Ren Chun-Feng, Zhao Ya-Xin, Hou Chun-Feng, Luan Luan, Duan Guo-Qing, Li Shu-Jie, Zhao Jian-Hong
Department of Rheumatism, The First People's Hospital of Jining, Jining, Shangdong 272000, P.R. China.
Department of Pharmaceutical, The First People's Hospital of Jining, Jining, Shangdong 272000, P.R. China.
Mol Med Rep. 2017 Jan;15(1):460-466. doi: 10.3892/mmr.2016.5968. Epub 2016 Nov 28.
The present study aimed to investigate the role of the soluble programmed death‑1 (sPD-1) protein, which is released by peripheral blood regulatory T cells (Treg) during the progression of rheumatoid arthritis (RA). From October 2012 to May 2014, 82 RA patients (RA group) and 90 healthy volunteers (healthy controls; HC) were recruited. Cluster of differentiation (CD)4, CD25 and forkhead/winged helix transcription factor p3 (Foxp3) and expression of cytotoxic T lymphocyte associated antigen 4 (CTLA-4) and Foxp3 were detected by flow cytometry. Expression of sPD‑1 in Treg was detected by western blot analysis. Immunosuppressive activity of CD4+CD25‑ Treg was measured via thiazolyl blue in an MTT assay. ELISA was used to detect interleukin‑10 (IL‑10), transforming growth factor beta (TGF-β), interleukin-4 (IL-4), interferon‑γ (IFN-γ) and nuclear factor of activated T cells (NF‑AT). It was observed that in peripheral blood, CD4+CD25-FOXP3+/CD4+ levels were reduced in the RA group (P<0.001), and sPD‑1 levels were markedly higher (P<0.001), compared with the HC group. Additionally, it was observed that relative sPD‑1 protein expression in the small interfering RNA (siRNA)-sPD-1 treated group was reduced compared with the untreated and scrambled siRNA groups (all P<0.0001). The mean fluorescence intensity of CTLA-4 and Foxp3 decreased markedly upon transfection with siRNA-sPD-1 (P<0.001). Compared with the normal CD4+CD25‑ T group, optical density (OD)540 values, IFN-γ/IL-4 concentration ratio and NF‑AT activity in siRNA untreated and scramble groups reduced significantly (all P<0.001). OD540 value, IFN-γ/IL-4 concentration ratio and NF‑AT activity in the siRNA‑sPD‑1 group were significantly upregulated (all P<0.001). Therefore, sPD-1 may suppress the level of CD4+CD25‑ Tregs in the peripheral blood of RA patients, and may be involved in a variety of immune processes mediated by CD4+CD25‑ Tregs.
本研究旨在探讨可溶性程序性死亡蛋白1（sPD-1）在类风湿关节炎（RA）进展过程中由外周血调节性T细胞（Treg）释放后所发挥的作用。2012年10月至2014年5月，招募了82例RA患者（RA组）和90名健康志愿者（健康对照组；HC）。采用流式细胞术检测分化簇（CD）4、CD25和叉头/翼状螺旋转录因子p3（Foxp3）以及细胞毒性T淋巴细胞相关抗原4（CTLA-4）和Foxp3的表达。通过蛋白质免疫印迹分析检测Treg中sPD-1的表达。采用噻唑蓝比色法（MTT法）测定CD4+CD25-Treg的免疫抑制活性。采用酶联免疫吸附测定法（ELISA）检测白细胞介素-10（IL-10）、转化生长因子β（TGF-β）、白细胞介素-4（IL-4）、干扰素-γ（IFN-γ）和活化T细胞核因子（NF-AT）。结果观察到，与HC组相比，RA组外周血中CD4+CD25-FOXP3+/CD4+水平降低（P<0.001），而sPD-1水平显著升高（P<0.001）。此外，观察到与未处理组和乱序小干扰RNA（siRNA）组相比，小干扰RNA（siRNA）-sPD-1处理组中sPD-1蛋白相对表达降低（所有P<0.0001）。用siRNA-sPD-1转染后，CTLA-4和Foxp3的平均荧光强度显著降低（P<0.001）。与正常CD4+CD25-T组相比，siRNA未处理组和乱序组的光密度（OD）540值、IFN-γ/IL-4浓度比值和NF-AT活性显著降低（所有P<0.001）。siRNA-sPD-1组的OD540值、IFN-γ/IL-4浓度比值和NF-AT活性显著上调（所有P<0.001）。因此，sPD-1可能抑制RA患者外周血中CD4+CD25-Treg的水平，并可能参与由CD4+CD25-Treg介导的多种免疫过程。
