Department of Veterinary and Biomedical Sciences, Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802, USA.
Ann Rheum Dis. 2013 Oct;72(10):1708-16. doi: 10.1136/annrheumdis-2012-202639. Epub 2013 Jan 24.
Rheumatoid Arthritis (RA) is a chronic inflammatory disease of unclear aetiology, which is associated with inflamed human fibroblast-like synoviocytes (HFLS). Epidemiological studies have identified a positive correlation between tobacco smoking (a rich source of aryl hydrocarbon receptor (AHR) agonists) and aggressive RA phenotype. Thus, we hypothesise that antagonism of AHR activity by a potent AHR antagonist GNF351 can attenuate the inflammatory phenotype of HFLS-RA cells.
Quantitative PCR was used to examine IL1B-induced mRNA expression in primary HFLS-RA cells. A structurally diverse AHR antagonist CH223191 and transient AHR repression using AHR small interfering RNA (siRNA) in primary HFLS-RA cells were used to demonstrate that effects observed by GNF351 are AHR-mediated. The levels of PTGS2 were determined by western blot and secretory cytokines such as IL1B and IL6 by ELISA. Chromatin-immunoprecipitation was used to assess occupancy of the AHR on the promoters of IL1B and IL6.
Many of the chemokine and cytokine genes induced by IL1B in HFLS-RA cells are repressed by co-treatment with GNF351 at both the mRNA and protein level. Pretreatment of HLFS-RA cells with CH223191 or transient gene ablation of AHR by siRNA confirmed that the effects of GNF351 are AHR-mediated. GNF351 inhibited the recruitment of AHR to the promoters of IL1B and IL6 confirming occupancy of AHR at these promoters is required for enhanced inflammatory signalling.
These data suggest that AHR antagonism may represent a viable adjuvant therapeutic strategy for the amelioration of inflammation associated with RA.
类风湿关节炎（RA）是一种病因不明的慢性炎症性疾病，与炎症性人成纤维样滑膜细胞（HFLS）有关。流行病学研究已经确定，吸烟（芳香烃受体（AHR）激动剂的丰富来源）与侵袭性 RA 表型之间存在正相关。因此，我们假设通过有效的 AHR 拮抗剂 GNF351 拮抗 AHR 活性可以减轻 HFLS-RA 细胞的炎症表型。
使用定量 PCR 检测原发性 HFLS-RA 细胞中 IL1B 诱导的 mRNA 表达。使用结构多样的 AHR 拮抗剂 CH223191 和原发性 HFLS-RA 细胞中的 AHR 短暂基因沉默（siRNA）来证明 GNF351 观察到的作用是 AHR 介导的。通过 Western blot 测定 PTGS2 的水平，通过 ELISA 测定 IL1B 和 IL6 等分泌细胞因子的水平。使用染色质免疫沉淀法评估 AHR 在 IL1B 和 IL6 启动子上的占据。
IL1B 在 HFLS-RA 细胞中诱导的许多趋化因子和细胞因子基因在与 GNF351 共同处理时在 mRNA 和蛋白质水平上均受到抑制。HLFS-RA 细胞的预处理用 CH223191 或通过 siRNA 短暂基因敲除 AHR 证实 GNF351 的作用是 AHR 介导的。GNF351 抑制了 AHR 募集到 IL1B 和 IL6 的启动子，证实了 AHR 在这些启动子上的占据是增强炎症信号所必需的。
这些数据表明，AHR 拮抗可能代表改善与 RA 相关炎症的可行辅助治疗策略。
