La majorité des individus exposés au virus de l’hépatite C (VHC) développent une infection chronique. Une réponse immunitaire adaptative forte et soutenue est associée avec la guérison spontanée du VHC, mais les mécanismes sous-jacents demeurent mal définis. Le rôle des cellules NK et des cellules dendritiques (DC) dans la guérison spontanée du VHC est encore méconnu. Les cellules NK sont la population effectrice la plus importante de l’immunité innée car elles tuent les cellules infectées et sécrètent diverses cytokines. Les DC reconnaissent des agents infectieux et elles sont les premières à initier et réguler l’immunité adaptative. Les cellules NK et les DC interagissent également entre elles afin de réguler l’immunité innée et adaptative. L’hypothèse du projet de doctorat est que l'activité des cellules NK pendant la phase aiguë de l'infection par le VHC module la fonction des DC afin que ces dernières puissent générer une réponse immunitaire adaptative capable d'éliminer le VHC. Le premier objectif était d’établir une corrélation entre l'activité des cellules NK et l'évolution de l'infection au VHC. Nous avons observé une augmentation de la cytotoxicité, mais une diminution de la sécrétion de cytokines par les cellules NK chez les patients chroniques et qui ont résolu spontanément pendant la phase aiguë en comparaison aux contrôles non infectés, démontrant alors une dissociation entre ces deux fonctions. Nos résultats suggèrent que les cellules NK sont activées pendant la phase aiguë indépendamment de l’évolution de l’infection. Le deuxième objectif était d’établir une corrélation entre le phénotype et la fonction des DC, et l'évolution de l'infection. Nous avons d’abord observé que les DC plasmacytoïdes de tous les patients infectés ont un phénotype plus immature que les contrôles, et que ce phénotype est plus prononcé chez les patients ayant résolu spontanément. De plus, en réponse à des stimulations, nous avons observé que pendant la phase aiguë précoce, les DC myéloïdes (mDC) de tous les patients infectés indépendamment de l’évolution de l’infection produisent davantage de cytokines en comparaison aux contrôles. Cependant, cette hyperréactivité n’est pas soutenue au cours de l’évolution chronique. Le troisième objectif était d’établir une corrélation entre les interactions NK/DC et l’évolution de l’infection. Nous avons étudié la capacité des cellules NK à lyser les DC potentiellement tolérogéniques, ainsi que la capacité des DC matures à activer les cellules NK, et nous avons observé aucune différence entre les patients infectés et les contrôles. Finalement, nous avons démontré pour la première fois la capacité des DC immatures à inhiber la fonction des cellules NK. En conclusion, nous avons démontré que les cellules NK sont activées pendant la phase aiguë de l’infection par le VHC indépendamment de l’évolution de l’infection. De plus, la capacité des cellules NK à éliminer les DC potentiellement tolérogéniques est intacte. Finalement, les mDC sont hyperréactives pendant la phase aiguë de l’infection, mais cette hyperréactivité n’est pas soutenue avec la persistance de l’infection. Cette perte d’hyperréactivité des mDC ne semble pas affecter la capacité des DC à activer les cellules NK, mais elle pourrait jouer un rôle dans l’inefficacité de l’immunité adaptative à éliminer le VHC. / The majority of individuals exposed to the hepatitis C virus (HCV) develop a chronic infection. It is known that a strong and sustained adaptive immune response is associated with the spontaneous clearance of HCV, however the underlying mechanisms are not well defined. The role of natural killer (NK) cells and dendritic cells (DCs) during the spontaneous resolution of HCV remains unknown. NK cells are the primary effector population of the innate immune response which are able to kill infected cells and secrete various cytokines. On the other hand, DCs are the first cell type to initiate and regulate adaptive immunity after recognizing infectious pathogens. NK cells and DCs can also interact reciprocally to further regulate innate and adaptive immunity. Our hypothesis is that NK cell activity during acute HCV will modulate DC function to prime a highly efficient adaptive immune response resulting in viral clearance. The first aim of my project was to establish a correlation between NK cell activity and the outcome of HCV infection. We observed increased NK cell cytotoxicity, but decreased cytokine secretion during acute HCV in patients with chronic evolution as well as spontaneous resolution, further demonstrating a dissociation between these two NK cell functions. Our results suggest that NK cells are activated during acute HCV infection regardless of infection outcome. The second aim was to establish a correlation between DC phenotype, function and the outcome of infection. We observed that plasmacytoid DCs (pDCs) from all HCV-infected patients have a more immature phenotype as compared to negative controls, yet this is more pronounced in spontaneous resolvers. Furthermore, we observed that during the early acute phase, myeloid DCs (mDCs) from all HCV-infected patients, regardless of outcome, have increased production of cytokines as compared to un-infected controls in response to stimulation. However, this hyperresponsiveness of mDCs is not sustained with chronic evolution. The third aim was to establish a correlation between the NK/DC cross-talk and infection outcome. We studied the capacity of NK cells to kill potentially tolerogenic DCs, as well as the capacity of mature DCs to activate NK cells, and we observed no major differences between different stages of HCV infection and un-infected controls. However, we obtained unprecedented data which suggests that immature DCs have the capacity to inhibit NK cell function. In conclusion, our results demonstrate that NK cells are activated during acute HCV infection regardless of its outcome. Furthermore, the capacity of NK cells to kill potentially tolerogenic DCs is intact for all groups of patients. Finally, mDCs are hyperresponsive during acute HCV, but this hyperresponsiveness is not sustained with persistence of viremia. The loss of mDC hyperresponsiveness does not seem to affect the capacity of DCs to activate NK cells, but might play a role in the capacity of DCs to prime a highly efficient adaptive immune response resulting in viral clearance.

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Typ 2-cytokiner, IL-4 och IL-13 är kända för att spela en viktig roll i airway hyperresponsiveness (AHR) eller luftvägshyperreaktivitet, där de glatta muskelcellerna (ASM) drar ihop sig för lätt och för mycket som svar på direkta eller indirekta stimuli. Detta gör AHR till en avgörande egenskap hos astmatiker. Det har gjorts studier som har visat involvering av typ 2-cytokiner i AHR, men deras specifika inflammatoriska mekanismer är ännu outforskade. Denna experimentella studie på glatta muskelceller från luftvägarna (ASM) syftade till att undersöka involveringen av typ 2-cytokin inducerad kontraktilitet genom att fokusera på receptoraktivering, receptoruttryck samt ge insikter om frisättning av proteiner relaterade till luftvägsfibros, så kallad remodelling. Dessutom studerar den också effekten av IL- 4/IL-13 receptor antagonisten Dupilumab (anti-IL4Ra) på cytokinbehandlade HASM. Efter stimulering av HASM med IL-13 under 24 timmar visade resultaten att IL-13 orsakar en ospecifik, icke-receptormedierad ökning av intracellulärt kalciumflöde som svar på kalciumjonoforen A23187. Detta skulle potentiellt kunna öka kontraktiliteten hos glatta muskelceller som svar på flera olika kontraktila stimuli. Denna forskning ger också preliminära resultat som tyder på att IL-13 och IL-4 också ökar kalciumflödet som svar på aktivering av receptorer för specifika kontraktila mediatorer (Histamin, Carbachol, Leukotriene D4 och Substance P), och att effekten förmedlas via IL-4/IL-13-receptorn vilket blockeras med dupilumab som verkade minska effekten. Närvaron av fler receptorer för kontraktila mediatorer kan också öka kontraktiliteten hos glatta muskelceller som svar på IL-4 och IL-13. Återigen sågs ökat uttryck av receptorer för kontraktila stimuli efter behandling med cytokinerna som inhiberades av dupilumab. Dessutom undersökte vi effekten av IL-13 och IL-4 på frisättning av prokollagen 1 (en prekursor för mogna kollagena former) från mänskliga glatta muskelceller i luftvägarna. Våra resultat visade inte några signifikanta effekter på frisättningen av just detta kollagenprotein. Sammantaget visar vi att typ 2 cytokiner kan på flera olika sätt öka kontraktiliteten av glatta muskelceller vilket ökar vår kunskap om mekanismerna som orsakar luftvägshyperreaktivitet hos astmatiker. / Type 2 cytokines, IL-4 and IL-13 are known to play an essential role in airway hyperresponsiveness (AHR) - in response to direct or indirect stimulus the smooth muscle cells (ASM) contract too easily and too heavily. This makes AHR a defining feature of asthma. There have been studies that have demonstrated involvement of type 2 cytokines in AHR. However, the specific mechanisms involved remain undefined. This experimental study in airway smooth muscle (ASM) was aimed to investigate the involvement of type 2 cytokine on AHR by focusing on the expression and activation of receptors for contractile mediators as well as provide insights on the release of proteins related to airway remodelling. Experiments were performed where human airway smooth muscle cells were treated with IL-4 and/or IL-13, with or without Dupilumab, an antagonist of the joint IL-4/IL-13 receptor (anti-IL4Ra). After stimulating HASMs with IL-13 for 24 hours, results showed that IL-13 caused a non-specific, non-receptor-mediated increase in intracellular calcium flux in response to the calcium ionophore A23187. This could potentially increase the contractility of smooth muscle cells in response to any contractile stimulus. This study also suggests that IL-13 and IL-4 increased calcium flux in response to activation of receptors for specific contractile mediators (Histamine, Carbachol, Leukotriene D4 and Substance P). The mechanism involved likely involves the common IL-4/IL-13 receptor as blocking this with dupilumab seemed to reduce the effect. The presence of more receptors for contractile mediators could also increase contractility of smooth muscle cells in response to IL-4 and IL-13. Preliminary results show that mRNA expression of receptors for Histamine (H1) and LTD4 (CYSLT1) were upregulated by type 2 cytokines and again, this upregulation appeared to be inhibited by dupilumab. Moreover, we examined the effect of IL-13 and IL-4 on release of procollagen 1 (a precursor of mature collagen forms) from human airway smooth muscle cells. Our results did not show any significant effects on the release of this particular collagen protein. Taken together these findings increase our understanding of the mechanisms whereby type 2 cytokines may increase the contractility of airway smooth muscle and provide a basis for follow-up investigations. Improved knowledge of the mechanisms underlying AHR could ultimately lead to improved treatment of asthma.