clc is co-expressed with clf or cntfr in developing mouse muscles

BACKGROUND: The ciliary neurotrophic factor (CNTF) receptor is composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor, associated with a non-signalling CNTF binding receptor α component (CNTFR). This tripartite receptor has been shown to play important roles in the development of motor neurons, but the identity of the relevant ligand(s) is still not clearly established. Recently, we have identified two new ligands for the CNTF receptor complex. These are heterodimeric cytokines composed of cardiotrophin-like cytokine (CLC) associated either with the soluble receptor subunit cytokine-like factor-1 (CLF) or the soluble form of the binding receptor itself (sCNTFR). RESULTS: Here we show that, during development, clc is expressed in lung, kidney, vibrissae, tooth, epithelia and muscles during the period of development corresponding to when motoneuron loss is observed in mice lacking a functional CNTF receptor. In addition, we demonstrate that it is co-expressed at the single cell level with clf and cntfr, supporting the idea that CLC might be co-secreted with either CLF or sCNTFR. CONCLUSION: This expression pattern is in favor of CLC, associated either with CLF or sCNTFR, being an important player in the signal triggered by the CNTF receptor being required for motoneuron development

Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF

BACKGROUND: The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures. RESULTS: Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFNγ and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF). As well as P-gp expression, the IL-6 type cytokines - but not IFNγ - stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFNγ produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF -which is internalised with its receptor - produced an overexpression of P-gp in CNTF-deficient astrocytes. CONCLUSIONS: These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF

Expression of HLA-G by mast cells is associated with hepatitis C virus-induced liver fibrosis.

International audienceBACKGROUND AND AIMS: Infection by hepatitis C virus is a worldwide health problem. An inadequate Th2 cytokine response promotes the fibrosis-cirrhosis fate. Immune-modulating molecules favoring a Th2 profile, such as HLA-G molecules of the HLA class Ib family, may play a role in chronic hepatitis. HLA-G contributes to the escape of tumors, and their involvement in viral infections has been increasingly described. The aim of this work was to study the expression of HLA-G in the liver, its cellular source and its regulation in cases of chronic C hepatitis. METHODS: HLA-G cells in blocks of liver derived from patients infected with HCV were labeled by immunohistochemistry and enumerated. Double immunofluorescence allowed the identification of the cellular source. HLA-G secretion by a human mast cell line was quantified by ELISA after various stimulations. After treatment with IFN-α real-time PCR was performed to determine the kinetics of cytokine expression profiles, followed by heat map clustering analysis. RESULTS: The number of HLA-G + cells was significantly associated with the area of fibrosis. For the first time, we identify the HLA-G+ cells as being mast cells. HLA-G secretion was significantly induced in human mast cells stimulated by IL-10 or interferons of class I. The transcriptome of the secretome of this cell line stimulated by IFN-α revealed that i) the HLA-G gene is upregulated late, ii) T lymphocytes and NK cells are recruited. CONCLUSIONS: These findings suggest an autocrine loop in the genesis of HCV liver fibrosis, based on mast cells expressing HLA-G

Invariant NKT Cells Drive Hepatic Cytokinic Microenvironment Favoring Efficient Granuloma Formation and Early Control of Leishmania donovani Infection

The development of inflammatory granulomas around infected Kupffer cells is necessary for hepatic parasite clearance during visceral leishmaniasis. Invariant NKT (iNKT) cells are predominant T cells in the mouse liver and can synthesize large quantities of IL-4 and IFN-γ, two cytokines involved in granuloma formation. This study analyzed the role of iNKT cells in the hepatic immune response during Leishmania donovani infection, using a murine model of wild-type (WT) and iNKT cell-deficient (Jα18-/-) C57BL/6 mice sacrificed 15, 30 or 60 days post-infection. We recorded hepatic parasite loads, cytokine expression, and analyzed granulomatous response by immunohistochemistry and hepatic immune cell infiltration by flow cytometry. Whereas WT animals rapidly controlled the infection and developed an inflammatory response associated with a massive influx of iNKT cells observed by flow cytometry, Jα18-/- mice had significantly higher parasitic loads on all time points. This lack of control of parasite burden was associated with a delay in granuloma maturation (28.1% of large granulomas at day 60 versus 50.7% in WT). Cytokine transcriptome analysis showed that mRNA of 90/101 genes encoding chemokines, cytokines and their receptors, was underexpressed in Jα18-/- mice. Detection of IL-4 and TNF-α by ELISA in liver extracts was also significantly lower in Jα18-/- mice. Consistent with flow cytometry analysis, cytokinome profile in WT mice showed a bias of expression towards T cell-chemoattractant chemokines on D15, and displayed a switch towards expression of granulocytes and/or monocytes -chemoattractant chemokines on D60. In Jα18-/- mice, the significantly lower expression of CXCL5, MIP-2 and CCL2 mRNA was correlated with a defect in myeloperoxidase positive-cell attraction observed by immunohistochemistry and with a lower granulocyte and monocyte infiltration in the liver, as shown by flow cytometry. These data indicate that iNKT cells play a role in early and sustained pro-inflammatory cytokine response warranting efficient organization of hepatic granulomas and parasite clearance

The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps

The long pentraxin (PTX) 3 is produced by macrophages and myeloid dendritic cells in response to Toll-like receptor agonists and represents a nonredundant component of humoral innate immunity against selected pathogens. We report that, unexpectedly, PTX3 is stored in specific granules and undergoes release in response to microbial recognition and inflammatory signals. Released PTX3 can partially localize in neutrophil extracellular traps formed by extruded DNA. Eosinophils and basophils do not contain preformed PTX3. PTX3-deficient neutrophils have defective microbial recognition and phagocytosis, and PTX3 is nonredundant for neutrophil-mediated resistance against Aspergillus fumigatus. Thus, neutrophils serve as a reservoir, ready for rapid release, of the long PTX3, a key component of humoral innate immunity with opsonic activity

Analyse fonctionnelle des récepteurs de l'oncostatine M et de l'interleukine 31

ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Génération et analyse fonctionnelle d'antagonistes de l'oncostatine M et de l'interleukine-31, cytokines impliquées dans l'inflammation cutanée et le développement tumoral

L'oncostatine M (OSM) et l'interleukine-31 (IL-31), cytokines de la famille de l'IL-6, sont impliquées dans l'inflammation cutanée associée à des pathologies telles que la dermatite atopique ou le psoriasis. Dans une perspective thérapeutique, deux premières études sont basées sur la génération d'antagonistes agissant comme des récepteurs solubles et destinés à piéger ces cytokines. Afin de disposer d'un nouveau modèle d'étude de l'IL-31 in vivo, nous avons également identifié et cloné cette protéine chez le rat. Dans une dernière étude, nous avons mis en évidence l'implication de l'IL-31 dans le développement tumoral en démontrant son activité cytostatique sur certaines lignées tumorales, de manière similaire à l'OSM. Cette étude nous a également amenés à analyser les voies de signalisation intracellulaires impliquées dans ces effets anti-tumoraux et nous avons ainsi démontré l'activation d'un nouveau facteur de transcription en réponse à ces deux cytokines..Oncostatin M (OSM) and interleukin-31 (IL-31), cytokines of the IL-6 family, are involved in inflammation associated with skin diseases such as atopic dermatitis or psoriasis. In a therapeutic perspective, two first studies were based on the generation of antagonists acting as soluble receptors and intended to trap these cytokines. In order to have a new model for the IL-31 in vivo study, we also identified and cloned this protein in rat. In a latest study, we highlighted IL-31 involvement in tumor development by demonstrating its cytostatic activity on tumor cell lines, in a similar way to OSM. This study has also led us to analyze the intracellular signaling pathways involved in these anti-tumor effects and we demonstrated the activation of a new transcription factor in response to these two cytokines.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Analyse structurale des interactions cytokines/récepteurs dans la famille de l'interleukine-6

Les cytokines sont des protéines multifonctionnelles qui régulent la croissance et la différenciation cellulaires dans des systèmes biologiques variés comme l'immunité, l'hématopoïèse, l'inflammation et le développement du système nerveux. La superfamille des cytokines est divisée en sous-familles en fonction de la longueur des hélices a. Chaque sous-famille est divisée en différentes catégories. La famille de l'interleukine-6 (IL-6) contient dix membres qui partagent tous la chaîne de transduction du signal gp130. Les récepteurs des cytokines de la famille de l'IL-6 sont tous homo- ou hétéromultimériques. Certains d'entre eux sont constitués d'une chaîne réceptrice de type a spécifique. La fixation de la cytokine sur le complexe récepteur entraîne l'activation de voies de signalisation intracellulaires (JAK/STAT, PI3-Kinase/AKT, ERK). Mon projet de thèse consistait à comprendre les interactions protéines/protéines dans cette famille grâce à deux voies complémentaires. La stratégie a consisté à allier la modélisation moléculaire à la mutagenèse dirigée afin de définir des règles physico-chimiques d'interaction. Nous avons ainsi pu montré que CLC interagissait avec le CNTFRa et CLF par deux sites différents pour former les deux cytokines composites CLC/CNTFRa et CLC/CLF. Les déterminants structuraux qui permettent l'interaction de CLC avec CLF sont les mêmes que ceux recrutés par la chaîne LIFRb. Cette stratégie nous a permis de mettre en évidence des différences significatives quant aux propriétés électrostatiques de l'IL-6 virale et de l'IL-6 humaine. Ceci peut être une explication structurale à la non nécessité pour l'IL-6 virale de fixer le récepteur IL-6Ra pour induire la prolifération des cellules infectées et permettre ainsi la survie du virus. Enfin, nous avons montré que les cytokines LIF, CT-1 et OSM partageaient les mêmes déterminants structuraux d'interaction vis-à-vis du LIFRb. Ces travaux de recherche nous ont permis de mieux comprendre comment les interactions cytokines/récepteurs permettaient de réguler les grandes voies fonctionnelles à l'intérieur de la cellule. Ils pourront être utiles pour de nombreuses études utilisant des molécules modifiées dans d'éventuelles approches thérapeutiques.Cytokines are multifunctional proteins regulating cell growth and differentiation in a large number of biological systems such as immunity, hematopoiesis, inflammation and in nervous system development. The cytokines superfamily is divided in sub-families regarding the length of each a-helices. Each sub-family is divided in different categories. The interleukine-6 (IL-6) family contains ten members all sharing the gp130 signal transduction chain. Cytokine receptors of the IL-6 family are homo- or heteromultimeric. Some involved a specific a-chain. Binding of the cytokine on the receptor complex induces the activation of cellular signaling pathways (JAK/STAT, PI3-Kinase/AKT, ERK). My PhD project deals with the understanding of protein/protein interactions in this family using two complementary methods. The strategy consisted to use molecular modeling and site-directed mutagenesis in order to define interaction rules. By this way, we have shown that CLC interacts with CNTFR? and CLF through two different sites to build two composite cytokines CLC/CNTFRa and CLC/CLF. The structural determinants allowing interaction of CLC with CLF are shared with LIFRb. This strategy allowed us to highlight differences in the electrostatics properties of viral and human IL-6. This may be a structural explanation for the loss of IL-6Ra recruitment by viral IL-6 to induce proliferation of the infected cells and sustain virus survival. Finally, we have shown that LIF, CT-1 and OSM shared the same structural determinants towards LIFRb chain. This study allowed us to understand a bit more how cytokine/receptor interactions regulate cellular functions. It can be useful for following studies involving modified proteins in putative therapeutics approaches.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Analyse structure fonction des récepteurs et cytokines de la famille de l'interleukine 6

ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Etude de la sécrétion et du mode d'action des cytokines de la famille de l'IL-6

Les cytokines de la famille de l'interleukine-6 (IL-6) sont aujourd'hui au nombre de huit, dont le ciliary neurotrophic factor (CNTF) et le cardiotrophin-like cytokine (CLC), récemment mis en évidence. L'existence d'un second ligand pour le CNTFR était pressentie depuis plusieurs années. Dans cette étude, nous montrons que CLC peut s'associer à un récepteur soluble jusqu'alors orphelin, le cytokine-like factor (CLF) ou à la forme soluble du CNTFR (sCNTFR) pour être sécrétée. Par ailleurs, nous avons montré que ces deux cytokines, CLC/CLF et CLC/sCNTFR étaient de nouveaux ligands pour le CNTFR. Nous avons ensuite analysé les voies de signalisation intracellulaires recrutées par ces deux nouvelles cytokines hétéromériques. Dans la dernière partie de ce travail, des études de modélisation moléculaire suivies de mutagenèse dirigée nous ont permis d'identifier les sites d'interaction des cytokines de la famille de l'IL-6 avec leurs récepteurs.The interleukin (IL)-6 type cytokines comprises ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC), recently identified. The existence of a second, developmentally important ligand for CNTFR was suspected for many years. The following studies show that the cellular release of functional CLC can be mediated by an orphan soluble receptor, cytokine-like factor (CLF) or soluble CNTFR (sCNTFR) to generate two new composite cytokines defining second ligands for CNTFR. We next examined the signaling pathways recruited by these two new composite cytokines. In the last part of this work, we maked site-directed mutagenesis on residues of receptors of IL-6 family identified after computer assisted modelling studies and identified binding sites of cytokines on their receptors.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF