Objective: Smad7 is an inhibitory Smad and plays a protective role in many inflammatory diseases. However, the roles of Smad7 in rheumatoid arthritis (RA) remain unexplored, which were investigated in this study.Methods: The activation of TGF-β/Smad signaling was examined in synovial tissues of patients with RA. The functional roles and mechanisms of Smad7 in RA were determined in a mouse model of collagen-induced arthritis (CIA) in Smad7 wild-type (WT) and knockout (KO) CD-1 mice, a strain resistant to autoimmune arthritis induction.Results: TGF-β/Smad3 signaling was markedly activated in synovial tissues of patients with RA, which was associated with the loss of Smad7, and enhanced Th17 and Th1 immune response. The potential roles of Smad7 in RA were further investigated in a mouse model of CIA in Smad7 WT/KO CD-1 mice. As expected, Smad7-WT CD-1 mice did not develop CIA. Surprisingly, CD-1 mice with Smad7 deficiency developed severe arthritis including severe joint swelling, synovial hyperplasia, cartilage damage, massive infiltration of CD3+ T cells and F4/80+ macrophages, and upregulation of proinflammatory cytokines IL-1β, TNFα, and MCP-1. Further studies revealed that enhanced arthritis in Smad7 KO CD-1 mice was associated with increased Th1, Th2 and, importantly, Th17 over the Treg immune response with overactive TGF-β/Smad3 and proinflammatory IL-6 signaling in the joint tissues.Conclusions: Smad7 deficiency increases the susceptibility to autoimmune arthritis in CD-1 mice. Enhanced TGF-β/Smad3-IL-6 signaling and Th17 immune response may be a mechanism through which disrupted Smad7 causes autoimmune arthritis in CD-1 mice.

IntroductionProline-rich tyrosine kinase 2 (PYK2) provides important signals during the activation of lymphocytes, which is essential in autoimmune diseases. Systemic lupus erythematosus (SLE) is a representative autoimmune disease, and lupus nephritis (LN) is one of its most severe complications. Although glucocorticoid-binding immuno-suppression is the first-line therapy for patients with LN, the common and severe side effects of such treatment call for new strategies to improve long-term prognosis and life quality for these patients. Curcumin has been used to treat autoimmune disease with good curative effect, but little is known about the effect of curcumin on LN patients. Our aim was to investigate the mechanism of curcumin for management of LN, specifically regarding the PYK2 pathways.Material and methodsFreshly isolated peripheral blood mononuclear cells (PBMCs) from 20 LN patients and 20 healthy individuals were cultured and stimulated with either PMA, PMA+TyrA9 (PYK2 specific inhibitor), or PMA+Curcumin, and with PBS as control. After 48 hours of incubation, cells were harvested and the expression of PYK2, p-PYK2, CD40L, CTLA-4, and PBMCs proliferation were measured. Then the expression and activation of PYK2 was evaluated using Western blot, the expression of costimulatory molecules CD40L and CTLA-4 protein was evaluated using flow cytometry, and PBMC proliferation was assessed using a [3H]-thymidine incorporation assay.ResultsCurcumin inhibited the expression and activation of PYK2 in PBMCs in patients with LN in vitro. The inhibition rate of curcumin was negatively correlated with the level of serum complement, but positively correlated with 24-h proteinuria. Curcumin also suppressed the expression of costimulatory molecules CD40L and CTLA-4, as well as PBMC proliferation. Interestingly, these effects were not reproduced on PBMC cultures of healthy subjects.ConclusionsThe inhibition of PYK2 signalling protein may be one of the mechanisms underlying the action of curcumin in LN treatment.

Objective: To evaluate the biological effect and mechanisms of C-reactive protein (CRP) on the activation of fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). Study design: To understand if CRP is involved in RA, expression of CRP and its receptors CD32/64 was examined in synovial tissues from RA patients and normal controls. In vitro, the potential role and mechanisms of CRP in FLS proliferation and invasion, expression of pro-inflammatory cytokines, and activation of signaling pathways were investigated in both RA-FLS and a normal human fibroblast-like synoviocyte line (HFLS). Results: Compared to normal controls, synovial tissues from 21 RA patients exhibited highly activated CRP signaling, particularly by FLSs as identified by 65% of CRP-expressing cells being CRP+vimentin+ and CD32/64+vimentin+ cells. In vitro, FLSs from RA patients, but not HFLS, showed highly reactive to CRP by largely increasing proliferative and invasive activities and expressing pro-inflammatory cytokines and chemokines, including CCL2, CXCL8, IL-6, and MMP2/9. All these changes were blocked largely by a neutralizing antibody to CD32 and, to a less extent by the anti-CD64 antibody, revealing CD32 as a primary mechanism of CRP signaling during synovial inflammation. Further studies revealed that CRP also induced synovial inflammation differentially via CD32/CD64-NF-κB or p38 pathways as blockade of CRP-CD32-NF-κB signaling inhibited CXCL8, CCL2, IL-6, whereas CRP induced RA-FLS invasiveness through CD32-p38 and MMP9 expression via the CD64-p38-dependent mechanism. Conclusions: CRP signaling is highly activated in synovial FLSs from patients with RA. CRP can induce synovial inflammation via mechanisms associated with activation of CD32/64-p38 and NF-κB signaling.

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ErMiao San (EMS) is composed of the Cortex Phellodendri chinensis and Atractylodes lancea, and it has the function of eliminating heat and excreting dampness in terms of traditional Chinese medicine to damp heat syndrome. Previous reports indicate that EMS possesses anti-inflammatory activity; however, its action on angiogenesis of rheumatoid arthritis (RA) has not been clarified. The present study aims to determine the antiangiogenic activity of EMS in collagen-induced arthritis (CIA) mice and in various angiogenesis models. Our data showed that EMS (5 g/kg) markedly reduced the immature blood vessels in synovial membrane tissues of inflamed joints from CIA mice. It also inhibited vascular endothelial growth factor (VEGF)-induced microvessel sprout formation ex vivo. Meanwhile, EMS suppressed VEGF-induced migration, invasion, adhesion, and tube formation of human umbilical vein endothelial cells (HUVECs). Moreover, EMS significantly reduced the expression of angiogenic activators including interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) in synovium of CIA mice. More interestingly, EMS blocked the autophosphorylation of VEGF-induced JAK1, STAT1, and STAT6 in CIA mice and VEGF-induced HUVECs. These findings suggest for the first time that EMS possesses the antiangiogenic effect in RA in vivo, ex vivo, and in vitro by interrupting the targeting of JAK/STAT activation.