Hakam Miya Soukaina, Miranda-Sayago Jose Maria, Hayrabedyan Soren, Todorova Krassimira, Spencer Patrick Simon, Jabeen Asma, Barnea Eytan R, Fernandez Nelson
School of Biological Sciences, University of Essex, Colchester, United Kingdom.
Department of Biochemistry, Molecular Biology and Immunology, Faculty of Medicine, University of Malaga, Malaga, Spain.
Cell Physiol Biochem. 2017;43(6):2277-2296. doi: 10.1159/000484378. Epub 2017 Oct 27.
BACKGROUND/AIMS: Pregnancy success requires mandatory maternal tolerance of the semi/ allogeneic embryo involving embryo-derived signals. Expression levels of PreImplantation Factor (PIF), a novel peptide secreted by viable embryos, correlate with embryo development, and its early detection in circulation correlates with a favourable pregnancy outcome. PIF enhances endometrial receptivity to promote embryo implantation. Via the p53 pathway, it increases trophoblast invasion, improving cell survival / immune privilege. PIF also reduces spontaneous and LPS-induced foetal death in immune naïve murine model. We examined PIF effect on gene expression of human leukocyte antigen (HLA-G, -E -F and -C) and the influence of PIF on local progesterone activity in JEG-3 choriocarcinoma cells.
PIF and progesterone (P4) effects on JEG-3 cells surface and intracellular HLA molecules was tested using monoclonal antibodies, flow cytometry, and Western blotting. PIF and IL17 effects on P4 and cytokines secretion was determined by ELISA. PIF and P4 effects on JEG-3 cells proteome was examined using 2D gel staining followed by spot analysis, mass spectrometry and bioinformatic analysis.
In cytotrophoblastic JEG-3 cells PIF increased intracellular expression of HLA-G, HLA-F, HLA-E and HLA-C and surface expression of HLA-G, HLA-E and HLA-C in dose and time dependent manner. In case of HLA-E, -F results were confirmed also by Western blot. Proteome analysis confirmed an increase in HLA-G, pro-tolerance FOXP3+ regulatory T cells (Tregs), coagulation factors and complement regulator. In contrast, PIF reduced PRDX2 and HSP70s to negate oxidative stress and protein misfolding. PIF enhanced local progesterone activity, increasing steroid secretion and the receptor protein. It also promoted the secretion of the Th1/Th2 cytokines (IL-10, IL-1β, IL-8, GM-CSF and TGF-β1), resulting in improved maternal signalling.
PIF can generate a pro-tolerance milieu by enhancing the expression of HLA molecules and by amplifying endogenous progesterone activity. A Fast-Track clinical trial for autoimmune disease has been satisfactorily completed. The acquired data warrants PIF use for the treatment of early pregnancy disorders.
背景/目的：成功怀孕需要母体对半同种异体/同种异体胚胎产生强制性耐受性，这涉及胚胎衍生信号。着床前因子（PIF）是一种由存活胚胎分泌的新型肽，其表达水平与胚胎发育相关，在循环中早期检测到PIF与良好的妊娠结局相关。PIF可增强子宫内膜容受性以促进胚胎着床。通过p53途径，它可增加滋养层细胞侵袭，提高细胞存活率/免疫特权。在免疫未致敏的小鼠模型中，PIF还可减少自发性和脂多糖诱导的胎儿死亡。我们研究了PIF对人白细胞抗原（HLA-G、-E、-F和-C）基因表达的影响以及PIF对JEG-3绒毛膜癌细胞局部孕酮活性的影响。
使用单克隆抗体、流式细胞术和蛋白质印迹法检测PIF和孕酮（P4）对JEG-3细胞表面和细胞内HLA分子的影响。通过酶联免疫吸附测定法测定PIF和白细胞介素17对P4和细胞因子分泌的影响。使用二维凝胶染色，随后进行斑点分析、质谱分析和生物信息学分析，研究PIF和P4对JEG-3细胞蛋白质组的影响。
在细胞滋养层JEG-3细胞中，PIF以剂量和时间依赖性方式增加HLA-G、HLA-F、HLA-E和HLA-C的细胞内表达以及HLA-G、HLA-E和HLA-C的表面表达。对于HLA-E、-F，蛋白质印迹法也证实了上述结果。蛋白质组分析证实HLA-G、促耐受性叉头框蛋白3阳性调节性T细胞（Tregs）、凝血因子和补体调节因子增加。相反，PIF可减少PRDX2和热休克蛋白70以消除氧化应激和蛋白质错误折叠。PIF增强局部孕酮活性，增加类固醇分泌和受体蛋白。它还促进Th1/Th2细胞因子（白细胞介素10、白细胞介素1β、白细胞介素8、粒细胞巨噬细胞集落刺激因子和转化生长因子β1）的分泌，从而改善母体信号传导。
PIF可通过增强HLA分子的表达和放大内源性孕酮活性来产生促耐受性环境。一项针对自身免疫性疾病的快速通道临床试验已圆满完成。所获得的数据证明PIF可用于治疗早期妊娠疾病。
