成纤维样滑膜细胞(FLS)活化出现异常迁移和侵袭是造成类风湿关节炎(rheumatoid arthritis,RA)关节炎症及破坏的重要原因。我们前期报道,RA-FLS高表达的过氧化物酶体增殖物激活受体γ辅激活因子(PGC1β)可促进RA-FLS分泌炎症因子(TNF-α、IL-6、IL-8)、基质金属蛋白酶(MMP)-3、MMP-13及RANKL,抑制RA-FLS凋亡。本研究首先通过临床病理学研究发现PGC1β在RA滑膜衬里层及衬里下层多种细胞的胞核中表达,RA滑膜PGC1β表达与滑膜局部及全身炎症水平、关节破坏程度呈正相关，提示RA滑膜PGC1β可能参与RA滑膜炎症及关节破坏。进一步体外培养原代RA-FLS,慢病毒转染RA-FLS沉默或上调PGC1β表达，通过划痕实验、体外Transwell小室迁移和Matrigel基质胶包被的Transwell小室侵袭实验结合鬼笔环肽染色、Western blot及G-LISA检测发现,PGC1β可通过激活Rho GTPase促进RA-FLS板状伪足及丝状伪足的形成进而促进其迁移和侵袭。应用荧光探针DCFH-DA结合流式细胞仪检测发现,RA-FLS中ROS水平显著高于非炎性关节病患者的FLS。在沉默或过表达PGC1β的同时，加入N-乙酰半胱氨酸(NAC)抑制ROS生成或加入过氧化氢(H2O2)促进ROS生成，发现PGC1β通过提高 ROS水平促进 RA-FLS迁移和侵袭。Western blot检测发现PGC1β可能通过上调ROS激活NF-κB信号通路发挥调控作用。最后，采用高度免疫缺陷模型NCG小鼠建立体内RA-FLS软骨侵袭模型，证实PGC1β在体内可促进RA-FLS侵袭软骨。本研究结果从临床研究、体外及体内实验的角度，全面证实PGC1β在RA滑膜炎症及关节破坏中的重要调控作用，并从免疫代谢的崭新角度进一步阐明PGC1β通过调控能量代谢促进RA-FLS活化的具体调控机制，为今后开发RA的治疗药物提供新靶点。 Activation of fibroblast-like synoviocytes(FLS)and its subsequent excessive migration and invasion cause synovitis and joint destruction in rheumatoid arthritis(RA).We previously reported that elevated transcription factor peroxisome proliferator-activated receptor gamma coactivator(PGC)1β in RA-FLS promoted the secretion of TNF-α,IL-6,IL-8,MMP-3,MMP-13 and RANKL,and inhibited apoptosis of RA-FLS.In this study,we further found PGC-1β was expressed in the nuclei of lining synoviocytes,sublining inflammatory cells and vascular endothelial cells of RA synovium.Synovial PGC-1β was positively associated with local and systemic inflammation as well as joint destruction,which implied that PGC1β might involve in the pathogenesis of synovitis and joint destruction in RA.In vitro,PGC1β was knockdown or overexpressed by transfection of lentivirus on RA-FLS.Wound healing assay,transwell migration and invasion assays showed that PGC1β promoted the migration and invasion of RA-FLS.Western blot and G-LISA assays showed that PGC1β increased Rho GTPases activity.F-actin staining showed that PGC-1β increased flat or ruffling lamellipodia and filopodia at the leading edges of RA-FLS.Furtherly,DCFH-DA using flow cytometry showed that the mean levels of ROS in RA-FLS were higher than that in Orth.A-FLS,while PGC1β up-regulated the level of ROS in RA-FLS.Supernumerary H2O2 could reverse the inhibitory effect of PGC1β knockdown on migration and invasion of RA-FLS.On the contrary,NAC could reverse the facilitating effect of PGC1β overexpression on the aggressive ability of RA-FLS,suggesting that the regulatory role of PGC1β is,at least in part,through the ROS pathway.Western blot showed that PGC1β promoted the expression of NF-κB p65 and NF-κB p-p65.In vivo study,RA-FLS transfected with PGC1 knockdown were implanted into the flanks of NCG mice which exhibited a significant reduction of invasion into cartilage with significantly lower invasion score.This study proved the critical role of PGC1β on promoting synovitis and joint destruction in RA and clarified the mechanism of PGC1β on regulating mitochondrial oxidative phosphorylation pathway,in order to develop a new therapeutic target for RA.

Background: Bilateral hands including proximal interphalangeal joints (PIPJs) are recommended on physical, X-ray radiographic, or ultrasonographic examination by clinical guidelines of rheumatoid arthritis (RA), but MRI still tends to examine unilateral wrists and/or MCPJs. We aimed to demonstrate the advantages of MRI examination on bilateral hands including PIPJs for disease assessment in early RA patients.Methods: Active early RA patients received 3.0T whole-body MRI examination with contrast-enhanced imaging on bilateral wrists, MCPJs, and PIPJs. MRI features were scored referring to the updated RAMRIS. Clinical assessments were conducted on the day of MRI examination.Results: The mean time of MRI examination was 24 +/- 3 min. MRI bone erosion in MCPJs would be missed-diagnosed in 23% of patients if non-dominant MCPJs were scanned unilaterally, while osteitis in MCPJs would be missed-diagnosed in 16% of patients if dominant MCPJs were scanned unilaterally. MRI synovitis severity was also asymmetric: 21% of patients showing severe synovitis unilaterally in non-dominant MCPJs/PIPJs and other 20% showing severe synovitis unilaterally in dominant MCPJs/PIPJs. Among these early RA patients, MRI tenosynovitis occurred the most frequently in wrist extensor compartment I, while MRI examination on bilateral hands demonstrated no overuse influence present. However, overuse should be considered in dominant PIPJ2, PIPJ4, and IPJ of thumb of which MRI tenosynovitis prevalence was respectively 18%, 17%, or 16% higher than the nondominant counterparts. Early MRI abnormality of nervus medianus secondary to severe tenosynovitis occurred either in dominant or non-dominant wrists; MRI of unilateral hands would take a risk of missed-diagnosis. Common MRI findings in PIPJs were synovitis and tenosynovitis, respectively in 87% and 69% of patients. MRI tenosynovitis prevalence in IPJ of thumb or PIPJ5 was much higher than the continued wrist flexor compartments. MRI synovitis or tenosynovitis in PIPJs independently increased more than twice probability of joint tenderness (OR = 2.09 or 2.83, both p < 0.001).Conclusions: In consideration of asymmetric MRI features in early RA, potential overuse influence for certain tenosynovitis in dominant hands, and high prevalence of MRI findings in PIPJs, MRI examination on bilateral hands including PIPJs is deserved for disease assessment in early RA patients.

Objective Activation of osteoclastogenesis at the bone site in rheumatoid arthritis (RA) is well established. The mechanisms by which circulating osteoclast precursors contribute are still unclear. Peroxisome proliferator-activated receptor gamma coactivator 1 beta (PGC-1 beta) is implicated in transcriptional regulation of osteoclastogenesis in mouse models. This study was undertaken to investigate the contribution of PGC-1 beta to circulating osteoclast precursors and its link to bone destruction in RA. Methods PGC-1 beta expression in RA peripheral blood CD14+ monocytes was increased and showed correlation with joint destruction shown on radiographs. Cells from RA patients or healthy controls were transfected with a lentivirus vector for PGC-1 beta gene silencing or overexpression and cultured with macrophage colony-stimulating factor and RANKL. Bone resorption activity, bone-degrading enzymes, and signaling molecules were measured in these mature osteoclasts. Results Increased nuclear accumulation of PGC-1 beta was observed in RA peripheral blood CD14+ monocytes, and these cells had stronger osteoclastogenesis than in healthy controls. PGC-1 beta protein expression was positively correlated with radiographic joint destruction (r = 0.396-0.413; all P < 0.05). PGC-1 beta knockdown suppressed (51-82% reduction) the expression of cathepsin K, tartrate-resistant acid phosphatase (TRAP), and matrix metalloproteinase 9 (MMP-9), as well as osteoclast differentiation and bone resorption activity. Conversely, PGC-1 beta overexpression increased these markers (by 1.5-1.8-fold) and osteoclastogenesis. VIVIT, an inhibitor of NFATc1 activation, inhibited the effect of overexpressed PGC-1 beta by reducing cathepsin K, TRAP, and MMP-9 expression. Chromatin immunoprecipitation assay and dual-luciferase reporter gene assay showed PGC-1 beta bound to NFATc1 promoter, leading to transcriptional activation. Conclusion Activation of the PGC-1 beta/NFATc1 pathway in circulating osteoclast precursors was associated with bone destruction in RA. This may represent a new treatment target.

IntroductionAnti-malarial drug artesunate can suppress inflammation and prevent cartilage and bone destruction in collagen-induced arthritis model in ratssuggesting it may be a potent drug for rheumatoid arthritis (RA) therapy. We aimed to investigate its effect on the invasive property of fibroblast-like synoviocytes (FLS) from patients with RA.MethodsSynovial tissues were obtained by closed needle biopsy from active RA patients, and FLS were isolated and cultured in vitro. RA-FLS were treated with artesunate at various concentrations, while methotrexate or hydroxychloroquine was employed as comparator drugs. Cell viability, proliferation, cell cycle, apoptosis, migration, invasion, and pseudopodium formation of RA-FLS were assessed by CCK-8 assays, EdU staining, Annexin V-FITC/PI staining, transwell assays, or F-actin staining, respectively. Further, relative changes of expressed proteases were analyzed by Proteome profiler human protease array and verified by quantitative real-time PCR (qPCR), Western blot, and ELISA. The expression of signaling molecules of MAPK, NF-B, AP-1, and PI3K/Akt pathways were measured by qPCR and Western blot. PDK-1 knockdown by specific inhibitor AR-12 or siRNA transfection was used to verify the pharmacological mechanism of artesunate on RA-FLS.ResultsArtesunate significantly inhibited the migration and invasion of RA-FLS in a dose-dependent manner with or without TNF- stimulation. The effect was mediated through artesunate inhibition of MMP-2 and MMP-9 production, and pre-treatment with exogenous MMP-9 reversed the inhibitory effect of artesunate on RA-FLS invasion. Artesunate had a stronger inhibitory effect on migration and invasion of RA-FLS as well as greater anti-inflammatory effect than those of hydroxychloroquine. Similar inhibitory effect was detected between artesunate and methotrexate, and synergy was observed when combined. Mechanistically, artesunate significantly inhibited PDK-1 expression as well as Akt and RSK2 phosphorylationin a similar manner to PDK-1-specific inhibitor AR-12 or PDK-1 knockdown by siRNA transfection. This inhibition results in suppression of RA-FLS migration and invasion as well as decreased MMP-2 and MMP-9 expression.ConclusionsOur study demonstrates artesunate is capable of inhibiting migration and invasion of RA-FLS through suppression of PDK1-induced activation of Akt and RSK2 phosphorylationsuggesting that artesunate may be a potential disease-modifying anti-rheumatic drug for RA.

In rheumatoid arthritis (RA), imbalanced T cells subsets play a critical role in sustaining chronic inflammatory responses in the synovium. Naive T cells in RA patients undergo maldifferentiation, including an increase in the effector Th1/Th17 lineage and a reduction in regulatory T (Treg) cells. Upon stimulation, naive CD4(+)CD45RO(-) T cells from RA patients exhibited insufficient expression of Foxp3, which induced a deficiency in Tregs production and an imbalance of Treg/Th17 differentiation. Further mechanistic study indicated that RA T cells failed to produce sufficient levels of the histone acetyltransferase Tip60, leading to reduced acetylation of Foxp3; this, in turn, decreased Foxp3 expression, impaired Treg commitment, and promoted Th17 production. Moreover, in human synovium chimeric mice, suppression of Tip60 activity in healthy T cells promoted tissue infiltration and arthritogenesis, while reconstitution of Tip60 in RA T cells suppressed synovitis and effector T cell infiltration. Our findings link T cell maldifferentiation and tissue infiltration with Tip60-mediated Foxp3 acetylation and identify Tip60 as a potential therapeutic target for suppression of tissue inflammation and autoimmunogenesis in RA.