Modulation Of TLR9 Signaling Pathway In Dendritic Cells And Its Application

Part Ⅰ. Mechanisms of CpG-induced CD40 expression on murine bone marrow-derived dendritic cellsDendritic cells (DCs), the most potent antigen presenting cells (APCs), play crucial roles in the initiation and coordination of the innate and adaptive immune responses. Aberrant CD40 expression by DCs, induced by microbial stimuli, such as CpG, contributes to the pathogenesis of many human/murine diseases, particularly autoimmune and inflammatory diseases. Given the importance of CD40 in these diseases, and the contribution of DCs to the diseases process, it is very important to investigate the mechanisms of CD40 expression induced by CpG on DCs. In this study, we made the observation that CpG-B is a potent inducer on CD40 expression on murine bone marrow-derived DCs. Based on this finding, we undertook an analysis of the molecular basis of CpG-induced CD40 expression on DCs. By using selective inhibitors, it was demonstrated that MAPKs (JNK and p38 MAPK, but not ERK) and NF-κB were involved in CpG-induced CD40 expression on DCs. In addition, RNA interference analysis revealed that IRF8 was a key transcription factor in the basal expression of CD40 upon CpG stimulation. Moreover, up-regulating miRNA-146a in DCs effectively decreased CD40 expression by targeting TRAF6 and IRAK1. Thus, our results have elucidated the molecular mechanisms underlying CpG-induced CD40 expression and DC maturation.Part Ⅱ. Chaetoglobosin F, a small molecule compound, possesses immunomodulatory properties on bone marrow-derived dendritic cells via TLR9 signaling pathwayChaetoglobosin F (Cha F), a novel small molecule compound, was obtained from the EtOAc extract of a solid culture of Chaetomium globosum IFB-E019. Dendritic cells (DCs), the most potent antigen presenting cells, are considered as the major target in the modulation of excessive immune responses. Recognition of CpG-DNA by Toll-like receptor 9 (TLR9) on DCs is an important step in the pathogenesis of autoimmune diseases. However, the effect of Cha F on the maturation and immunostimulatory function of CpG-stimulated DCs remains unclear. This study investigated the effects of Cha F on bone marrow (BM)-derived DCs. We found that Cha F inhibits the CpG-induced DCs maturation and function by suppressing the expression of surface molecules (CD40, CD80, CD86 and MHC-Ⅱ), reducing the production of cytokine and chemokine (IL-12 and CXCL-10), inhibiting the CpG-induced DCs-elicited allogeneic T-cell proliferation, and impairing the migration ability to chemokines. The Cha F-treated DCs were highly efficient at Ag capture, via mannose receptor-mediated endocytosis. Additionally, Cha F was also demonstrated to inhibit CpG-induced activation of MAPKs (p38 and JNK, but not ERK) and the nuclear translocation of NF-κB and STAT1. Furthermore, we confirmed that Cha F was able to suppress TLR9 expression of CpG-induced DCs. Collectivelly, these findings provide novel insight into the immunopharmacological functions of Cha F, especially with regard to their impact on CpG-induced DCs. These immunosuppressive properities of Cha F may prove useful in controlling DCs-associated autoimmune and/or inflammatory diseases.Part III. A novel small molecule compound FC-99 modulates TLRs-induced DCs activation and alleviates lupus-like syndrome in MRL/lpr miceTLR3, TLR4 and TLR9 are constitutively expressed in/on murine mDCs and signaling initiated by these pathways implicated in the pathogenesis of autoimmune diseases such as SLE. Thus, modulation of TLR signaling might be a potential strategy to regulate DCs excessive activation and ultimately inflammatory atouimmune diseases. Here, a novel small molecule compound FC-99 was evaluated for the anti-inflammatory activity induced by TLRs (TLR3,4 and 9) ligand using DCs-based assay. Our results indicate, for the first time, that FC-99 was a potent inhibitor of the maturation of mDCs induced by the TLR3 agonist poly I:C, the TLR4 agonist LPS and the TLR9 agonist CpG. FC-99 down-regulate the expression of CD40 and MHC-II on DCs and inhibit IL-6, IL-12, CXCL-10 and IFN-beta production by DCs following TLR3, TLR4 and TLR9 stimulation. Our results also show that FC-99 inhibits the activation of NF-κB and up-regulates the expression of Tnfaip3, which is a key negative regulatory gene in the downstream of TLR3, TLR4 and TLR9. These suggest that FC-99 alters DCs maturation in response to TLR3, TLR4 or TLR9 through a mechanism involving NF-κB activation and Tnfaip3 transcriptional activity. We also investigated the effect of FC-99 in MRL/lpr mice, which develop a lupus-like disease similar to human SLE. Our data showed that FC-99 treatment reversed signs of disease and resulted in reductions in cytokine production, autoantibody levels, along with significant improvement in renal function. Moreover, we demonstrated that FC-99 actively suppresses the population of mDCs and the expression of CD40 in inguinal lymph nodes (ILN), mesenteric lymph nodes (MLN), and spleen. The anti-inflammatory and immunosuppressive effects of FC-99 may be related, in part, to its’ability to control the maturation and functions of DCs. Interestingly, FC99 treatment significantly inhibits the activation of IκB-a and improves the expression of Tnfaip3 in PBMCs compared to the vehicle-treated mice. These results strongly suggest FC-99 therapy alleviating SLE-like symptoms in MRL/lpr mice. Therefore, our results suggest that FC-99 has the potential to attenuate SLE in humans.