Chantal Rabourdin-Combe

Hepatitis C virus infection protein network

A proteome‐wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. A total of 314 protein–protein interactions between HCV and human proteins was identified by yeast two‐hybrid and 170 by literature mining. Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HCV are enriched in highly central and interconnected proteins. A global analysis on the basis of functional annotation highlighted the enrichment of cellular pathways targeted by HCV. A network of proteins associated with frequent clinical disorders of chronically infected patients was constructed by connecting the insulin, Jak/STAT and TGFβ pathways with cellular proteins targeted by HCV. CORE protein appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins.

CD40 signaling in human dendritic cells is initiated within membrane rafts

Despite CD40s role in stimulating dendritic cells (DCs) for efficient specific T‐cell stimulation, its signal transduction components in DCs are still poorly documented. We show that CD40 receptors on human monocyte‐derived DCs associate with sphingolipid‐ and cholesterol‐rich plasma membrane microdomains, termed membrane rafts. Following engagement, CD40 utilizes membrane raft‐associated Lyn Src family kinase, and possibly other raft‐associated Src family kinases, to initiate tyrosine phosphorylation of intracellular substrates. CD40 engagement also leads to a membrane raft‐restricted recruitment of tumor necrosis factor (TNF) receptor‐associated factor (TRAF) 3 and, to a lesser extent, TRAF2, to CD40s cytoplasmic tail. Thus, the membrane raft structure plays an integral role in proximal events of CD40 signaling in DCs. We demonstrate that stimulation of Src family kinase within membrane rafts initiates a pathway implicating ERK activation, which leads to interleukin (IL)‐1α/β and IL‐1Ra mRNA production and contributes to p38‐dependent IL‐12 mRNA production. These results provide the first evidence that membrane rafts play a critical role in initiation of CD40 signaling in DCs, and delineate the outcome of CD40‐mediated pathways on cytokine production.

Measles Virus Induces Functional TRAIL Production by Human Dendritic Cells

ABSTRACT Measles virus infection induces a profound immunosuppression that can lead to serious secondary infections. Here we demonstrate that measles virus induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA and protein expression in human monocyte-derived dendritic cells. Moreover, measles virus-infected dendritic cells are shown to be cytotoxic via the TRAIL pathway.

Hepatocytes induce functional activation of naive CD8+ T lymphocytes but fail to promote survival

Intraperitoneal peptide injection of TCR‐transgenic mide or expression of antigen in hepatocytes leads to an accumulation in the liver of specific apoptotic CD8+ T cells expressing activation markers. To determine whether liver cells are capable of directly activating naive CD8+ T cells, we have studied the ability of purified hepatocytes to activate TCR‐transgenic CD8+ T cells in vitro. We show that hepatocytes which do not express CD80 and CD86 co‐stimulatory molecules are able to induce activation and effective proliferation of specific naive CD8+ T cells in the absence of exogenously added cytokines, a property only shared by professional antigen‐presenting cells (APC). Specific T cell proliferation induced by hepatocytes was comparable in magnitude to that seen in response to dendritic cells and was independent of CD4+ T cell help or bystander professional APC co‐stimulation. During the first 3 days, the same number of divisions was observed in co‐cultures of CD8+ T cells with either hepatocytes or splenocytes. Both APC populations induced expression of early T cell activation markers and specific cytotoxic T lymphocyte (CTL) activity. However, in contrast to T cells activated by splenocytes, T cells activated by hepatocytes lost their cytolytic function after 3 days of co‐culture. This correlated with death of activated T cells, suggesting that despite efficient activation, proliferation and transient CTL function, T cells activated by hepatocytes did not survive. Death could be prevented by adding antigen‐expressing splenocytes or exogenous IL‐2 to the co‐culture, indicating that hepatocytes are not involved in direct killing of CD8+ T cells but rather fail to promote survival. Dying cells acquired a CD8low TCRlow B220+ phenotype similar to the one described for apoptotic intrahepatic T cells, suggesting an alternative model to account for the origin of these cells in the liver. The importance of these findings for the understanding of peripheral tolerance and the ability of liver grafts to be accepted is discussed.

Linking innate and acquired immunity: divergent role of CD46 cytoplasmic domains in T cell–induced inflammation

CD46 is a widely expressed transmembrane protein that was initially identified as binding and inactivating C3b and C4b complement products. We used mice that were transgenic for one of two human CD46 isoforms that differ in their cytoplasmic domains (termed CD46-1 and CD46-2) to analyze the effect of CD46 stimulation on the immune response. We show here that CD46 can regulate inflammatory responses, either by inhibiting (CD46-1) or increasing (CD46-2) the contact hypersensitivity reaction. We found that engagement of CD46-1 or CD46-2 differentially affected CD8+ T cell cytotoxicity, CD4+ T cell proliferation, interleukin 2 (IL-2) and IL-10 production as well as tyrosine phosphorylation of Vav in T lymphocytes. These results indicate that CD46 plays a role in regulating the T cell–induced inflammatory reaction and in fine-tuning the cellular immune response by bridging innate and acquired immunity.

Measles Virus Induces Abnormal Differentiation of CD40 Ligand-Activated Human Dendritic Cells

Measles virus (MV) infection induces a profound immunosuppression responsible for a high rate of mortality in malnourished children. MV can encounter human dendritic cells (DCs) in the respiratory mucosa or in the secondary lymphoid organs. The purpose of this study was to investigate the consequences of DC infection by MV, particularly concerning their maturation and their ability to generate CD8+ T cell proliferation. We first show that MV-infected Langerhans cells or monocyte-derived DCs undergo a maturation process similarly to the one induced by TNF-α or LPS, respectively. CD40 ligand (CD40L) expressed on activated T cells is shown to induce terminal differentiation of DCs into mature effector DCs. In contrast, the CD40L-dependent maturation of DCs is inhibited by MV infection, as demonstrated by CD25, CD69, CD71, CD40, CD80, CD86, and CD83 expression down-regulation. Moreover, the CD40L-induced cytokine pattern in DCs is modified by MV infection with inhibition of IL-12 and IL-1α/β and induction of IL-10 mRNAs synthesis. Using peripheral blood lymphocytes from CD40L-deficient patients, we demonstrate that MV infection of DCs prevents the CD40L-dependent CD8+ T cell proliferation. In such DC-PBL cocultures, inhibition of CD80 and CD86 expression on DCs was shown to require both MV replication and CD40 triggering. Finally, for the first time, MV was shown to inhibit tyrosine-phosphorylation level induced by CD40 activation in DCs. Our data demonstrate that MV replication modifies CD40 signaling in DCs, thus leading to impaired maturation. This phenomenon could play a pivotal role in MV-induced immunosuppression.

Autophagy Induction by the Pathogen Receptor CD46

Autophagy is a highly regulated self-degradative mechanism required at a basal level for intracellular clearance and recycling of cytoplasmic contents. Upon intracellular pathogen invasion, autophagy can be induced as an innate immune mechanism to control infection. Nevertheless, pathogens have developed strategies to avoid or hijack autophagy for their own benefit. The molecular pathways inducing autophagy in response to infection remain poorly documented. We report here that the engagement of CD46, a ubiquitous human surface receptor able to bind several different pathogens, is sufficient to induce autophagy. CD46-Cyt-1, one of the two C-terminal splice variants of CD46, is linked to the autophagosome formation complex VPS34/Beclin1 via its interaction with the scaffold protein GOPC. Measles virus and group A Streptococcus, two CD46-binding pathogens, induce autophagy through a CD46-Cyt-1/GOPC pathway. Thus, upon microorganism recognition, a cell surface pathogen receptor can directly trigger autophagy, a critical step to control infection.

Langerhans cell histiocytosis reveals a new IL-17A- dependent pathway of dendritic cell fusion

IL-17A is a T cell–specific cytokine that is involved in chronic inflammations, such as Mycobacterium infection, Crohns disease, rheumatoid arthritis and multiple sclerosis. Mouse models have explained the molecular basis of IL-17A production and have shown that IL-17A has a positive effect not only on granuloma formation and neurodegeneration through unknown mechanisms, but also on bone resorption through Receptor activator of NF-κB ligand (RANKL) induction in osteoblasts. Langerhans cell histiocytosis (LCH) is a rare disease of unknown etiology, lacking an animal model, that cumulates symptoms that are found separately in various IL-17A–related diseases, such as aggressive chronic granuloma formation, bone resorption and soft tissue lesions with occasional neurodegeneration. We examined IL-17A in the context of LCH and found that there were high serum levels of IL-17A during active LCH and unexpected IL-17A synthesis by dendritic cells (DCs), the major cell type in LCH lesions. We also found an IL-17A–dependent pathway for DC fusion, which was highly potentiated by IFN-γ and led to giant cells expressing three major tissue-destructive enzymes: tartrate resistant acidic phosphatase and matrix metalloproteinases 9 and 12. IFN-γ expression has been previously documented in LCH and observed in IL-17A–related diseases. Notably, serum IL-17A–dependent fusion activity correlates with LCH activity. Thus, IL-17A and IL-17A–stimulated DCs represent targets that may have clinical value in the treatment of LCH and other IL-17A–related inflammatory disorders.

Mechanism of Measles Virus–Induced Suppression of Inflammatory Immune Responses

Measles virus (MV) causes profound immunosuppression, resulting in high infant mortality. The mechanisms are poorly understood, largely due to the lack of a suitable animal model. Here, we report that particular MV proteins, in the absence of MV replication, could generate a systemic immunosuppression in mice through two pathways: (1) via MV-nucleoprotein and its receptor FcgammaR on dendritic cells; and (2) via virus envelope glycoproteins and the MV-hemagglutinin cellular receptor, CD46. The effects comprise reduced hypersensitivity responses associated with impaired function of dendritic cells, decreased production of IL-12, and the loss of antigen-specific T cell proliferation. These results introduce a novel model for testing the immunosuppressive potential of anti-measles vaccines and reveal a specific mechanism of MV-induced modulation of inflammatory reactions.

Cutting Edge: CD46, a New Costimulatory Molecule for T Cells, That Induces p120CBL and LAT Phosphorylation

The widely expressed transmembrane molecule CD46 is the complement regulatory receptor for C3b as well as the receptor for several pathogens. Beside its binding functions, CD46 is also able to transduce signals. We showed that CD46 aggregation on human T cells induces p120CBL and linker for activation of T cells (LAT) phosphorylation. These two proteins are adaptor proteins known to regulate TCR signaling. p120CBL is a complex adaptor protein involved in negatively regulating signaling events, whereas LAT is a transmembrane adaptor protein found in glycolipid-enriched microdomains essential for T cell activation. Therefore, we investigated if a CD46/TCR costimulation would affect T cell activation. Indeed, CD46/CD3 costimulation strongly promotes T cell proliferation. Therefore, we propose that CD46 acts as a potent costimulatory molecule for human T cells.