Autophagy promotes citrullination of VIM (vimentin) and its interaction with major histocompatibility complex class II in synovial fibroblasts
Autophagy promotes citrullination of VIM (vimentin) and its interaction with major histocompatibility complex class II in synovial fibroblasts
复制标题
DOI：
10.1080/15548627.2019.1664144
复制
发表时间：
2019-09-08
期刊：
影响因子：
13.3
通讯作者：
Atsumi, Tatsuya
中科院分区：
文献类型：
--
作者：
Sugawara, Eri;Kato, Masaru;Atsumi, Tatsuya
我们旨在通过VIM （vimentin）瓜氨酸化及其与滑膜成纤维细胞（SFs） MHC II类的相互作用，研究类风湿性关节炎（RA）自身免疫中巨噬/自噬的参与。IFNG/ ifn - γ（干扰素γ）处理后，流式细胞术分析SFs细胞表面MHC II类和B7共刺激分子的表达。以抗瓜氨酸肽抗体（ACPA）阳性患者血清为一抗，采用免疫印迹法分析sf细胞内瓜氨酸化自身抗原。sf在无血清培养基中孵育或用蛋白酶体抑制剂MG132处理以诱导自噬。使用自噬抑制剂3-甲基ladenin （3-MA）。采用免疫印迹和免疫细胞化学方法评价胞内瓜氨酸化VIM (cVIM)。采用共免疫沉淀和邻近结联法（PLA）评估MHC II类与cVIM的相互作用。我们证明，在IFNG治疗后，MHC II类、CD274/B7-H1和PDCD1LG2/B7-DC在SFs上表达，而CD276/B7-H3在SFs上检测到，无论IFNG是否存在。acpa阳性血清可识别SFs中的54 kDa蛋白。通过免疫沉淀，发现RA血清识别的54 kDa蛋白为cVIM。诱导自噬后，SFs细胞内cVIM增加，但这种作用被3-MA抵消。通过共免疫沉淀证实MHC II类与cVIM之间的相互作用。此外，PLA显示自噬诱导后MHC ii类- cvim相互作用显著增加。我们的研究结果表明，sf可能通过VIM的瓜氨酸化及其与自噬促进的MHC II类的相互作用，参与RA的自身免疫。
We aimed to investigate the involvement of macroautophagy/autophagy in autoimmunity in rheumatoid arthritis (RA) through citrullination of VIM (vimentin) and its interaction with MHC class II in synovial fibroblasts (SFs). The cell surface expression of MHC class II and B7 costimulatory molecules on SFs was analyzed by flow cytometry after treatment with IFNG/IFN-gamma (interferon gamma). Intracellular citrullinated autoantigens in SFs were analyzed by immunoblotting using serum from anti-citrullinated peptide antibodies (ACPA)-positive patient as a primary antibody. SFs were incubated in serum-free medium or treated with proteasome inhibitor MG132 to induce autophagy. An autophagy inhibitor 3-methyladenin (3-MA) was used. Intracellular citrullinated VIM (cVIM) was evaluated by immunoblotting and immunocytochemistry. The interaction between MHC class II and cVIM was evaluated with co-immunoprecipitation and proximity ligation assay (PLA). We demonstrated that MHC class II, CD274/B7-H1 and PDCD1LG2/B7-DC were expressed on SFs following treatment with IFNG whereas CD276/B7-H3 was detected on SFs regardless of the presence of IFNG. ACPA-positive sera recognized a 54 kDa protein in SFs. By immunoprecipitation, the 54 kDa protein recognized by RA sera was revealed to be cVIM. Following induction of autophagy, intracellular cVIM was increased in SFs but the effect was canceled by 3-MA. The interaction between MHC class II and cVIM was demonstrated by co-immunoprecipitation. Furthermore, PLA revealed the significant increase of MHC class II-cVIM interaction following induction of autophagy. Our findings suggest that SFs may contribute to the autoimmunity in RA through citrullination of VIM and its interaction with MHC class II promoted by autophagy.