Department of General Surgery, Tianjin Medical University General Hospital, 154 Anshan Road, Heping District, Tianjin, 300052, China.
Tianjin General Surgery Institute, Tianjin, China.
Stem Cell Res Ther. 2021 Oct 15;12(1):541. doi: 10.1186/s13287-021-02604-2.
Autoimmune hepatitis (AIH) is a T cell-mediated immune disease that activates abnormally against hepatic antigens. We have previously reported that endometrial regenerative cells (ERCs) were a novel source of adult stem cells, which exhibiting with powerful immunomodulatory effects. Galectin-9 (Gal-9) is expressed in ERCs and plays an important role in regulating T cell response. This study aims to explore the role of ERCs in attenuation of AIH and to determine the potential mechanism of Gal-9 in ERC-mediated immune regulation.
ERCs were obtained from menstrual blood of healthy female volunteers. In vitro, ERCs were transfected with lentivirus vectors carrying LGALS9 gene and encoding green fluoresce protein (GFP-Gal-9-LVs) at a MOI 50, Gal-9 expression in ERCs was detected by ELISA and Q-PCR. CD4 T cells isolated from C57BL/6 mouse spleen were co-cultured with ERCs. The proliferation of CD4 T cells was detected by CCK-8 kit and the level of Lck/zap-70/LAT protein was measured by western blot. Furthermore, AIH was induced by ConA in C57BL/6 mice which were randomly assigned to untreated, unmodified ERC-treated and Gal-9 high-expressing ERC-treated groups. Histopathological score, liver function, CD4/CD8 cell infiltration in liver tissues, the proportion of immune cells in the spleen and liver, and ERC tracking were performed accordingly to assess the progression degree of AIH.
After transfecting with GFP-Gal-9-LVs, Gal-9 expression in ERCs was significantly increased. Additionally, Gal-9 high-expressing ERCs effectively inhibited CD4 T cell proliferation and downregulated CD4 T cell active related proteins p-Lck/p-ZAP70/p-LAT in vitro. Furthermore, treatment with Gal-9 high-expressing ERCs restored liver function, ameliorated liver pathological damage, inhibit CD4 and CD8 T cell proliferation and suppress Th1 and Th17 cell response in the hepatitis mice. In addition, Gal-9 high-expressing ERCs further markedly enhanced the level of IL-10 but reduced the levels of IFN-γ, TNF-α, and IL-4 in mouse sera and liver. Cell tracking also showed that ERCs could migrate to the damaged liver organs.
The results suggested that Gal-9 was an essential modulator, which was required by ERCs in regulating T cell response and attenuating ConA-induced experimental hepatitis. And also, it provides a novel idea for the clinical treatment of AIH.
自身免疫性肝炎（AIH）是一种 T 细胞介导的免疫性疾病，其异常激活针对肝抗原。我们之前报道过，子宫内膜再生细胞（ERCs）是一种新的成体干细胞来源，具有强大的免疫调节作用。半乳糖凝集素-9（Gal-9）在 ERCs 中表达，并在调节 T 细胞反应中发挥重要作用。本研究旨在探讨 ERCs 在减轻 AIH 中的作用，并确定 Gal-9 在 ERC 介导的免疫调节中的潜在机制。
从健康女性志愿者的月经血中获得 ERCs。在体外，将携带 LGALS9 基因并编码绿色荧光蛋白（GFP-Gal-9-LVs）的慢病毒载体以 MOI50 转染 ERCs，通过 ELISA 和 Q-PCR 检测 ERCs 中 Gal-9 的表达。将从小鼠脾脏分离的 CD4 T 细胞与 ERCs 共培养。通过 CCK-8 试剂盒检测 CD4 T 细胞的增殖，通过 Western blot 检测 Lck/zap-70/LAT 蛋白的水平。此外，用 ConA 诱导 C57BL/6 小鼠的 AIH，将其随机分为未处理组、未经修饰的 ERC 处理组和高表达 Gal-9 的 ERC 处理组。进行组织病理学评分、肝功能、肝组织中 CD4/CD8 细胞浸润、脾和肝中免疫细胞的比例以及 ERC 追踪，以评估 AIH 的进展程度。
转染 GFP-Gal-9-LVs 后，ERC 中 Gal-9 的表达明显增加。此外，高表达 Gal-9 的 ERCs 可有效抑制 CD4 T 细胞增殖，并下调体外 CD4 T 细胞活性相关蛋白 p-Lck/p-ZAP70/p-LAT。此外，用高表达 Gal-9 的 ERCs 处理可恢复肝功能，改善肝病理损伤，抑制肝炎小鼠 CD4 和 CD8 T 细胞增殖，并抑制 Th1 和 Th17 细胞反应。此外，高表达 Gal-9 的 ERCs 还可显著提高小鼠血清和肝脏中 IL-10 的水平，降低 IFN-γ、TNF-α和 IL-4 的水平。细胞追踪还显示 ERCs 可迁移到受损的肝器官。
结果表明，Gal-9 是 ERCs 调节 T 细胞反应和减轻 ConA 诱导的实验性肝炎所必需的重要调节剂。这为 AIH 的临床治疗提供了新的思路。
