| Immune response is a highly complicated and strictly regulated process. While efficient initiation of immune responses is essential for host to eliminate the invading pathogens, a flexible and exact negative regulatory network is critical to the control of the strength and duration of immune responses to avoid the tissue damage due to excessive immune response. As the most potent professional antigen presenting cells, dendritic cells (DCs) play a critical role in the initiation of immune response and also maintenance of immune tolerance. It has been shown that DC can induce the generation of or determine the differentiation of certain T cell subsets such as Th17, regulatory T cells (Treg), however, the detailed mechanisms for the regulation of T cell subsets by DC remain to be fully understood. DC express various kinds of pathogen recognition receptors (PRR) including Toll-like receptors (TLR), which can sense the invading pathogens or recognize microbial components and subsequently trigger the innate immune response against pathogens. Overactivation of TLR response may lead to immunological disorders such as septic shock and autoimmune diseases. Therefore, much attention has been paid to identify and functionally investigate the negative regulators of TLR signaling.RHBDD (Rhomboid domain-containing protein) belongs to a family of proteins with Rhomboid domain. Up to now, three members of RHBDD family, Rhbddl, Rhbdd2和Rhbdd3, have been identified. RHBDD3 is firstly cloned from pituitary tumor and found to able to induce apoptosis of pituitary tumor cells, thus designated as pituitary tumor apoptosis gene (PTAG). Rhbdd3 is the mouse homolog of human RHBDD3, which encodes 385 AA. RHBDD3 is predicated to have signal peptide in N terminal, and then four transmembrane domain, one UBA domain in C terminal. Up to now, there are very few reports about the function of this molecule. Rhbdd3-deficient mice (Rhbdd3-/-) have been successfully generated. Interestingly, higher serum autoantibodies and massive lymphocytes infiltration in the immune organs were observed in the elderly Rhbdd3-/-mice, indicating the Rhbdd3-/- mice are prone to the pathogenesis of autoimmune diseases. On the basis of these observations, in this study, we investigated whether or not RHBDD3 can control autoimmune immunity and subsequently prevent the pathogenesis of autoimmune diseases. Then we went further to investigate the cellular and molecular mechanisms for the negative regulation of immune response by RHBDD3. We demonstrate that RHBDD3 is a new potent negative regulator of TLR signaling, which prevents Th-17 mediated pathogenesis of autoimmune diseases by controlling DC maturation and suppressing DC-derived IL-6 production.1. RHBDD3 controls DC maturation and suppresses TLR-triggered production of proinflammatory cytokines (IL-6 and TNF-a)We first examined the DC phenotype and function in Rhbdd3-/-mice. Bone marrow-derived DC (BMDC) expressed higher level of co-stimulatory molecules and produced more IL-6 and TNF-a in response to LPS, CpG ODN and poly(I:C) stimulation, as compared with their littermates. When co-cultured with OT-ⅡT cells in the presence of OVA323-339 peptide, Rhbdd3-/- BMDCs could induce proliferation of T cells and the differentiation of IFN-y-secreting Thl and IL-17-secreting Th17 more significantly, as compared with their littermates. We constructed lentivirus vector carrying Rhbdd3 gene and overexpressed RHBDD3 in BMDC. Overexpression of Rhbdd3 could inhibit the phenotype maturation and inflammatory cytokine production of BMDCs upon LPS stimulation. Overexpression of RHBDD3 in Rhbdd3-/- BMDCs rescued their overactivation both in phenotype and inflammatory cytokine production.We next assessed phenotypic and functional change of the freshly isolated splenic cDC in Rhbdd3-/=mice. Rhbdd3-/- splenic CD11c+ cDC expressed higher I-Ab, produced higher IL-6 and TNF-a in the presence or absence of TLR agonist stimulation, and could also induce proliferation of T cells and the differentiation of Thl and Th17 more significantly, as compared with their littermates. We also detected higher level of IL-6 and TNF-a in the sera of Rhbdd3-/- mice challenged with LPS as compared with wild-type mice. Altogether, these results indicate that RHBDD3 is a negative regulator of DC maturation and TLR-triggered production of proinflammatory cytokines (IL-6 and TNF-a).2. RHBDD3 promotes induction of Treg but inhibit differentiation of Thl and Th17 by controlling IL-6 production from mature DCWe observed that the proportion of CD4+CD25+and CD4+FoxP3+Treg cells in spleen and MLN was significantly lower than that in wild type control. CD4+T cells from Rhbdd3-/- spleen and MLN produced more IFN-y and IL-17 in response to the activatory signals (CD3/CD28). Upon in vivo injection of LPS, Rhbdd3-/- mice produced higher IFN-y and IL-17 in serum than their littermates. These results indicate that RHBDD3 is involved in the controlling of T subset differentiation in vivo. Then we investigated whether the disorders of T cell subsets in Rhbdd3-/- mice were secondary to the overmaturation of DC and overproduction of IL-6. It is reported that IL-6 plays an important role in inhibiting the generation of Treg while favoring the Th17 differentiation. We then explored the role of IL-6 in immune abnormity of Rhbdd3-/- mice through the generation of Rhbdd3-/-Il6-/-hybrid mice. Rhbdd3-/-Il6-/- mice showed impaired T cell-stimulating activity by DC, and suppressed differentiation of Thl and Th17 but rescue of Treg generation in vivo, as compared with Rhbdd3-/-Il6+/+ mice. These results indicate that the IL-6, which is overproduced in the Rhbdd3-/- mice, is the key mediator derived from Rhbdd3-/- DCs in inducing the proliferation and differentiation of Thl and Th17.3. RHBDD3 suppresses LPS-triggered NF-κB activation by interacting with IKKy via its UBA domainWe next investigated the molecular mechanisms for how RHBDD3 negatively regulates TLR signaling and subsequently suppresses the IL-6 production in DC. We found that Rhbdd3-/- BMDCs showed significantly enhanced activation of NF-κB pathway in response to LPS as compared with WT control. In contrast, the phosphorylation of JNK, ERK, p38 and IRF-3 was intact. Overexpression of RHBDD3 significanly inhibited the activation of NF-κB signaling in 293 cells. Furthermore, we found that RHBDD3 was located in endosome and interacted with IKKy via its UBA domain in 293 cells upon TNF-a stimulation. Above data suggest that RHBDD3 suppresses the activation of NF-κB pathway in response to LPS by interacting with IKKy via its UBA domain.4. RHBDD3 plays an important role in preventing pathogenesis of autoimmune diseasesRhbdd3-/- mice spontaneously showed autoimmune disease-like symptoms. Rhbdd3-/-mice developed more severe clinical symptoms and higher incidence than wild type control in two murine models of autoimmune diseases, TNBS-induced colitis and MOG-induced EAE. In TNBS-induced colitis, Rhbdd3-/- mice showed higher I-Ab, CD40, CD80, and CD86 expression in DC, and higher CD44 expression in T cells, as compared with their littermates. These data suggest that RHBDD3 is required to prevent excessive immune responses and Th-17-mediated autoimmune diseases.Together, we demonstrate that RHBDD3 contributes to the maintenance of the balance of Treg, Thl and Th17 through controlling DC maturation and IL-6 production from mature DC by inhibiting NF-κB activation, which subsequently prevents the development of autoimmune diseases. Our research sheds new light on the pathogenic mechanism of autoimmune diseases and may provide a potential target for the design of immunotherapy of inflammatory autoimmune diseases.
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