背 景

        自噬是免疫细胞发育、存活和作用的关键机制，而自噬体途径失调也与包括类风湿性关节炎(RA)在内的自体免疫疾病的发病机理有关。实际上，自噬似乎与瓜氨酸肽的生成有关，最终导致RA耐受损伤。此外，研究发现RA滑膜组织的自噬水平有所上升，细胞凋亡相关的分子出现减少，因此有研究提出TNF诱导的自噬对RA病程发展有一定影响。

目 的

        本研究旨在分析TNF和抗TNF抑制剂依那西普对RA发病细胞中的自噬和凋亡的作用。

方 法

        在TNF存在和去血清(饥饿)状态下培养外周血单个核细胞(PBMCs)和从RA患者分离出的成纤维样滑膜细胞4个小时，然后加入浓度为15ug/mL的依那西普继续培养。24个小时后，用免疫印迹分析细胞的自噬分子标记物LC3-II的水平并用流式细胞术分析膜联蛋白V-阳性凋亡细胞百分比。

结 果

        正如预期，培养24小时后(所有实验状态下p<0.05)，RA PBMC and FLS中的TNF和饥饿诱导的自噬产生剂量依赖。此外，在这两种促自噬刺激(p<0.05)后，添加依那西普导致LC3-II水平显著下降(p=0.004)以及细胞凋亡率上升(p=0.002)。

结 论

        本研究首次证实依那西普对RA发病机理中细胞自噬激活的抑制作用，此外研究结果还证明自噬在RA细胞存活中的重要作用。

原 文

In vitro inhibitory effect of etanercept on autophagy: a new mechanism of action of tnf inhibitors in rheumatoid arthritis

        M Vomero1, A Capozzi2, C Barbati1, T Colasanti1, V Manganelli2, F Ceccarelli1, FR Spinelli1, F Conti1, C Perricone1, A Finucci1, M Pendolino1, R Scrivo1, R Misasi2, M Sorice2, G Valesini1, C Alessandri1

        Citation

        Vomero M, Capozzi A, Barbati C, et al FRI0028 In vitro inhibitory effect of etanercept on autophagy: a new mechanism of action of tnf inhibitors in rheumatoid arthritis Annals of the Rheumatic Diseases 2017;76:490.

        Publication history

        Published in print 1 June 2017.

        Published online 15 June 2017.

ABSTRACT

Background

        Autophagy has emerged as a key mechanism in the development, survival and function of immune cells and dysregulation of autophagic pathway has been implicated in the pathogenesis of several autoimmune diseases including Rheumatoid Arthritis (RA) (1). In fact, autophagy seems to be involved in the generation of citrullinated peptides, with consequent breakage of tolerance in RA (2). Moreover, increased autophagy levels and a reduction of apoptosis-related molecules have been found in RA synovial tissues and a role of TNF-induced autophagy in RA development has been proposed (3).

Objectives

        The aim of the study was to analysed the effect of TNF and anti-TNF inhibitor etanercept on autophagy and apoptosis in cells involved in RA pathogenesis.

Methods

        Peripheral blood mononuclear cells (PBMCs) and fibroblast-like synoviocytes (FLS) isolated from RA patients were cultured in presence of TNF and in serum deprivation state (starvation) for 4 hours and then etanercept, at concentration of 15 ug/mL, were added to the culture. After 24h cells were analyzed for levels of autophagy marker LC3-II by western blot and for percentage of annexin V-positive apoptotic cells by flow cytometry.

Results

        As expected, TNF and starvation induced autophagy on RA PBMC and FLS in dose-dependent manner after 24h of culture (p<0.05 in all experimental conditions). Moreover, the adding of etanercept caused a significant reduction of LC3-II levels (p=0.004) and an increase of apoptosis rate (p=0.002) after both pro-autophagic stimuli (p<0.05).

Conclusions

        We demonstrated for the first time an inhibitory effect of etanercept on autophagy activation of cells involved in RA pathogenesis. In addition, our findings suggest a crucial role of autophagy in RA cells survival.