of the immune response. Viral engagement of pattern recognition receptors (PRR), such as toll-like receptors (TLRs) and cytosolic nucleic acids sensors, initiates the host immune response through activation of elaborate signalling programs. The ensuing inflammatory response is further sustained and amplified through cytokines, such as IL-1β, activating signalling pathways greatly overlapping those utilized by TLRs. The central hypothesis of this thesis is that a viral counter-measure by murine CMV (MCMV) involves specific targeting of TLR- and IL-1β-induced signalling along the MyD88 to NF-κB pathway. To test this hypothesis MCMV inhibition of IL-1β signalling was initially investigated in a fibroblast cell line. It was demonstrated that in MCMV infected cells IL-1β-induced IκBα degradation is largely inhibited. Comparison of productive and non-productive infection showed this modulation requires de-novo viral gene expression beyond the immediate early region. Further investigations utilising a ORF M45 deletion mutant identified viral gene M45 as necessary for mediating the observed modulation of IL-1β- induced IκBα degradation. To further test the hypothesis, studies were extended to include TLR stimulation in the context of bone marrow-derived macrophages (BMDM) infection. It was found that TLR7/9-induced NF-κB activation is inhibited in MCMV infected BMDM. Overall, data presented in this study demonstrate a previously unrecognised MCMV inhibition of IL-1β- and TLR7/9-induced NF-κB activation, and indicate a role for viral gene M45 in mediating this effect.

IgA is the dominant antibody isotype found at mucosal surfaces during homeostasis. However, genetic variation in Fcγ receptors (FcγRs), a family of receptors that mediate immune cell activation by IgG, influences susceptibility to inflammatory bowel disease (IBD), suggesting that IgG may be important during gut inflammation. IBD is a chronic relapsing condition with two major subtypes, Crohn’s disease (CD) and ulcerative colitis (UC), both driven by aberrant immune responses to commensals. In the first part of this thesis, we sought to investigate anti-commensal IgG responses in patients with UC and to determine the mechanism by which local IgG might contribute to intestinal inflammation. We found that UC and murine dextran sodium sulfate (DSS)-induced colitis are associated with a significant increase in anti-commensal IgG and local enrichment of FcγR signalling pathway genes. The genes most robustly correlated with FCGR2A, an activating FcγR associated with UC susceptibility, were IL1B andCXCL8. Ex vivo stimulation of human and murine lamina propria mononuclear cells with IgG immune complexes (IC) resulted in an increase in these cytokines/chemokines. In vivo manipulation of the macrophage FcγR A/I ratio in transgenic mice determined IL-1β and Th17 cell induction. Finally, IL-1β blockade in mice with a high FcγR A/I ratio reduced IL-17 and IL-22-producing T cells and the severity of colitis. Our data reveal that commensal-specific IgG contributes to intestinal inflammation via FcγR-dependent, IL-1β-mediated Th17 activation. In this thesis, we have also addressed the interplay between IgG and group 3 innate lymphoid cells (ILC3s). ILC3s are closely related to natural killer cells, which are known to express FcγRs, and are characterised by their production of Th17 cytokines. Here, we have shown that ILC3s express FcγRs, that ICs drive IL-22 production and MHC class II expression by ILC3s, and FcγR signalling induces a transcriptional programme that reinforces ILC3 maintenance and functionality. These results represent a new paradigm for ILC activation, with direct regulation by the adaptive immune response. Finally, we have begun to address the role played by ILC3-derived cytokines in the regulation of local tissue-resident immune cells. We have demonstrated that ILC depletion significantly alters the activation state of intestinal macrophages, resulting in detrimental bacterial outgrowth following C. rodentium infection but protection from overwhelming DSS-induced inflammation. We have shown that GM-CSF promotes macrophage IL-1β and IL-23 production, which in turn act to reinforce ILC3-derived GM-CSF and IL-22 secretion in vitro, respectively. Therefore, ILC3s are essential coordinators of the local inflammatory response within the gut through activation and possible recruitment of immune cells, and their modulation may be beneficial in the treatment of IBD.

Inflammation plays a crucial role in protecting the host from infection and tissue injury. However, uncontrolled inflammation contributes to the pathogenesis of major auto-inflammatory diseases. Interleukin-1 (IL-1), a pleiotropic pro-inflammatory cytokine, is a pivotal mediator of many of these diseases. The best characterised IL-1 family members, IL-1α and IL-1β, are produced as precursor forms of 31 kDa in size. Both precursors are cleaved and secreted, activating transmembrane IL-1 receptors on IL-1-responsive cells. Many studies that focused on IL-1α have shown that the precursor and processed mature Ct peptide, as well as its N terminus (Nt) form, can elicit a signal. However, with IL-1β, only the processed mature Ct form is known to elicit an inflammatory response and no immunological activity has been attributed to Nt fragments of pro-IL-1β. Therefore, the first objective of this study was to produce recombinant human Nt-IL-1β fragments in bacterial and mammalian expression system to investigate their possible immunomodulatory functions. Recombinant His-tagged N-terminus fragments (10 and 14 kDa) of pro-IL-1β were cloned into the bacterial expression vector pET-22(+) and expressed in E. coli BL21(DE3) followed by purification using three consecutive columns (IMAC, SEC and AEC). Purification analysis of eluted proteins from columns indicated that the recombinant proteins were always co-purified with some other bacterial proteins. The Nt fragments of pro-IL-1β were cloned into the mammalian expression plasmid, pcDNA3.1(+). Expression of these proteins was monitored by transfection of two mammalian cell lines: Human Embryonic Kidney (HEK) 293 cells and monkey kidney cells (COS-7). No protein expression was observed with either construct. These limitations urged us to investigate the expression and degradation of endogenous IL-1 in vitro. Previous studies have shown that the transcription of cytokine genes in response to lipopolysaccharide (LPS) is usually rapid and begins to decline within a few hours after stimulation. The proteasome is the major cellular proteolytic apparatus and controls the turn-over of cellular proteins. We investigated the intracellular stability of IL-1α and IL-1β in LPS-stimulated mouse J774 macrophages and primary mouse bone marrow derived macrophages (BMDMs). Exposure of LPS-stimulated J774 and BMDMs to three different classes of proteasome inhibitors (peptide alhedyde (ALLN), peptide boronate (MG262) and non-peptide inhibitor (β-lactone)) prevented the degradation of intracellular IL-1α and IL-1β in a concentration and time dependent manner. Furthermore, the release of IL-1 into the culture media was not affected by any of these inhibitors in LPS-stimulated J774 cells. However, in LPS-stimulated BMDMs, β-lactone increased the release of both IL-1α and IL-1β and ALLN only increased IL-1α release into culture supernatant compared to control. MG262 had no effect on the release of either. These data suggest that the proteasome plays an important role in controlling the amount of IL-1α and IL-1β by restricting the intracellular levels of these cytokines in activated monocytes and macrophages. Therefore, this study provides evidence in support of the hypothesis that the proteasome is involved in the degradation of IL-1α and IL-1β and may offer a potential therapeutic target in inflammatory diseases.

We previously reported that Prunella vulgaris aqueous extract (PVAE) promotes hepatic glycogen synthesis and decreases postprandial hyperglycemia in ICR mice. Inflammatory cytokines play a critical role in the pathogenesis of diabetes. This study was designed to examine whether PVAE has a protective effect on IL-1β-induced apoptosis in INS-1 cells. INS-1 pancreatic β cells were plated at 2×10 6/ml and treated with PVAE (100 μg/ml) 30 min before the cells were challenged with IL-1β (10 ng/ml). Untreated INS-1 cells served as control. INS-1 cell cytotoxicity was examined by MTT and lactate dehydrogenase (LDH) activity assays. Caspase-3 activity and activation of the apoptotic signaling pathway were analyzed by western blotting. NF-κB binding activity was examined by EMSA. The levels of inflammatory cytokines in the supernatant were measured by ELISA. IL-1β treatment significantly induced INS-1 cell death by 49.2%, increased LDH activity by 1.5-fold and caspase-3 activity by 7.6-fold, respectively, compared with control cells. However, PVAE administration significantly prevented IL-1β-increased INS-1 cell death and LDH activity and attenuated IL-1β-increased caspase-3 activity. Western blot data showed that PVAE also significantly attenuated IL-1β-increased Fas, FasL and phospho-JNK levels in the INS-1 cells. In addition, PVAE treatment significantly attenuated IL-1β-increased NF-κB binding activity and prevented IL-1β-increased TNF-α and IL-6 expression in INS-1 cells. Our data suggest that PVAE has a protective effect on IL-1β-induced INS-1 cell apoptosis. PVAE also attenuates IL-1β-increased NF-κB binding activity and inflammatory cytokine expression in INS-1 cells. PVAE may have a benefit for type I diabetic patients.

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L’arthrose ou ostéoarthrose (OA) est l’affection rhumatologique la plus fréquente au monde. Elle est caractérisée principalement par une perte du cartilage articulaire et l’inflammation de la membrane synoviale. L’interleukine (IL)-1ß, une cytokine pro-inflammatoire, joue un rôle très important dans la pathogenèse de l’OA. Elle exerce son action en induisant l’expression des enzymes cyclo-oxygénase 2 (COX-2), prostaglandine E synthétase microsomale 1 (mPGES-1) et l’oxyde nitrique synthétase inductible (iNOS) ainsi que la production de la prostaglandine E2 (PGE2) et de l’oxyde nitrique (NO). Ces derniers (PGE2 et NO) contribuent à la synovite et la destruction du cartilage articulaire par leurs effets pro-inflammatoires, pro-cataboliques, anti-anaboliques, pro-angiogéniques et pro-apoptotiques. Les modifications épigénétiques, telles que la méthylation de l’ADN, et l’acétylation et la méthylation des histones, jouent un rôle crucial dans la régulation de l’expression des gènes. Parmi ces modifications, l’acétylation des histones est la plus documentée. Ce processus est contrôlé par deux types d’enzymes : les histones acétyltransférases (HAT) qui favorisent la transcription et les histones déacétylases (HDAC) qui l’inhibent. L’objectif de ce travail est d’examiner le rôle des enzymes HDAC dans la régulation de l’expression de la COX-2, mPGES-1 et iNOS. Nous avons montré qu’au niveau des chondrocytes, les inhibiteurs des HDAC (iHDAC), trichostatine A (TSA) et butyrate de sodium (NaBu), suppriment l’expression de la COX-2 et iNOS au niveau de l’ARNm et protéique, ainsi que la production de la PGE2 et du NO, induites par l’IL-1ß. L’effet inhibiteur à lieu sans affecter l’activité de liaison à l’ADN du facteur de transcription NF-κB (nuclear factor κ B). La TSA et le NaBu inhibent également la dégradation induite par l’IL-1ß des protéoglycanes au niveau du cartilage. Nous avons également montré, qu’au niveau des fibroblastes synoviaux, les iHDAC, TSA, NaBu et acide valproïque (VA), suppriment l’expression de la mPGES-1 ainsi que la production de la PGE2 induites par l’IL-1ß. En utilisant diverses approches expérimentales, nous avons montré que HDAC4 est impliquée dans l’induction de l’expression de la mPGES-1 par l’IL-1ß. HDAC4 exerce son action, via son activité déacétylase, en augmentant l’activité transcriptionnelle de Egr-1 (early growth factor 1), facteur de transcription principal de l’expression de la mPGES-1. L’ensemble de ces résultats suggère que les inhibiteurs des HDAC pourraient être utilisés dans le traitement de l’OA. / Osteoarthritis (OA) is the most common form of arthritic diseases in the world. It is primarily characterized by the loss of articular cartilage and inflammation of the synovial membrane. Interleukin (IL)-1ß is a pro-inflammatory cytokine that plays a major role in the pathogenesis of OA. It induces the expression of cyclo-oxygenase 2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), inducible nitric oxide synthase (iNOS), as well as the production of prostaglandin E2 (PGE2) and nitric oxide (NO). The later (PGE2 and NO) contribute to articular cartilage destruction and synovitis through their pro-inflammatory, pro-catabolic, anti-anabolic, pro-angiogenic and pro-apoptotic effects. Epigenetic modifications such as DNA methylation, histone acetylation and methylation play a crucial role in gene expression. Among these modifications, histone acetylation is the most studied. Histone acetylation is determined by two types of enzymes: histone acetyltransferases (HAT) and histone deacetylases (HDAC) which activate and repress transcription, respectively. The purpose of these studies is to examine the role of HDAC enzymes in the regulation of COX-2, mPGES-1, and iNOS expression. We demonstrated that HDAC inhibitors (HDACi), trichostatin A (TSA) and sodium butyrate (NaBu), suppressed the Il-1ß-induced transcription and translation of COX-2 and iNOS, as well as the production of PGE2 and NO in chondrocytes. The inhibitory effect of HDACi on transcription does not affect the binding activity of NF-κB (nuclear factor κ B) to DNA. Treatment with TSA and NaBu also inhibited the Il-1ß-induced degradation of proteoglycan in cartilage explants. We also showed that HDACi, TSA, NaBu and valproic acid (VA), suppressed IL-1-induced-mPGES-1 expression and the production of PGE2 in synovial fibroblasts. Our data indicated that HDAC4 is involved in Il-1ß-induced expression of mPGES-1. HDAC4, through its deacetylase activity, up-regulated the transcriptional activity of Egr-1 (early growth factor-1), a principal transcription factor for the expression of mPGES-1. From our studies we propose that HDAC inhibitors can be used in the treatment of OA.

No description available.

No description available.