Table_4_Pregnancy-Induced Changes in microRNA Expression in Multiple Sclerosis.xlsx

Pregnancy affects the disease course in multiple sclerosis (MS), particularly in the third trimester, where the relapse rate is reduced by as much as two thirds. This study aimed at identifying changes in microRNA (miRNA) and immune cell phenotypes in pregnant MS patients. Discovery and validation studies to detect differentially expressed miRNAs were performed with quantitative real-time PCR on peripheral blood mononuclear cells (PBMC). Flow cytometry analysis was performed on PBMC stained with antibodies directed against surface markers of antigen presenting cells (APCs), NK-cells, NKT cells, CD4+ and CD8+ T cells and subsets of these cell types, including PDL1 and PDL2 expressing subsets. RNA was extracted from whole blood, monocytes, and NK-cells to investigate expression and correlation between regulated miRNAs and mRNAs. In total, 15 miRNAs were validated to be differentially expressed between third trimester pregnant and postpartum MS patients (Benjamini-Hochberg false discovery rate from p = 0.03–0.00004). Of these, 12 miRNAs were downregulated in pregnancy and 6 of the 15 miRNAs were altered by more than ±2-fold (+2.99- to -6.38-fold). Pregnant MS patients had a highly significant increase in the percentage of monocytes and a decrease of NK-cells and myeloid dendritic cells compared to non-pregnant MS patients. We confirm previous reports of a relative increase in CD56-bright NK-cells and a decrease in CD56-dim NK-cells in third trimester of pregnancy and report an increase in non-committed follicular helper cells. PDL1 and PDL2 expression was increased in pregnant patients together with IL10. Also, in monocytes IL10, PDL1, and PDL2 were upregulated whereas miR-1, miR-20a, miR-28, miR-95, miR-146a, miR-335, and miR-625 were downregulated between pregnant and untreated MS patients. IL10, PDL1, and PDL2 were predicted targets of MS pregnancy-changed miRNAs, further supported by their negative correlations. Additionally, previously identified pregnancy-regulated mRNAs were identified as predicted targets of the miRNAs. PDL1 and PDL2 bind PD-1 expressed on T cells with an inhibitory effect on T-cell proliferation and increase in IL10 production. These results indicate that some of the effects behind the disease-ameliorating third trimester of pregnancy might be caused by changed expression of miRNAs and immunoregulatory molecules in monocytes.

妊娠可影响多发性硬化（multiple sclerosis, MS）的疾病进程，尤以妊娠第三期（third trimester）为显著，此阶段疾病复发率可降低多达三分之二。本研究旨在探究妊娠多发性硬化患者体内微小RNA（microRNA, miRNA）及免疫细胞表型的变化。研究通过对外周血单个核细胞（peripheral blood mononuclear cells, PBMC）进行实时定量聚合酶链反应（quantitative real-time PCR），开展了差异表达miRNA的筛选与验证实验。通过使用针对抗原呈递细胞（antigen presenting cells, APCs）、自然杀伤细胞（NK-cells）、自然杀伤T细胞（NKT cells）、CD4阳性及CD8阳性T细胞及其亚群（包括表达PDL1与PDL2的亚群）表面标志物的抗体对PBMC进行染色，随后开展流式细胞术分析。研究人员从全血、单核细胞及自然杀伤细胞中提取RNA，以探究受调控的miRNA与信使RNA（mRNA）之间的表达情况及相关性。最终，共计15种miRNA被验证在妊娠第三期多发性硬化患者与产后多发性硬化患者之间存在差异表达（本雅明-霍赫伯格错误发现率对应的p值介于0.03至0.00004之间）。其中12种miRNA在妊娠状态下呈下调表达，15种miRNA中有6种的表达变化幅度超过±2倍（变化范围为+2.99倍至-6.38倍）。与未妊娠的多发性硬化患者相比，妊娠多发性硬化患者的单核细胞占比显著升高，而自然杀伤细胞及髓系树突状细胞占比则出现下降。本研究证实了此前关于妊娠第三期CD56亮型自然杀伤细胞相对占比升高、CD56暗型自然杀伤细胞占比下降的报道，并新发现未定向滤泡辅助细胞占比出现升高。妊娠患者体内PDL1、PDL2及白细胞介素10（IL10）的表达均出现上调。此外，在单核细胞中，IL10、PDL1与PDL2均呈上调表达，而miR-1、miR-20a、miR-28、miR-95、miR-146a、miR-335及miR-625则呈下调表达，该差异存在于妊娠患者与未治疗多发性硬化患者之间。IL10、PDL1及PDL2被预测为妊娠相关多发性硬化患者体内差异表达miRNA的靶点，二者的负相关表达进一步佐证了这一结论。此外，此前已报道的妊娠调控mRNA也被鉴定为上述miRNA的预测靶点。PDL1与PDL2可与T细胞表面表达的PD-1结合，对T细胞增殖产生抑制作用，并促进IL10的产生。上述结果表明，妊娠第三期所带来的疾病改善效应，部分可能源于单核细胞内miRNA及免疫调节分子的表达变化。