Sorice Maurizio, Iannuccelli Cristina, Manganelli Valeria, Capozzi Antonella, Alessandri Cristiano, Lococo Emanuela, Garofalo Tina, Di Franco Manuela, Bombardieri Michele, Nerviani Alessandra, Misasi Roberta, Valesini Guido
Dipartimento di Medicina Sperimentale.
Reumatologia, Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Roma, Italy.
Rheumatology (Oxford). 2016 Aug;55(8):1374-85. doi: 10.1093/rheumatology/kew178. Epub 2016 Apr 12.
Autophagy may represent a functional processing event that creates a substrate for autoreactivity. In particular, autophagy may play a role in the pathogenesis of RA, since autophagy is a key cellular event involved in the generation of citrullinated peptides, with consequent breakage of tolerance. Thus, in RA, autophagy may be the common feature in several situations (including smoking, joint injury and infection) that may drive the adaptive responses to citrullinated self-proteins. The aim of this study was the analysis, in vitro, of the role of autophagy in the generation of citrullinated peptides and, in vivo, of the relationship between autophagy and the production of anti-CCP antibodies (Abs).
For autophagy induction, fibroblast-like synoviocytes, primary fibroblasts and monocytes were stimulated with tunicamycin or rapamycin. Peptidyl arginine deiminase activity was tested by enzyme-linked immunosorbent assay, and protein citrullination was evaluated by western blotting. The main citrullinated RA candidate antigens, vimentin, α-enolase and filaggrin, were demonstrated by immunoprecipitation. The relationship between autophagy and anti-CCP Abs was analysed in 30 early-active RA patients.
Our results demonstrated in vitro a role for autophagy in the citrullination process. Cells treated with tunicamycin or rapamycin showed peptidyl arginine deiminase 4 activation, with consequent protein citrullination. Immunoblotting and immunoprecipitation experiments, using specific Abs, identified the main citrullinated proteins: vimentin, α-enolase and filaggrin. In vivo, a significant association between levels of autophagy and anti-CCP Abs was observed in treatment-naïve early-active RA patients.
These findings support the view that the processing of proteins in autophagy generates citrullinated peptides recognized by the immune system in RA.
自噬可能代表一种功能性加工事件，为自身反应性创造底物。特别是，自噬可能在类风湿关节炎（RA）的发病机制中起作用，因为自噬是参与瓜氨酸化肽生成的关键细胞事件，从而导致耐受性破坏。因此，在RA中，自噬可能是几种情况（包括吸烟、关节损伤和感染）中的共同特征，这些情况可能驱动对瓜氨酸化自身蛋白的适应性反应。本研究的目的是在体外分析自噬在瓜氨酸化肽生成中的作用，以及在体内分析自噬与抗环瓜氨酸肽抗体（Abs）产生之间的关系。
为诱导自噬，用衣霉素或雷帕霉素刺激成纤维样滑膜细胞、原代成纤维细胞和单核细胞。通过酶联免疫吸附测定法检测肽基精氨酸脱亚氨酶活性，并通过蛋白质印迹法评估蛋白质瓜氨酸化。通过免疫沉淀法证实了主要的瓜氨酸化RA候选抗原波形蛋白、α-烯醇化酶和丝聚蛋白。分析了30例早期活动型RA患者中自噬与抗CCP Abs之间的关系。
我们的结果在体外证明了自噬在瓜氨酸化过程中的作用。用衣霉素或雷帕霉素处理的细胞显示肽基精氨酸脱亚氨酶4激活，从而导致蛋白质瓜氨酸化。使用特异性抗体的免疫印迹和免疫沉淀实验鉴定出主要的瓜氨酸化蛋白：波形蛋白、α-烯醇化酶和丝聚蛋白。在体内，在未接受治疗的早期活动型RA患者中观察到自噬水平与抗CCP Abs之间存在显著关联。
这些发现支持这样一种观点，即自噬中的蛋白质加工产生了RA中被免疫系统识别的瓜氨酸化肽。
