IRF3-IL27-IL17信号轴调控移植肾缺血再灌注损伤及修复的机制

In kidney transplantation, ischemia-reperfusion (IR) injury has been clearly recognized as the key cause of delayed graft function but also as a cause of acute and chronic rejection. To date, no effective treatment is available. Toll like receptors (TLR) signaling plays pivotal role in mediating kidney IR injury. While IRF3-IL27-IL17 axis is an important branch of TLR signaling pathways, its function in kidney IR injury remains undetermined. We have reported that IRF3-dependent events downstream of TLR4 control the IL-23/IL-17 axis in the liver and this regulatory role of IRF3 is relevant to liver IR injury. As previous studies always focus on the early stage of kidney IR injury and less care about the repair process. We intend to use syngeneic kidney transplantation cold ischemia model and bilateral kidney warm ischemia model, to study the regulatory mechanism of IRF3-IL27-IL17 in the full process of kidney IR injury and repair. IRF3, TLR4, TRIF, IFAR and IL-17F/IL-17C deficient mice will be used for in vivo study, while primary tubular epithelium for in vitro study. Techniques including flow cytometry analysis/sorting and confocal immunofluorescence will be applied. Chimeric mouse will be generated to study the IRF3-IL27-IL17 signal in parenchymal and none parenchymal cells. This study may provide new therapeutic targets for future clinical graft kidney IR injury treatment.

缺血再灌注（IR）损伤严重影响移植肾功能恢复，目前缺乏有效治疗方法。TLR信号通路在肾脏IR损伤过程中发挥关键作用，IRF3-IL27-IL17轴是TLR下游的一个重要分支，但其在肾脏IR损伤中的作用仍不清楚。申请人曾首次报道了该信号轴负相调控肝脏IR后晚期致伤作用。鉴于前期研究对侧重肾脏IR的早期致伤机制，且对修复机制重视不足。本项目拟采用IRF3、TLR4、TRIF、IFAR、IL-17F/IL-17C等基因缺陷小鼠，联用小鼠同系肾移植冷缺血损伤模型和双肾IR热缺血损伤模型，通过嵌合小鼠、细胞过继回输、流式细胞分析/分选以及免疫荧光共聚焦技术进行研究，并在体外采用野生或缺陷的肾小管上皮细胞模拟IR，通过下调IL27/IL17表达或加入细胞因子刺激，探究肾脏实质及非实质细胞的IRF3-IL27-IL17信号轴在移植肾IR损伤与修复的调控机制，为临床治疗移植肾IR损伤提供新的治疗靶点。

本课题通过分别采用小鼠肾脏热缺血IR模型和肾移植冷缺血IR模型研究了IRF3信号通路在IR损伤及修复中的作用及潜在机制。在IR后IRF3表达升高。IRF3缺陷（IRF3-/-）小鼠在IR后存活率低于野生型（WT）组。在IR后的前48hIRF3-/-小鼠和WT小鼠无明显差异，但在后期损伤明显加重，且伴有较多的单个核细胞在肾脏间质浸润。炎症因子分析发现IRF3-/-小鼠在IR之后TNF-α先呈下调趋势，随后逐渐升高，IL-1β、IL-23、IL-17A、IL-17C均较WT小鼠显著升高。体外模拟IR结果亦与之一致。IRF3-/-小鼠单核巨噬细胞、T细胞和中性粒细胞等炎症细胞在IR后肾组织中浸润明显。其浸润细胞的凋亡程度水平显著高于WT组小鼠。IRF3-/-小鼠在IR之后CD3+T细胞浸润增加，其中浸润的CD4/CD8比例下调。但IRF3-/-小鼠肾间质的CD4+T细胞计数仍然显著高于WT组。这些浸润的Th细胞70%以上为CD44highCD62L-的记忆性T细胞。IRF3-/-小鼠在IR之后浸润的Th17细胞显著高于WT小鼠。在再灌注后7d，IRF3-/-小鼠的TEC增殖情况显著低于WT小鼠，而凋亡情况显著高于WT小鼠。骨髓嵌合小鼠研究发现实质和非实质细胞的IRF3信号通路均参与肾脏IR后的炎症反应调控。抗体阻断IL-17A之后，WT及IRF3-/-小鼠在IR后的肾脏损伤均有显著减轻，IRF3-/-小鼠肾功能和病理损伤较注射Control-Ig的对照组明显改善。延伸研究发现IL17C-/-小鼠移植后存活时间显著低于野生型小鼠。IL17C-/-小鼠移植肾损伤较野生型小鼠明显加重。IL17C-/-小鼠移植肾组织中Granzyme B、IFN-γ 、TNF-α、IL-6的mRNA表达水平显著高于WT小鼠。IL17C-/-小鼠移植肾组织浸润的中性粒细胞和单核细胞浸润较野生型小鼠明显增加，而CD45+Ly6Chi F4/80+巨噬细胞浸润无明显变化。综上，本课题研究结果表明IRF3通过调控IL-23和IL17信号通路介导的炎症因子的表达以及炎症细胞浸润，调控肾脏IR后的损伤和修复过程，肾脏实质和非实质细胞的IRF3通过均参与这种调控。阻断IL-17A可以逆转IRF3缺陷所造成的损伤加重。