Department of Rheumatology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, 145 Shandong C Rd, Shanghai, 200001, China.
Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, 320 Yueyang Rd, Shanghai, 200031, China.
Arthritis Res Ther. 2019 Apr 11;21(1):92. doi: 10.1186/s13075-019-1874-2.
This study aimed to explore the molecular mechanism and clinical relevance of iguratimod in the regulation of human B cell terminal differentiation.
An in vitro human antibody-secreting cell (ASC) differentiation system was established to test the effect of iguratimod. B cell phenotype and key transcription factors (TFs) relevant to ASC differentiation were analyzed through flow cytometry and qPCR. The COX-2 activity was measured by enzyme immunoassay (EIA). RNA sequencing was used to identify potential targets of iguratimod. We enrolled six treatment-naive rheumatoid arthritis (RA) patients whose blood samples were collected for phenotypic and molecular studies along with 12-week iguratimod monotherapy.
Iguratimod inhibited human ASC generation without affecting B cell activation and proliferation. Iguratimod showed only weak COX-2 activity. Gene set enrichment analysis (GSEA) identified that protein kinase C (PKC) pathway was targeted by iguratimod which was confirmed by PKC activity detection. Furthermore, early growth response 1 (EGR1), a target of PKC and a non-redundant TF for ASC differentiation, was found to be the most downregulated gene in iguratimod-treated B cells. Lastly, iguratimod monotherapy decreased peripheral ASCs and was associated with improved disease activity. The expression of major ASC-related TFs, including EGR1, was similarly downregulated in patient blood samples.
Iguratimod inhibits ASC differentiation both in vitro and in RA patients. Our study suggests that PKC/EGR1 axis, rather than COX-2, is critically involved in the inhibitory effect by iguratimod on human ASC differentiation. Iguratimod could have a broader application to treat B cell-related autoimmune diseases in clinics.
本研究旨在探讨依那西普调节人 B 细胞终末分化的分子机制和临床相关性。
建立体外人抗体分泌细胞（ASC）分化系统来检测依那西普的作用。通过流式细胞术和 qPCR 分析 B 细胞表型和与 ASC 分化相关的关键转录因子（TFs）。通过酶免疫测定（EIA）测量 COX-2 活性。使用 RNA 测序鉴定依那西普的潜在靶标。我们招募了六名未经治疗的类风湿关节炎（RA）患者，他们的血液样本用于表型和分子研究，同时进行 12 周的依那西普单药治疗。
依那西普抑制人 ASC 的产生，而不影响 B 细胞的激活和增殖。依那西普仅表现出较弱的 COX-2 活性。基因集富集分析（GSEA）确定蛋白激酶 C（PKC）途径是依那西普的靶标，通过 PKC 活性检测得到证实。此外，早期生长反应 1（EGR1），PKC 的靶标和 ASC 分化的非冗余 TF，被发现是依那西普处理的 B 细胞中下调最明显的基因。最后，依那西普单药治疗减少外周 ASC，并与改善疾病活动度相关。患者血液样本中主要 ASC 相关 TF 的表达，包括 EGR1，也相似地下调。
依那西普在体外和 RA 患者中均抑制 ASC 分化。我们的研究表明，PKC/EGR1 轴而不是 COX-2，在依那西普抑制人 ASC 分化的作用中起着至关重要的作用。依那西普在临床上可能有更广泛的应用来治疗 B 细胞相关的自身免疫性疾病。
