TLR3预刺激的间充质干细胞通过免疫代谢对克罗恩病肠道纤维化的调节及机制研究

Intestinal fibrosis is the starting link of intestinal obstruction, which seriously affects long-term prognosis and quality of life in patients with Crohn’s diseases (CD). Lacking safe and effective anti-fibrosis means limits the efficacy of intestinal obstruction treatment. Mesenchymal stem cells (MSCs) transplantation is expected to be a new CD therapy. Our early founding showed that Toll-like receptor 3 (TLR3) pre-condition MSCs (TLR3-MSCs) optimized the efficacy of MSCs and alleviated acute colitis of trinitrobenzesulfonic acid (TNBS)-induced CD mice model through immunoregulation. However, there is insufficient evidence for the efficacy of TLR3-MSCs in intestinal fibrosis treatment. We recently confirmed that TLR3-MSCs transplantation alleviated the intestinal obstruction in chronic CD mice models. The proportion of CD4+T lymphocyte reduced and the expression of pro-fibrotic cytokines was decreased. Recent studies have found that the immune balance could be affected by nutrient metabolism of immune cells. We also found that TLR3-MSCs transplantation affected the glycometabolism of CD4+T cells in chronic CD mice models. Based on our previous data, we proposed to test the hypothesis that TLR3-MSCs transplantation alleviates intestinal fibrosis in CD through the rebuilt of immunologic balance which was mediated by immunometabolism. We will test our hypothesis by applying TLR3-MSCs in TNBS-induced colitis mice models and in-vitro coculture system, then analyzing the regulation of TLR3-MSCs in intestinal fibrosis treatment. We aim to delineate the mechanism of TLR3-MSCs in alleviating intestinal fibrosis. As such, the anticipated results will provide novel therapeutic approaches for CD, explore new target of therapeutic intervention and improve the long-time clinical outcomes of CD patients.

肠壁纤维化是肠梗阻等并发症的起始环节，严重影响克罗恩病(CD)中远期疗效。缺乏安全有效的抗纤维化手段是攻克该“顽症”的瓶颈。间充质干细胞（MSCs）移植有望成为新的CD治疗手段。前期摸索发现，TLR3预刺激能够优化MSCs疗效，通过免疫调节治疗急性结肠炎小鼠，但对肠壁纤维化的疗效证据不足。我们新近证实，TLR3-MSCs移植改善慢性CD小鼠肠梗阻，治疗过程中下调CD4+T细胞比例，降低促纤维化细胞因子表达。近年研究发现，免疫细胞的物质代谢影响免疫平衡。我们也发现TLR3-MSCs移植影响小鼠CD4+T细胞的糖代谢水平。据此提出假说：TLR3-MSCs通过免疫代谢调节CD4+T细胞免疫平衡，改善肠纤维化。本研究拟在动物、细胞、分子多层面，以慢性结肠炎小鼠为研究载体，应用体外共培养体系，阐明TLR3-MSCs改善CD肠纤维化的疗效及机制，预期为CD纤维化治疗提供新的治愈性手段并深入了解其机制。

肠纤维化是克罗恩病的常见并发症，其发生与α-SMA(+)肌成纤维细胞产生过量细胞外基质有关。间充质干细胞（MSCs）或可通过免疫调节改善纤维化。但MSCs体内生存时间短等限制了临床疗效。TLRs被激活的MSCs，其体内存活时间、增殖、迁移及免疫调节能力均可提升。本研究建立TNBS诱导的小鼠慢性结肠炎模型，探讨TLR3预处理的MSCs（TLR3-MSCs）是否较未处理的MSCs（U-MSCs）更有效地改善小鼠肠道慢性炎症和纤维化，并探索其可能机制。TNBS诱导慢性结肠炎小鼠模型，其体重增长缓慢，死亡率和DAI较对照组明显升高，并出现结肠缩短、炎症相关组织学变化评分升高、黏膜层及黏膜下层胶原大量沉积的病理表现。慢性结肠炎小鼠的结肠组织中多种纤维化相关基因异常表达，血清和结肠组织中多种纤维化相关细胞因子异常分泌。TLR3-MSCs较U-MSCs更显著地改善慢性结肠炎小鼠的低体重、高死亡率和高疾病活动指数，并可有效减轻结肠长度变短和肠黏膜慢性炎症。TLR3-MSCs可更明显地减轻模型小鼠结肠黏膜的胶原沉积、改善结肠收缩功能，并显著抑制纤维化相关基因Collagen Ⅰ、Collagen Ⅲ、MMP1、MMP2、MMP3和α-SMA表达异常增多，以及TIMP1表达异常减少。此外，TLR3-MSCs较U-MSCs更显著地抑制模型小鼠血清中促纤维化因子IL-13、IL-17A增多和抗纤维化因子IL-10减少，并促进血清和结肠组织中抗纤维化因子IFNγ的生成，下调TNF-α和TGF-β的表达。流式细胞术分析各组小鼠脾脏和肠系膜淋巴结CD4+细胞显示，与TNBS组相比，U-MSCs治疗组和TLR3-MSCs治疗组均可下调Th17细胞亚群的比例，并上调Tregs细胞亚群的比例，且TLR3-MSCs治疗对Th17和Tregs细胞亚群比例的调节较U-MSCs更为显著。与iTregs细胞共培养的小鼠肠纤维母细胞，其α-SMA(+)细胞的比例明显低于未共培养的纤维母细胞，提示iTregs细胞可显著抑制小鼠结肠α-SMA(-)成纤维细胞向α-SMA(+)肌成纤维细胞转化。本研究首次发现TLR3-MSCs通过调节Th17及Tregs细胞亚群分化，较U-MSCs更好地改善小鼠慢性结肠炎及肠纤维化，这可能与Tregs抑制α-SMA(-)成纤维细胞向α-SMA(+)肌成纤维细胞转化有关。