MSC抑制ALI过度炎症反应的新机制——Wnt/β-catenin 信号途径活化诱导调节性DC

Severely uncontrolled inflammation in acute lung injury (ALI) is the main pathophysiologic characteristic and the key treatment target. Mesenchymal stem cells (MSCs) as a promising treatment for ALI has been confirmed recently powerful to inhibit the inflammation and thus ameliorating the pulmonary impairment. Therefore exploration of the underling mechanisms, which are still unclear, is critical for improving their therapeutic effects. Dendritic cells (DCs), one of the most important immunocytes for human, the phenotype and function of which can influence the early inflammation and tissue repair in ALI. Regulatory DCs (DCreg), one subset of DCs to induce immune tolerance, has potential anti-inflammatory effect in ALI and can be induced by MSC from immature or mature DC in vitro. It was found that the activation of β-catenin/wnt signaling in DC was involved in the generation and immunotolerance of DCreg.So, it can be hypothesized that MSCs influencing the generation and functions of DCreg through the activation of their β-catenin/Wnt pathway in vivo partly mediate their anti-inflammation and protective effect in ALI. So, in this study we will firstly evaluate the effect of MSC on DC, DCreg and T cells in lung or spleen of ALI mice. Then the mechanisms of β-catenin/ Wnt pathway in the induction of DCreg via MSCs was explored in a co-culture system consisted of MSCs and mature DCs in vitro.Finally the effect of MSCs with Wnt3a or DKK 1 transfection in ALI mice was evaluated through the level of DCreg, T cells, Treg, inflammatory cytokines, neutrophile infiltration, pulmonary pathology and lung wet weight after the administration into ALI mice.In this way, this study may contribute to the new and effective treatment to ALI.

失控的炎症反应是ALI发生发展的根本及治疗靶点，MSC能有效抑制ALI炎症，但机制尚不清楚。我们前期发现DC影响ALI早期炎症反应，其中具有免疫抑制作用的调节性DC在抑制ALI炎症中具有潜在作用，MSC可诱导调节性DC产生，但调节性DC介导的MSC抑制ALI炎症反应的作用及机制亟待阐明。基于此及经典Wnt途径可能促进调节性DC形成，我们假设经典Wnt途径介导了MSC诱导调节性DC产生，继而抑制ALI炎症反应。本项目首先在体证实MSC对ALI小鼠调节性DC的影响；然后在体外MSC诱导调节性DC产生的基础上，激活或抑制经典Wnt途径以明确该通路在MSC诱导调节性DC产生和免疫耐受中的作用及分子机制；最后采用慢病毒转染，建立高表达Wnt3a或DKK1的MSC，并考察其对ALI小鼠DC、T细胞、炎症等的影响，以阐明该通路在调节性DC介导MSC抑制ALI炎症反应中的意义，为改善ALI治疗提供新思路。

间充质干细胞（MSC）能有效抑制急性肺损伤（ALI）失控的炎症反应，是其治疗ALI的关键，但机制尚不清楚。MSC可诱导调节性树突状细胞（DCreg）产生，结合我们前期发现树突状细胞（DC）对ALI早期炎症反应具有重要调节作用，因而具有免疫抑制作用的调节性DC可能在介导MSC抑制ALI炎症反应中起重要作用。为证实该假设并进一步探讨MSC诱导调节性DC产生的机制，(1) 我们通过体外MSC与成熟DC（mDC）共培养，发现导致DC表达MHCII及共刺激分子下降，吞噬能力增强，刺激T淋巴细胞增殖能力减低，同时分泌抑制性炎症因子增多；(2) 同时通过在体实验观察MSC注入LPS气道注射诱导ALI小鼠后对肺及脾脏DC亚群的影响，发现MSC能够减少肺及脾脏中DCs的数量，抑制其成熟，使脾脏DC吞噬功能增强，而诱导T淋巴细胞增殖的能力下降；(3) 我们将体外诱导分化的DCreg注入ALI小鼠体内，发现DCreg和MSC一样，均能减轻LPS诱导的ALI小鼠肺损伤和肺血管内皮肺泡上皮通透性，减轻肺泡灌洗液中炎症细胞数量和促炎因子水平，而上调抗炎因子；(4) 我们在体外MSC与mDC共培养诱导DCreg生成基础上，检测DC中Wnt通路变化，发现MSC经典Wnt信号通路相关蛋白水平上调，同时用DKK1抑制DC经典Wnt信号通路，发现抑制经典Wnt信号通路后，DC表达共刺激分子及分泌促炎因子增加，而分泌抑炎因子减少，吞噬能力下降，刺激CD4+T淋巴细胞增殖能力增强。综上可以证明：(1) 体外MSC共培养及ALI小鼠在体注入MSC均能诱导DC向DCreg分化；(2) MSC通过诱导DCreg的生成减轻ARDS小鼠肺部炎症反应；(3) 经典Wnt信号通路活化是促进mDC向DCreg分化的重要机制。