Regulation Of Immune Responses By E3Ubiquitin Ligases CHIP And Nrdp1, And The Underlying Mechanisms

Ubiquitin modification plays important roles in the regulation of immunity, and is widely involved in the development of immune system and during the immune response. Recently, how E3ubiquitin ligases regulate immune responses and identifying new E3s for the regulation of immune response and what's their molecular mechanism are the frontiers of immunological research. Considering the important functions of E3ligases in immune response, we examined the gene expression profile of E3ligases in mice-derived dendritic cells (DC) and peritoneal macrophages by GeneChip assays. Based on the GeneChip results and our previous studies, we selected the E3ligases CHIP and Nrdp1for further research. CHIP, carboxyl terminus of Hsc70-interacting protein, is a U box-containing E3that are associated with chaperones. However, its functions and molecular mechanisms of actions during innate immunity have not been fully elucidated. Herein we investigated the roles of CHIP in regulating TLR signaling pathway, and explored the molecular mechanisms for its action. Knockdown of CHIP can significantly inhibit TLR2/4/7/9-triggered production of proinflammatory cytokines and type Ⅰ interferon (e.g. IL-6, TNF-α and IFN-β) in RAW264.7macrophages, peritoneal macrophages, bone marrow-derived dendritic cells (BMDC) and plasmacytoid dendritic cells (pDC). and significantly inhibit the production of IL-12p70and phenotypic maturation of BMDC after LPS treatment. In antigen-presenting cells, CHIP knockdown inhibits LPS/CpG-induced activation and nuclear translocation of NF-kB and IRF3/7, leading to reduced activation of IL-6, IFN-β and CCL5reporters. In contrast, CHIP overexpression could reverse the above effects. GST Pull-down and coimmunoprecipitation experiments show that CHIP can interact with HSP70, TLR4/9. Src and PKCζ, which is further confirmed by confocal microscopy. In Raw264.7macrophages stably overexpressing CHIP-HA, LPS/CpG-induced activation of Src/PKCζ is enhanced; while CHIP knockdown can significantly impair LPS/CpG-induced activation of Src/PKCζ. The in vitro kinase assays show that CHIP knockdown inhibits the kinase activity of IRAKI and TBK1in Raw264.7cells, and CHIP-HA overexpression promotes the activity of TBK1and IRAK1. In vivo and vitro ubiquitination assays show that CHIP can mediate the K63-linked polyubiquitination of Src/PKCζ. We conclude that CHIP can recruit and polyubiquitinate Src/PKOζ after LPS/CpG ligation, activate NF-kB and TBK1/IRAK1-IRF3/7, and finally promote the production of pro-inflammatory cytokines and type I interferon. Our study has thus elucidated a new function of CHIP in regulating the recognition of pathogen-associated molecular patterns, revealed the molecular mechanisms for the regulation of TLR4/9signaling pathway by CHIP-Src/PKC complex, improved and enriched the understanding of TLR signaling pathway. The novel finding that Src/PKC can activate TBK1/IRAK1and IRF3/7may represent a new step for the regulation of type I interferon production.Our previous studies indicated the important roles of Nrdpl involving in the regulation of innate immunity; however, its roles and effects in adaptive immunity have not been elucidated. Here, we showed that Nrdpl was preferentially expressed in CD8+T cells, indicating that Nrdpl may be involved in the functional regulation of CD8+T cells. Meanwhile, dominant negative Nrdpl can promote the production of IFN-γ, perforin and granzymes in CD8+T cells. In vivo, dominant negative Nrdpl can hinder the progression of experimental allergic encephalomyelitis (EAE) and accelerate the regression. To further demonstrate the roles of Nrdpl in EAE, we transfused DN-Nrdpl CD8+T cells into wild type mice and find that the pathogenesis of EAE was significantly improved. Our results suggest that Nrdpl possibly regulates the function of CD8+T cells, and thus may be involved in the pathogenesis of autoimmune diseases mediated by T cells, and the mechanisms are different to the other E3ubiquitin ligases. Our study about the roles and mechanism of Nrdpl in the regulation of CD8+T cells may provide new mechanistic explanations for adaptive immunity and autoimmune diseases redulated by E3ubiquitin ligases, and bring a new potential target for the treatment of autoimmune diseases.