Tahara M, Tsuboi H, Segawa S, Asashima H, Iizuka-Koga M, Hirota T, Takahashi H, Kondo Y, Matsui M, Matsumoto I, Sumida T
Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki.
Department of Internal Medicine, Fureai Higashitotsuka Hospital, Yokohama, Kanagawa, Japan.
Clin Exp Immunol. 2017 Feb;187(2):213-224. doi: 10.1111/cei.12868. Epub 2016 Oct 11.
We showed recently that M3 muscarinic acetylcholine receptor (M3R)-reactive CD3 T cells play a pathogenic role in the development of murine autoimmune sialadenitis (MIS), which mimics Sjögren's syndrome (SS). The aim of this study was to determine the effectiveness and mechanism of action of retinoic acid-related orphan receptor-gamma t (RORγt) antagonist (A213) in MIS. Splenocytes from M3R knockout (M3R ) mice immunized with murine M3R peptide mixture were inoculated into recombination-activating gene 1 knockout (Rag-1 ) mice (M3R →Rag-1 ) with MIS. Immunized M3R mice (pretransfer treatment) and M3R →Rag-1 mice (post-transfer treatment) were treated with A213 every 3 days. Salivary volume, severity of sialadenitis and cytokine production from M3R peptide-stimulated splenocytes and lymph node cells were examined. Effects of A213 on cytokine production were analysed by enzyme-linked immunosorbent assay (ELISA) and on T helper type 1 (Th1), Th17 and Th2 differentiation from CD4 T cells by flow cytometry. Pretransfer A213 treatment maintained salivary volume, improved MIS and reduced interferon (IFN)-γ and interleukin (IL)-17 production significantly compared with phosphate-buffered saline (PBS) (P < 0·05). These suppressive effects involved CD4 T cells rather than CD11c cells. Post-transfer treatment with A213 increased salivary volume (P < 0·05), suppressed MIS (P < 0·005) and reduced IFN-γ and IL-17 production (P < 0·05). In vitro, A213 suppressed IFN-γ and IL-17 production from M3R-stimulated splenocytes and CD4 T cells of immunized M3R mice (P < 0·05). In contrast with M3R specific responses, A213 suppressed only IL-17 production from Th17 differentiated CD4 T cells without any effect on Th1 and Th2 differentiation in vitro. Our findings suggested that RORγt antagonism is potentially suitable treatment strategy for SS-like sialadenitis through suppression of IL-17 and IFN-γ production by M3R-specific T cells.
我们最近发现，M3毒蕈碱型乙酰胆碱受体（M3R）反应性CD3 T细胞在小鼠自身免疫性涎腺炎（MIS）的发病过程中起致病作用，MIS可模拟干燥综合征（SS）。本研究的目的是确定维甲酸相关孤儿受体γt（RORγt）拮抗剂（A213）在MIS中的有效性及作用机制。用小鼠M3R肽混合物免疫的M3R基因敲除（M3R⁻）小鼠的脾细胞接种到患有MIS的重组激活基因1基因敲除（Rag-1⁻）小鼠（M3R⁻→Rag-1⁻）体内。免疫后的M3R⁻小鼠（转移前治疗）和M3R⁻→Rag-1⁻小鼠（转移后治疗）每3天用A213治疗一次。检测唾液分泌量、涎腺炎严重程度以及M3R肽刺激的脾细胞和淋巴结细胞产生的细胞因子。通过酶联免疫吸附测定（ELISA）分析A213对细胞因子产生的影响，并通过流式细胞术分析其对CD4⁺ T细胞向1型辅助性T细胞（Th1）、17型辅助性T细胞（Th17）和2型辅助性T细胞（Th2）分化的影响。与磷酸盐缓冲盐水（PBS）相比，转移前A213治疗可维持唾液分泌量、改善MIS并显著降低干扰素（IFN）-γ和白细胞介素（IL）-17的产生（P < 0.05）。这些抑制作用涉及CD4⁺ T细胞而非CD11c⁺细胞。转移后用A213治疗可增加唾液分泌量（P < 0.05）、抑制MIS（P < 0.005）并降低IFN-γ和IL-17的产生（P < 0.05）。在体外，A213可抑制M3R刺激的免疫M3R⁻小鼠的脾细胞和CD4⁺ T细胞产生IFN-γ和IL-17（P < 0.05）。与M3R特异性反应不同，A213在体外仅抑制Th17分化的CD4⁺ T细胞产生IL-17，而对Th1和Th2分化无任何影响。我们的研究结果表明，通过抑制M3R特异性T细胞产生IL-17和IFN-γ，RORγt拮抗作用可能是治疗类干燥综合征涎腺炎的合适策略。
