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Hepatitis C virus (HCV) infection is a global health problem characterized by a high rate of chronic infection, which may in part be due to a defect in myeloid dendritic cells (mDCs). This defect appears to be remedied by treatment with interferon-α (IFN-α) -based antiviral therapies; however, the molecular mechanisms underlying mDC dysfunction in HCV infection and restoration by IFN-α treatment are unclear. The ubiquitin-editing protein A20 plays a crucial role in controlling the maturation, cytokine production and immunostimulatory function of mDCs. We propose that the expression of A20 correlates with the function of mDCs during HCV infection and IFN-α therapy. In this study, we observed that A20 expression in mDCs isolated from chronically HCV-infected subjects was significantly higher than healthy subjects or subjects achieving sustained virological responses (SVR) following antiviral treatment. Notably, A20 expression in mDCs from HCV patients during IFN-α treatment was significantly lower than for untreated patients, SVR patients, or healthy subjects. Besides, A20 expression in mDCs stimulated by polyI:C differed between HCV patients and healthy subjects, and this difference could be abrogated by the treatment with IFN-α in vitro. Additionally, A20 expression by polyI:C-activated mDCs, with or without IFN-α treatment, negatively correlated with the expression of HLA-DR, CD86 and CCR7, and the secretion of interleukin-12 (IL-12), but positively associated with the production of IL-10. Importantly, silencing A20 expression using small interfering RNAs increased the production of IL-12 in mDCs of chronically HCV-infected individuals. These findings suggest that A20 plays a crucial role in negative regulation of innate immune responses during chronic viral infection.

Diverses études ont souligné le rôle prépondérant du dialogue entre les cellules NK et les cellules dendritiques au cours des réponses immunes. Cependant, les récepteurs impliqués dans ce processus restent incertains. Au cours de ce travail, nous nous sommes attachés à identifier les récepteurs mis en jeu lors de la reconnaissance virale à l’aide de modèles humains et murins. Pour cela, nous avons mimé l’infection virale en utilisant deux ARN bicaténaires synthétiques – poly(AU) et poly(IC) – et montré qu’ils sont tous deux capables d’activer TLR3 mais que seul poly(IC) engage les hélicases RIG-I et MDA5. Les deux ARN induisent l’activation des cellules NK au sein des PBMC humaines, mais seul poly(IC) induit la production d’IFN-gamma. Les DC myéloïdes (mDC) sont requises pour cette activation sans nécessité d’un contact cellulaire entre les cellules NK et les mDC. En outre, les IFN de type I et l’IL-12 secrétés par les DC sont respectivement nécessaires à l’initiation du potentiel lytique et à la production d’IFN-gamma. Poly(IC), au contraire de poly(AU), a une action synergique avec l’IL-12 produite par les mDC pour induire la production d’IFN-gamma en agissant directement sur les cellules NK. Enfin, l’activation conjointe de TLR3 et des hélicases RLR sur les mDC et RIG-I sur les cellules NK, nécessaire à la production d’IFN-gama en réponse à l’ARN bicaténaire, a été confirmée à l’aide de souris déficientes pour TLR3 et Cardif et d’un ligand spécifique de RIG-I. En conclusion, nous rapportons pour la première fois la nécessité pour un composé microbien d’engager deux familles de récepteurs sur deux populations cellulaires distinctes pour induire une réponse innée éfficace. / Crosstalk between NK cells and DC is critical for the response to the microbial mimic poly(IC) but the dsRNA receptors involved in each cell types remained to be defined. We show herein that two dsRNA, poly(AU) and poly(IC), similarly engaged TLR3 while only poly(IC) triggered the RIG-I and MDA-5 helicases. Both dsRNA triggered NK cell activation within PBMC but only poly(IC) induced IFN-gamma. mDC were required for NK cell activation by the two dsRNA, suggesting that they triggered at least TLR3 on mDC. DsRNA induction of cytolytic potential and IFN-gamma production in NK cells did not require contact with mDC but was dependent on the secretion of type I IFN and IL-12, respectively. Poly(IC) but not poly(AU) synergized with mDC-derived IL-12 for high IFN-gamma production by acting directly on NK cells. Finally, the requirement of TLR3 and the RLR on mDC and the involvement of the RIG-I but not TLR3 on NK cells for the production of IFN-gamma induced by dsRNA was confirmed using TLR3 and Cardif deficient mice and RIG-I specific activator. This cooperation was further confirmed using inactivated FLU virus infected-target cells both in human and mouse system demonstrating that NK cells were able to sense viral material by a direct transfer from infected cells likely through lytic immunological synapse without prior infection of NK cells. Thus, we report for the first time the requirement of cotriggering

<h4> </h4> / <p>Asthma is an inflammatory disorder of the airways, and there has been growing insight into the cellular and molecular mechanisms underlying the inflammatory basis of this disease. Research into the inflammatory mechanisms of asthma has progressively shifted focus from downstream effectors, such as mast cells and eosinophils, up to Th2 lymphocytes and their proallergic cytokines. Even more upstream in the allergic cascade are dendritic cells (DCs), potent APCs that orchestrate immune responses. Evidence supporting a role of DCs in regulating airway allergic inflammation is derived mainly from animal studies. In animal models of asthma, myeloid DCs (mDCs) induce and maintain airway inflammation, while plasmacytoid DCs (pDCs) mediate tolerance and lung homeostasis. It remains uncertain, however, whether this concept of pro-allergic mDCs and anti-allergic pDCs translates from animal to human models. The overall objective of this thesis was to investigate the biology of DC subsets in allergen-induced asthma in asthmatic subjects. Initially, we demonstrate that both mDCs and pDCs increase in the airways of subjects with mild asthma after allergen inhalation. Next, we describe a distinct subpopulation of mDCs, called mDC2s, and demonstrate their association with allergy and asthma severity. Expanding on these findings, we show that mDC2s increase in the airways of mild asthmatics after allergen challenge. Lastly, we explore the potential of pharmacological therapies, anti-OX40L MAb and anti-TSLP MAb, to affect DCs in subjects with mild asthma, and demonstrate no effect of either drug on circulating DC subsets. The studies presented here provide evidence for multiple DC subtypes being involved in the regulation of allergen-induced inflammatory responses, and support continued investigations into the biology of different DC subsets in allergen-induced asthma.</p> / Doctor of Philosophy (Medical Science)

Allergy, as one of the worldwide most frequent pathologies, belongs to illnesses with constantly growing incidence among young children. In genetically predisposed individuals, dendritic cells are able to polarize the immune response of Th2 in contact with the allergen. Postnatal probiotic supplementation could be one the preventive measure to prevent the development of allergic diseases. It has been shown that introduction of selected probiotic strains or mixtures can prevent development of allergy. In this diploma thesis, the capacity of probiotic strain Escherichia coli O83:K24:H31 (E. coli O83) to support maturation of dendritic cells (DC) and polarization of immune responses was tested. Introduction of this probiotic vaccine called Colinfant Newborn appears to be suitable preventive measure, lowering allergy incidence in children with predisposition to development of allergy. The aim of this diploma thesis was to observe capacity of E. coli O83 to support maturation of the two main subpopulations of dendritic cells (myeloid dendritic cells - mDC and plasmacytoid dendritic cells - pDC) in cord blood of newborns of healthy mothers (children with relatively low risk for allergy development) and allergic mothers (children with relatively high risk for allergy development). To achieve this goal,...

Allergy as one of the most frequent pathologies worldwide belongs to illnesses with constantly increasing incidence even amongst young children. It develops in genetically predisposed individuals whose dendritic cells (DC) are, after contact with allergen, able to polarize the immune response predominantly to Th2, while Th1 response is supressed. One of the possible preventive measures to avoid an allergic disease developement could be an early postnatal supplementation of chosen probiotic bacterial strains or their mixtures. One of them is a well characterized strain Escherichia coli O83:K24:H31 (E. coli O83). Administering of this probiotic vaccine called Colifant Newborn is showing to be an effective prophylaxis to decrese the incidence of allergies in children with predisposition to their developement. The aim of my diploma thesis was to experimentally confirm the capacity of E. coli O83 to support maturation of two main subpopulations of newborn DC available from cord blood: myeloid dentritic cells - mDC and plasmacytoid dentritic cells - pDC. This DC subpopulations were isolated from cord blood of children born to healthy (non-allergic) mothers who had a low risk of allergy development or from children of allergic mothers who had an increased risk of allergy development. Subsequently, after...

Allergy, as one of the worldwide most frequent pathologies, belongs to illnesses with constantly growing incidence among young children. Identification of prognostic markers pointing to increased risk of allergy development, allows introduction of early preventive measures. Probiotic supplementation could be one the preventive measure. It has been shown that introduction of selected probiotic strains or mixtures can prevent development of allergy. In this diploma thesis, the capacity of probiotic strain Escherichia coli O83:K24:H31 (E. coli O83) to support maturation of dendritic cells and polarization of immune responses was tested. Introduction of this probiotic vaccine called Colinfant Newborn appears to be suitable preventive measure, lowering allergy incidence in children with predisposition to development of allergy. The capacity of E. coli O83 to support maturation of the two main subpopulations of dendritic cells (myeloid dendritic cells - mDC and plasmacytoid dendritic cells - pDC) in cord blood of newborns of healthy mothers (children with relatively low risk for allergy development) and allergic mothers (children with relatively high risk for allergy development) was measured by flow cytometry. The presence of cytokines and transcription factors characteristic for particular...

<p> Virus-induced modulation of dendritic cell function is thought to be an effective mechanism for viral-immune evasion. The severe-acute respiratory syndrome coronavirus (SARS-CoV) has been shown to infected human myeloid dendritic cells (MDCs) and directly modulate the cellular cytokine production. The ability of other human coronaviruses to infect MDCs and impair cell immune function has not been assessed. </p> <p> This thesis describes the infection of human MDCs with coronavirus 229E, NL63, and OC43. 229E showed productive, but limited genomic replication, nucleocapsid protein synthesis and infectious progeny release in MDCs. 229E infection stimulated IFN-α, IL-6 and MCP-1 production in MDCs, but little to no IL-12, TNF-α, IL-8, IP-10, or RANTES . 229E-infected MDCs showed poor CD80 expression, down-regulated CD86 and HLA-DR expression and were poor stimulators of CD4+ T cell proliferation. In contrast to 229E, OC43 showed persistent and productive genomic replication, nucleocapsid protein synthesis and infectious progeny release in MDCs. OC43 infection stimulated IFN-α, IL-12, IP-10 and MCP-1 production in MDCs, but little to no TNF-α, IL-6, IL-8 or RANTES . The up-regulation of maturation molecules and CD4+ T cell stimulatory capacity in OC43-infected MDCs was donor cell-dependent. In contrast to 229E and OC43, NL63 infection of MDCs was non-productive, showing no viral genomic replication, protein production or infectious progeny release. NL63 infection stimulated strong cytokine (IFN-α, IL-12, TNF-β and IL-6) and chemokine (IL-8, IP-10, RANTES and MCP-1) responses in MDCs. NL63-infected MDCs showed up-regulated CD80, CD83, CD86 and HLA-DR expression and were efficient stimulators of CD4+ T cell proliferation. </p> <p> This study provides the first evidence that human coronaviruses other than SARSCo V can abrogate MDC immune effector function. It also provides the first side-by-side comparison of 229E, NL63 and OC43 and identifies the potential of 229E and OC43 to impair MDC cytokine production and T cell stimulation as a mechanism of immune response evasion. <p> / Thesis / Doctor of Philosophy (PhD)