L’infection par le virus de l’hépatite E (VHE) peut entraîner une hépatite aiguë chez l’homme évoluant en hépatite fulminante dans 1-4% des cas, voire 20% chez les femmes enceintes dans les régions endémiques. Le VHE, transmis par voie entérique, est responsable de grandes épidémies d’origine hydrique dans les pays en voie de développement et de nombreux cas sporadiques d’origine zoonotique dans des pays industrialisés. La description récente de cas d’hépatites E chroniques ou d’atteintes neurologiques graves souligne l’importance de caractériser les interactions du VHE avec son hôte. L’objectif de ce projet de thèse était de caractériser les interactions entre le VHE et la réponse immune innée de l’hôte et en particulier avec le système interféron de type I (IFN-I).La première partie de ce projet a consisté en l’étude de la modulation des voies de signalisation de l’IFN-I par la polyprotéine non-structurale ORF1 du VHE. Celle-ci est constituée de plusieurs domaines fonctionnels putatifs tels qu’une methyltransférase (Met) ou une protéase à cystéine de type papaïne (PCP) dont les fonctions sur la signalisation IFN-I restent encore peu connues. Les résultats obtenus ont montré que le domaine MetPCP de l’ORF1 est capable d’inhiber l’activation du promoteur de l’IFN-β et de celui des gènes sous le contrôle de l’IFN contenant des éléments de réponse à l’IFN (ISRE), ainsi que l’expression de certains des gènes induits par l’IFN (ISGs). En recherchant le mécanisme impliqué dans l’inhibition du promoteur ISRE, nous avons montré que le domaine MetPCP inhibe la phosphorylation de STAT1 et sa relocalisation nucléaire. Nous avons également montré que le domaine MetPCP n’inhibe pas la phosphorylation de STAT2. Le mécanisme d’action du domaine MetPCP reste encore à préciser. La deuxième partie de ce projet a été de déterminer si l’infection par le VHE entraîne la production d’IFN-I par les cellules dendritiques plasmacytoïdes (pDCs). En effet, les pDCs sont la principale source d’IFN-I et jouent un rôle crucial dans la réponse immunitaire innée et adaptative. Les résultats obtenus suggèrent que les pDCs ne produisent que modérément de l’IFN-I lorsqu’elles sont co-cultivées avec des cellules infectées par le VHE. L’ensemble des résultats obtenus pendant ce travail de thèse suggère que le VHE utilise plusieurs mécanismes pour moduler la signalisation IFN-I de l’hôte. / Hepatitis E virus (HEV) is the causative agent of acute hepatitis in humans that can lead to fulminant hepatitis in 1-4% of cases, and in 20% of pregnant women in endemic regions. HEV is an enterically-transmitted virus responsible for large waterborne epidemics in developing countries and numerous cases of zoonotic hepatitis E in industrialized countries. The recent description of cases of chronic hepatitis E and severe neurological disorders highlight the importance to characterize the interactions between HEV and the host. The objective of this PhD project was to characterize the interactions between HEV and the host innate immune response and particularly with the type I interferon system (IFN-I).The first part of this project aimed to study the ability of the ORF1 non-structural polyprotein of HEV to modulate the IFN-I signalling pathways. HEV ORF1 contains several putative functional domains including a methyltransferase (Met) and a papain-like cysteine protease (PCP) whose functions on the IFN-I signalling remain poorly understood. The results obtained showed that the MetPCP domain of ORF1 inhibits IFN-β and IFN-stimulated response element (ISRE) promoter activation and the expression of some IFN-stimulated genes (ISGs). We then investigated the mechanism involved in this inhibition of ISRE promoter activation. We showed that the MetPCP domain inhibits STAT1 phosphorylation and nuclear translocation. In contrast, we found that the MetPCP domain does not inhibit STAT2 phosphorylation. However, the mode of action of MetPCP remains to be fully characterised. The second part of this project aimed to determine the ability of plasmacytoid dendritic cells (pDCs) to produce IFN-I in response to HEV infection. Indeed, pDCs are the main IFN-I source and play a crucial role in innate and adaptive responses. The results obtained suggest that pDCs produce IFN-I moderately when co-cultured with HEV-infected cells. Taken together, the results obtained during this PhD project suggest that HEV has evolved different mechanisms to modulate the IFN-I host response.

Host sensors that recognize pathogen associated molecular patterns and the mechanisms of innate immune response to mouse polyomavirus (MPyV) infection were the main topics of current work. We found that MPyV did not induce interferon (IFN) production during early events of infection, but induced interleukin-6 (IL-6) and other cytokine production without inhibiting virus multiplication. Cytokine microenvironment changed the phenotype of adjacent non infected fibroblasts toward the cancer-associated fibroblast (CAF)-like phenotype. We identified Toll-like receptor 4, a sensor of the innate immunity system, to be responsible for infection dependent IL-6 production. In an effort to determine whether and where virions are released from endosomal compartments into the cytosol, we found that the hydrophobic domains of minor capsid proteins, exposed on the surface of virions after their partial disassembly in the ER, play an important role in effective escape of virions from the lumen part of endoplasmic reticulum into the cytosol, Although naked, partially disassembled virions appear before translocation to the nucleus in the cytosol, viral DNA is not recognized by cytosolic sensors at this phase of infection Sensing of MPyV resulting in IFN production occurs first during viral replication. Mutant virus,...

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We have studied the effect of interferon α (IFN-α) on MHC class II expression by human buccal epithelial cells (BEC), and mRNA expression by BEC and mucosal-associated mononuclear cells (MAMC). In 6 experiments, freshly collected BEC were suspended at a concentration of 1.0 x 105/ml in RPMI 1640 and incubated in the presence of 0-10000 IU/ml of human lymphoblastoid IFN-α (HuIFN-α). Zero and six hour samples were analyzed by single color flow cytometry using FITC-labeled murine IgG1 monoclonal antibody to HLA-DR. Preparations were also analyzed for expression of cytokine transcripts (IL-2 IL-4, IL-5, IL-6, IL-8, IFN-γ, GM-CSF) by reverse transcriptase polymerase chain reaction (RT-PCR). Increasing concentrations of IFN-α resulted in proportionate increases in the percentage of HLA-DR + BEC (r = 0.7897, p = 0.0627) and in the percentage of HLA-DR + staining at higher intensities (101 to 102 log fluorescence intensity) (LFI) (r = 0.40l0, p = 0.0424). The percentage of HLA-DR + BEC rose from a mean of 1.5% with no IFN-α to 7% with 10000 IU/ml IFN-α (p < 0.05). The percentage of HLA-DR + BEC staining at 101 to 102 LFI rose from a mean of 8.3% with no added IFN-α to 19.2% with 10000 IU/ml IFN-α (p <0.05). Unstimulated BEC constitutively expressed IL-8 and GM-CSF. IFN-α stimulated preparations also expressed IFN-γ, possibly due to the presence of MAMC, which comprised 2-9% of the total cell population. These data indicated that HuIFN-α upregulates MHC class II expression by human BEC, possibly by enhancing IFN-γ production by MAMC present in the culture preparations.

The dichotomy between type 1 and 2 conventional DCs under steady-state conditions is well defined. Bosteels et al. demonstrate that, upon inflammation, cDC2s acquire a hybrid inf-cDC2 phenotype, sharing phenotype, gene expression, and function with cDC1s and monocyte-derived cells, to optimally boost CD4 and CD8 immunity via Fc receptors.

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Urinvägsinfektioner (UVI) är ett vanligt hälsoproblem som drabbar miljontals människor. Den allvarligaste formen, akut pyelonefrit (APN) kan ge svåra komplikationer. Urinvägspatogena Escherichia coli (UPEC) som orsakar APN uttrycker P fimbrier som specifikt binder till glykosphingolipider på ytan av uroepitelceller. Det sätter igång en toll-like receptor 4 (TLR4) beroende men LPS-oberoende immunreaktion. Den roll som interferon beta (IFN-β) spelar vid bakterieinfektioner är inte helt klarlagd men studier som gjorts på IFN-β knockoutmöss visade på en ökad infektionsbenägenhet och svåra njursymptom vid infektion med UPEC. IFN-β uttrycket i uroepitelceller som svar på bakterieinfektion undersöktes. För att ta reda på om uttrycket är P fimbrieberoende infekterades humana A498 njurcarcinomceller med den P fimbrieförsedda pyelonefritstammen CFT073 eller den icke-virulenta asymtomatisk bakterieuristammen E. coli 83972 och inkuberades i 1,5 respektive 4 timmar. Som kontroll användes celler som enbart behandlats med PBS. Uttrycket av IFN-β analyserades med immunofluorescens (IF) och konfokalmikroskopi, samt med Western blot. Resultaten från konfokalmikroskopi visade att celler som exponerats för CFT073 under 4 timmar uttryckte mest IFN-β medan cellerna som utsatts för E. coli 83972 visade på ett omvänt förhållande. Western blot visade på högst uttryck i de E. coli 83972-behandlade cellerna. IFN-β uttrycktes i alla celler, inklusive kontrollcellerna, i någon utsträckning. Det kan betyda att IFN-β även induceras på någon alternativ väg och/eller att det uttrycks konstitutivt av njurepitelceller. / Urinary tract infections (UTI) are a common health concern and affect millions of people. The most severe form of UTI, acute pyelonephritis (APN) is associated with serious complications. Uropathogenic Escherichia coli (UPEC) that cause APN express P fimbriae which specifically bind to glycosphingolipid molecules on the surface of urothelial cells. This triggers a toll-like receptor 4 (TLR4) mediated but LPS-independent innate immune response. The role of interferon beta (IFN-β) in bacterial infections is not well known but experiments with IFN-β knockout mice have shown an increased susceptibility and severe kidney pathology when infected with UPEC. IFN-β induction in urothelial cells in response to bacterial infection was investigated. To find out whether this response is P fimbriae dependent, A498 human kidney carcinoma epithelial cells were exposed to the P fimbriated CFT073 pyelonephritis strain or the non-virulent E. coli 83972 asymptomatic bacteriuria strain and incubated for 1.5 and 4 hours. For control, cells were treated with PBS alone. The IFN-β expression was analysed using immunofluorescence (IF) and confocal microscopy, and Western blot. Confocal microscopy results showed that the response to bacteria was both time- and dose-dependent. The highest IFN-β expression was detected in cells exposed to CFT073 for 4 hours, while cells exposed to E. coli 83972 showed an inverse relationship. Western blot analysis revealed that the highest expression was in the E. coli 83972 stimulated cells. IFN-β was expressed in all cells to some degree, including control cells. This could imply that IFN-β is induced by some other means and/or is constitutively expressed by kidney epithelial cells.