CD147 Participates In The Pathogenesis Of Rheumatoid Arthritis By Regulating Th17 Cell Responses

ObjectiveRheumatoid arthritis(RA) is a common autoimmune disease characterized by the infiltration of inflammatory cells into the joints, synovial hyperplasia, and systemic inflammation. However, the precise etiology and pathogenesis of RA are not completely understood. From the recent reports, Th17 cells and their secreted cytokines play important roles in the development of RA. Th17 cell is a T cell subset characterized by the production of IL-17, which could be differentiated from CD4+ naive T cells(Tn) and CD4+ memory T cells(Tm). Although Th17 cells are implicated in the pathogenesis of RA, but the in vivo conditions of human Th17 cell responses are still not fully understood, and the majority of researchs are focused on the regulatory effect of cytokines in vitro. In human, the immune network is complicated, and a variety of immune cells, such as antigen presenting cells(APC), paly important roles in Th17 cell differentiation. A recent study has reported that APC from joint of RA patients preferentially promoted Th17 cell responses in CD4+ Tm. Further, Th17 cell responses in this process rely on cell contact, suggesting a critical role for cell-to-cell signals, but the exact mechanism is still unknown. Therefore, it is important to explore the specific membrane-derived signals that required for APC-induced Th17 cell responses in RA.CD147 is one of the transmembrane glycoprotein that is expressed on the surface of all immune cells, and strongly up-regulated on activated T cells. Importantly, evidence supports a functional role for CD147 at T cell activity, such as T cell development, activation, proliferation, migration, adhesion, and invasion. In addition, it has been reported that CD147 is involved in the immune synapse formation between APC and T cells, and thus regulated T cell activation. Recently, it was reported that CD147 negatively regulates Th17 cell differentiation by suppressing STAT3 signal in mice. Since there seems to be substantial differences between murine and human Th17 cell development, its exact roles regulating signal pathways in Th17 cell responses remain to be identified in human, especially in RA.Therefore, in order to explore whether CD147 plays an important role in Th17 cell responses and RA pathogenesis, we investgate the regulating effect of CD147 in APC-induced Th17 cell responses and its possible molecular mechanisms in RA patients. MethodsPeripheral blood(PB) or synovial fluid(SF) samples were obtained from RA patients and health controls(HCs). Then the surface phenotype and cytokine production of cells were analyzed by flow cytometry, including the percentages of CD4+CD161+ T cells and its intracellular cytokine expression of IL-17 and/or IFN-γ; the expression of CD147 on CD4+ T cells, CD8+ T cells, CD19+ B cells, CD14+ monocytes(Mo), CD4+CD161+ T cells and CD4+CCR6+ T cells; and the relationship of CD147 expression with IL-17 and IFN-γ production. The plasma levels of related cytokines(including IL-17, IL-1β, IL-6, IL-21 and IFN-γ) were assessed by Luminex technology. Meanwhile, the basic information, disease activity and clinical parameters of subjects were collected.Purification of CD14+ Mo and CD4+ T cells(including CD4+ Tn and CD4+ Tm) were performed by magnetic cell sorting. Then T cells were cocultured with Mo to induce Th17 cell responses under optimal conditions. The percentages of IL-17 and/or IFN-γ production in T cells and the phosphorylation levels of pSTAT3, pAKT(T308), pAKT(S473), pS6 and p70S6 K were analyzed by flow cytometry. Also, IL-17 and IFN-γ levels in supernatants of cocultures were determined by Luminex technology.C57BL/6 mice were immunized with collagen II to induce collagen-induced arthritis(CIA). After anti-CD147 mAb treatment, arthritis score and pathological staining of paraffin sections were conducted. Meanwhile, the PB, spleen and draining lymph nodes of limbs were obtained from CIA, and the percentages of IL-17 and/or IFN-γ production in T cells were analyzed by flow cytometry.Data analyses were performed using SPSS version 17.0 and GraphPad Prism version 5.0. A two-sided p-value < 0.05 was considered significant. Results1. In RA patients, IL-17 production cells were mainly contained within CD4+CD161+ T cell subsets. And the percentages of CD161+ Th17 cells were significantly higher than Th17 cells, further, there was a positive correlation between the percentages of CD161+ Th17 cells and Th17 cells. In addition, the percentages of circulating CD4+CD161+ T cells and CD161+ Th17 cells in RA patients increased significantly and correlated positively with disease activity index.2. The expression of CD147 on Th17 cells and their marked cells(CD4+CCR6+ and CD4+CD161+ T cells) were increased both in HCs and RA patients. And an increased percentages of Th17 cells were observed with increasing levels of CD147 expression.3. The optimum condition inducing Th17 cells was found when CD4+ Tm were stimulated in the presence of LPS-activated monocytes(5:1) and anti-CD3 mAb for 3 days. Next, using the established coculture system, we found that anti-CD147 mAb strongly decreased the percentages of Th17 cells and IL-17 secretion. Moreover, our data showed that anti-CD147 mAb impairs the phosphorylation of pSTAT3, pAKT(T308), pS6 K and p70S6 in CD4+ Tm.4. In vivo-activated monocytes, from the local sites of inflammation of RA patients, spontaneously promoted Th17 cell responses inducing from autologous CD4+ Tm. Further, anti-CD147 mAb specifically decreased in vivo-activated monocytes induced Th17 cell responses.5. In CIA mice, the treatment of anti-CD147 mAb specifically decreased the levels of Th17 cells in spleen and draining lymph nodes. Meanwhile, anti-CD147 mAb significantly ameliorates the symptoms of joint swelling, the mean arthritic index, intra-articular infiltration of inflammatory cells, synovial hyperplasia, and cartilage destruction. ConclusionThis study further supports that Th17 cells participate in the pathogenesis of RA, and suggests that circulating CD4+CD161+ T cells and CD161+ Th17 cells are potential cellular biomarkers of RA disease activity. CD147 expression on CD4+ T cells was closely correlated with Th17 cells, and CD147 plays a specific role in the regulation of APC-induced Th17 cell responses in RA patients. Moreover, these findings indicate that a pivotal role of CD147-AKT- mTORC1-STAT3 pathway in Th17 cell responses. And these results from CIA mice indicate that anti-CD147 mAb treatment suppressed the development of CIA by reducing Th17 cell responses. Thus, this study highlights the importance role of CD147 in APC-induced Th17 cell responses, and reveals its signal transduction pathway, which will provide a theoretical basis for the treatment of RA and other Th17-mediated autoimmune diseases by targeting CD147.