Department of General Surgery, Tianjin Medical University General Hospital, 154 Anshan Road, Heping District, Tianjin, 300052, China.
Tianjin General Surgery Institute, Tianjin Medical University General Hospital, Tianjin, 300052, China.
Stem Cell Res Ther. 2023 Sep 29;14(1):277. doi: 10.1186/s13287-023-03505-2.
As a kind of mesenchymal-like stromal cells, endometrial regenerative cells (ERCs) have been demonstrated effective in the treatment of Concanavalin A (Con A)-induced hepatitis. However, the therapeutic mechanism of ERCs is not fully understood. Ecto-5`-nucleotidase (CD73), an enzyme that could convert immune-stimulative adenosine monophosphate (AMP) to immune-suppressive adenosine (ADO), was identified highly expressed on ERCs. The present study was conducted to investigate whether the expression of CD73 on ERCs is critical for its therapeutic effects in Con A-induced hepatitis.
ERCs knocking out CD73 were generated with lentivirus-mediated CRISPR-Cas9 technology and identified by flow cytometry, western blot and AMPase activity assay. CD73-mediated immunomodulatory effects of ERCs were investigated by CD4 T cell co-culture assay in vitro. Besides, Con A-induced hepatitis mice were randomly assigned to the phosphate-buffered saline treated (untreated), ERC-treated, negative lentiviral control ERC (NC-ERC)-treated, and CD73-knockout-ERC (CD73-KO-ERC)-treated groups, and used to assess the CD73-mediated therapeutic efficiency of ERCs. Hepatic histopathological analysis, serum transaminase concentrations, and the proportion of CD4 T cell subsets in the liver and spleen were performed to assess the progression degree of hepatitis.
Expression of CD73 on ERCs could effectively metabolize AMP to ADO, thereby inhibiting the activation and function of conventional CD4 T cells was identified in vitro. In addition, ERCs could markedly reduce levels of serum and liver transaminase and attenuate liver damage, while the deletion of CD73 on ERCs dampens these effects. Furthermore, ERC-based treatment achieved less infiltration of CD4 T and Th1 cells in the liver and reduced the population of systemic Th1 and Th17 cells and the levels of pro-inflammatory cytokines such as IFN-γ and TNF-α, while promoting the generation of Tregs in the liver and spleen, while deletion of CD73 on ERCs significantly impaired their immunomodulatory effects locally and systemically.
Taken together, it is concluded that CD73 is critical for the therapeutic efficiency of ERCs in the treatment of Con A-induced hepatitis.
作为一种间充质样基质细胞，子宫内膜再生细胞（ERCs）已被证明在治疗伴刀豆球蛋白 A（Con A）诱导的肝炎方面有效。然而，ERC 治疗的机制尚不完全清楚。外核苷酸酶（CD73）是一种可以将免疫刺激性单磷酸腺苷（AMP）转化为免疫抑制性腺苷（ADO）的酶，已被鉴定在 ERC 上高表达。本研究旨在探讨 ERC 上 CD73 的表达是否对其在 Con A 诱导的肝炎中的治疗效果至关重要。
利用慢病毒介导的 CRISPR-Cas9 技术生成敲除 CD73 的 ERCs，并通过流式细胞术、Western blot 和 AMPase 活性测定进行鉴定。通过体外 CD4 T 细胞共培养试验研究 ERC 中 CD73 的免疫调节作用。此外，将 Con A 诱导的肝炎小鼠随机分为磷酸盐缓冲液处理组（未处理）、ERC 处理组、负性慢病毒对照 ERC（NC-ERC）处理组和 CD73 敲除 ERC（CD73-KO-ERC）处理组，用于评估 ERC 中 CD73 介导的治疗效率。进行肝组织病理学分析、血清转氨酶浓度以及肝和脾中 CD4 T 细胞亚群的比例，以评估肝炎的进展程度。
体外鉴定出 ERC 上 CD73 的表达可有效将 AMP 代谢为 ADO，从而抑制常规 CD4 T 细胞的激活和功能。此外，ERC 可显著降低血清和肝转氨酶水平，减轻肝损伤，而 ERC 上 CD73 的缺失则减弱了这些作用。此外，基于 ERC 的治疗方法可减少肝内 CD4 T 和 Th1 细胞的浸润，并减少系统 Th1 和 Th17 细胞的数量以及 IFN-γ和 TNF-α等促炎细胞因子的水平，同时促进肝和脾中 Treg 的生成，而 ERC 上 CD73 的缺失则显著削弱了其局部和全身的免疫调节作用。
综上所述，CD73 对于 ERC 在治疗 Con A 诱导的肝炎中的治疗效果至关重要。
