Department of Rheumatology and Immunology, Xiangya Hospital, Central South University, Hunan Province, Changsha, 410008, People's Republic of China.
Arthritis Res Ther. 2018 Nov 28;20(1):263. doi: 10.1186/s13075-018-1759-9.
The cholinergic anti-inflammatory pathway (CAP) has a strong anti-inflammatory effect on collagen-induced arthritis (CIA), a classic animal model of rheumatoid arthritis (RA). However, the underlying immune regulatory mechanism remains unclear. Here, we investigated the effect of the CAP on arthritis development and the involvement of dendritic cells (DCs).
Forty DBA/1 mice were randomly divided into five groups: a control group (sham vagotomy+ phosphate-buffered saline; shamVGX+PBS), a CIA group (shamVGX+CIA + PBS), a vagotomy group (VGX + CIA + PBS), a GTS-21 (4 mg/kg) group (shamVGX+CIA + GTS-4), and a GTS-21 (8 mg/kg) group (shamVGX+CIA + GTS-8). The vagotomy group underwent left cervical vagotomy 4 days before arthritis induction, whereas the sham-vagotomy group underwent vagus nerve exposure. Mice were pretreated with GTS-21 by intraperitoneal injection on the day of surgery. The degree of arthritis was measured by using the arthritis score, hematoxylin and eosin staining, and TRAP (tartrate-resistant acid phosphatase) staining. Flow cytometry was used to detect the expression of CD80 and major histocompatibility complex II (MHC II) on CD11c DCs in the spleen. Luminex was used to detect the serum concentration of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFα), and IL-10. Immunohistochemistry was used to detect CD11c expression in the synovium. The effects of GTS-21 on DC differentiation and maturation were examined in vitro by treating bone marrow-derived DCs with GTS-21 and assessing differentiation and maturation. Flow cytometry was used to analyze CD80 and MHC II expression on the surface of DCs.
GTS-21 treatment ameliorated clinical arthritis in a mouse model of CIA in vivo, decreasing the secretion of pro-inflammatory cytokines in the serum and downregulating CD80 and MHC II expression on DCs in the spleen of CIA mice. GTS-21 treatment strongly suppressed the infiltration of DCs into the synovium. Vagotomy itself did not exacerbate the severity of arthritis in CIA mice. In vitro, GTS-21 (10 μmol/L) significantly downregulated CD80 and MHC II in bone marrow-derived immature DCs and this effect was blocked by the α7-nicotinic acetylcholine receptor antagonist methyllycaconitine (MLA). However, GTS-21 had no effects on mature DCs.
The present study provides new insight into the mechanism underlying the effects of the CAP on RA and indicates that the immunosuppressive effect of GTS-21 may be mediated by the inhibition of DC differentiation.
胆碱能抗炎途径（CAP）对胶原诱导性关节炎（CIA）具有很强的抗炎作用，CIA 是类风湿关节炎（RA）的经典动物模型。然而，其潜在的免疫调节机制尚不清楚。在这里，我们研究了 CAP 对关节炎发展的影响及其与树突状细胞（DC）的关系。
将 40 只 DBA/1 小鼠随机分为五组：对照组（假手术+磷酸盐缓冲液； shamVGX+PBS）、关节炎组（假手术+ CIA+PBS）、迷走神经切断组（VGX+CIA+PBS）、GTS-21（4mg/kg）组（shamVGX+CIA+GTS-4）和 GTS-21（8mg/kg）组（shamVGX+CIA+GTS-8）。迷走神经切断组在关节炎诱导前 4 天进行左侧颈迷走神经切断术，而假手术组仅进行迷走神经暴露。手术当天通过腹腔注射 GTS-21 对小鼠进行预处理。采用关节炎评分、苏木精和伊红染色和抗酒石酸酸性磷酸酶（TRAP）染色来测量关节炎的严重程度。采用流式细胞术检测脾内 CD11c DC 上 CD80 和主要组织相容性复合体 II（MHC II）的表达。采用 Luminex 检测血清中白细胞介素 6（IL-6）、肿瘤坏死因子-α（TNFα）和 IL-10 的浓度。采用免疫组织化学检测滑膜中 CD11c 的表达。通过用 GTS-21 处理骨髓来源的树突状细胞并评估分化和成熟，在体外研究 GTS-21 对 DC 分化和成熟的影响。采用流式细胞术分析 DC 表面 CD80 和 MHC II 的表达。
GTS-21 治疗可改善 CIA 小鼠体内的临床关节炎，降低血清中促炎细胞因子的分泌，并下调 CIA 小鼠脾内 DC 的 CD80 和 MHC II 表达。GTS-21 治疗强烈抑制 DC 浸润滑膜。迷走神经切断本身不会加重 CIA 小鼠关节炎的严重程度。在体外，GTS-21（10μmol/L）可显著下调骨髓来源未成熟 DC 中的 CD80 和 MHC II，这种作用被α7-烟碱型乙酰胆碱受体拮抗剂甲基牛扁亭碱（MLA）阻断。然而，GTS-21 对成熟 DC 没有影响。
本研究为 CAP 对 RA 影响的机制提供了新的见解，并表明 GTS-21 的免疫抑制作用可能是通过抑制 DC 分化介导的。
