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Graphical abstract DCs act as sentinels for the immune system. DCs capture antigens locally and become mature, characterized by up-regulation of chemokine receptors (CCR7 and CXCR4), adhesion molecule (L-selectin), co-stimulator molecules (CD40, CD80 and CD86) and MHC II. Under high concentration of CCL21 in draining lymph nodes (LNs), mature DCs traffic to LNs via afferent lymph vessels and present antigens to T cells, resulting in the development of arthritis. HCQ impaires DC maturation via blocking TLR9 signaling, retrains DC migration to LNs, and prevents the initiation and progression of RA., Highlights • HCQ efficiently inhibited DC phenotypic and functional maturation stimulated by serum from RA patients. • HCQ prevented progression of arthritis by inhibiting DC maturation and migration from peripheral blood to LNs. • HCQ inhibited CpG ODN 1826-activated BMDC maturation and migration. • The effect of HCQ on DCs was related to the block in TLR9 signaling. • The development of arthritis was impaired in TLR9−/− mice., Hydroxychloroquine (HCQ) is one of the most commonly prescribed immune-suppressants in treating rheumatoid arthritis (RA). Our previous research showed that HCQ suppressed RA development by inhibiting T follicular helper (Tfh) cells directly. Dendritic cells (DCs) serve as the link between innate and acquired immunity. Whether HCQ suppressed Tfh cell through DCs was not clear. In current study, we found that HCQ efficiently inhibited CD86, chemokine (C-X-C motif) receptor 4 (CXCR4) expression and interferon-α (IFN-α) secretion of healthy donor derived purified DCs stimulated by RA patient serum. To mimic RA, collagen-induced arthritis (CIA) mouse model was used and treated with HCQ daily for fifty-four days prior to sacrifice. We found HCQ inhibited DC maturation and migration to lymph nodes (LNs), manifested as down-regulated expression of CD40, CD80, CD86, MHCII (I-Aq) on LN DCs. In addition, HCQ reduced the level of chemokine receptor 7 (CCR7) and L-selectin on peripheral blood DCs and diminished percentage of LN DCs. Of note, HCQ only inhibited CpG ODN 1826-induced IL-12 secretion by bone marrow DCs (BMDCs) stimulated by various toll like receptor (TLR) agonists. Mechanistically, HCQ down-regulated the expression of TLR9 not only in healthy donor PBMC-derived DCs stimulated by RA patient serum, but also in LN DCs of CIA mice and CpG-activated BMDCs. Furthermore, arthritis scores in TLR9−/− mice were much lower than that in wild type mice with impaired maturity and migration capability of DCs. Collectively, activation of DCs contributes to the pathogenesis of RA and HCQ shows protective effects on RA by inhibition of DC activation via blocking TLR9.

Hydroxychloroquine (HCQ) is an immunosuppressive agent widely used in rheumatoid arthritis (RA). T follicular helper (Tfh) cells play a vital role in the pathogenesis of RA. However, whether HCQ suppresses arthritis development through interfering with Tfh cells have never been reported. To address this issue, we investigated the percent of Tfh cells in newly diagnosed RA patients and found that they were up-regulated in peripheral blood. Importantly, in ex vivo experiments of peripheral blood mononuclear cells (PBMCs) from healthy volunteers, we proved that the percentage of Tfh cells in PBMCs and purified CD4+ T cells were decreased after HCQ treatment. In in vivo experiments of collagen-induced arthritis (CIA) model, we discovered that HCQ suppressed the incidence and score of arthritis, reduced the secretion of proinflammatory cytokines in serum. Similar to ex vivo study, the ratio of Tfh cells in HCQ treated CIA mice declined to the level of vehicle-treated group. Further research demonstrated that HCQ inhibited the generation of Tfh cells stimulated by IL-12 and IL-21. In conclusion, our study indicates a previously unrecognized mechanism of HCQ in RA, that HCQ directly suppresses the generation of Tfh cells by blocking IL-12 and IL-21 signaling pathways probably.