IRAK-M基因介导巨噬细胞亚群参与心肌梗死后瘢痕修复的机制研究

Exaggerated and prolonged inflammation after myocardial infarction (MI) accelerates cardiac rupture and left ventricular remodeling by impairing scar healing. Inflammatory pathways may present a therapeutic target to prevent post-MI heart failure. Monocytes/macrophages are critical modulating cells in inflammatory responses. Two subpopulations of macrophages, defined as Ly6Chigh and Ly6Clow cells, have been proved to play distinct roles in this pro-and anti-inflammatory processes following cardiac injuries. Ly6Chigh cells exhibit pro-inflammatory properties, while the Ly6Clow subset shows significant activities on inflammatory suppression, angiogenic enhancement and collagen synthesis. However, the mechanisms why Ly6Clow macrophages exert an protective effect on injured tissues are largely unknown.Toll-Like receptor (TLR) and interleukin (IL)-1 signaling has been demonstrated to be critically involved in the post-infarction negative inflammatory responses. Our preliminary data showed that interleukin-1 receptor-associated kinase (IRAK)-M and type II IL1 receptor (IL1RII), both known as endogenous inhibitory moleculars of TLR/IL pathways, were upregulated in Ly6Clow cells after MI. The depletion of IRAK-M gene resulted in a reduced anti-inflammatory function of Ly6Clow cells. Therefore, we hypothesized that the inflammatory suppression of Ly6Clow macrophages may mediated by TLR/IL inhibitory signals. In the present study, IRAK-M gene knockout mice will be used to analyze the TLR/IL1R pathways and possible mechanisms by which Ly6Clow macrophages act as both a negative regulator of inflammation and a positive mediator of scar formation. Collagen formation and cross-linking will be measured by co-culturing ly6Clow macrophages and myo-fibroblasts isolated from infarcts after 7 days of ischemia and reperfusion procedures. Anti-inflammatory properties will be thoroughly investigated in Ly6Clow and myo-fibroblasts cell lines which over-express IRAK-M. Specific and timely clearance of macrophages by clodronate liposomes will be performed to illustrate the modulating effect of IRAK-M in Ly6Clow macrophages for cardiac remodeling after myocardial infarction. Our study will demonstrate for the first time the protective functions of Ly6Clow macrophages through endogenous inflammatory factor IRAK-M in infarcted hearts and may identify specific cellular effectors and mechanisms accelerating cardiac repair.

心肌梗死后过强的炎症反应导致瘢痕形成受阻，造成心脏破裂或心肌重塑。心肌梗死修复期Ly-6Clow细胞高表达白细胞介素相关激酶M（IRAK-M），并具有促胶原形成作用，但机制不清。本研究采用小鼠冠状动脉缺血再灌注模型，分离野生型和IRAK-M基因敲除小鼠心梗修复期心肌组织内Ly6Clow细胞，对TLR/IL1R信号通路的各阶段及其可能调节机制进行系统研究。并将心肌成纤维细胞与Ly6Clow细胞共培养，研究IRAK-M基因缺失对胶原形成、交联的影响。构建小鼠IRAK-M过表达的Ly6Clow细胞系和成纤维细胞系，对比各类细胞抑炎特性及TLR/IL1R信号通路的差异，寻找可能调控炎症向瘢痕转化的关键因子。通过巨噬细胞清除技术，在体研究心梗修复期高表达IRAK-M的Ly6Clow细胞亚群对心脏重塑的影响。本研究的意义在于找到心梗后促瘢痕修复的特异性靶细胞和相应机制，为改善心梗预后提供新的治疗位点。

心梗后过强的炎症反应会导致不良心肌重塑，白细胞介素受体相关激酶M（IRAK-M） 在单核巨噬细胞 Toll 样蛋白受体（TLR）介导的炎症信号通路中起抑制作用。本研究采用小鼠冠状动脉结扎模型，分离野生型和IRAK-M基因敲除小鼠心梗修复期心肌组织内Ly6Clow细胞，对TLR/IL1R信号通路可能的调节机制进行系统研究；并与成纤维细胞进行共培养研究IRAK-M基因缺失对胶原形成、交联的影响；进一步分离鉴定心梗修复期肌成纤维细胞，研究IRAK-M在肌成纤维细胞激活中的功能和调控机制；构建小鼠IRAK-M过表达的单核/巨噬细胞系和成纤维细胞系，对比各类细胞抑炎特性及TLR/IL1R信号通路的差异，寻找可能调控炎症向瘢痕转化的关键因子。研究发现对于Ly6Clow/int 细胞亚群， IRAK-M-/-小鼠较野生型小鼠表达 IL1β、TGFβ、MMP2 水平较低， 表达 TNFα、MCP-1、IL-10 水平较高，表达IRAK-1、IRAK-4、TRAF6、 IKK-γ、ERK、NF-κB、p38水平较低，而IκBα表达较高；相比于野生单核/巨噬细胞，与 IRAK-M 高表达单核/巨噬细胞体外共培养的成纤维细胞表达α-SMA、I 型胶原、III型胶原显著增加；较野生型小鼠，IRAK-M-/-小鼠心梗后7天肌成纤维细胞的收缩能力和α-SMA的表达量均下降，MMPs明显升高（以MMP9为主），而TIMPs的表达量明显下降（以TIMP1为主），给予TGF-β刺激后MMP9表达量下降，TIMP1表达量上升；进一步研究发现IRAK-M-/-小鼠肌成纤维细胞中的iNOS表达明显增加，而给予TGF-β1刺激后，iNOS表达量下降，与MMP9的变化趋势基本一致；心梗修复期肌成纤维细胞中NF-κB信号通路和TGF-β信号通路中的信号分子均有改变，可能涉及IRAK-M参与心梗后心肌修复的调控机制。IRAK-M 缺失影响心梗修复期 Ly6Clow 细胞亚群炎症特性，单核/巨噬细胞 IRAK-M 高表达可能影响成纤维细胞的胶原表达，肌成纤维细胞中IRAK-M通过抑制iNOS信号，下调MMP9并上调TIMP1，来促进α-SMA表达，从而调控心梗修复期纤维化过程。本研究有助于找到心梗后促瘢痕修复的特异性靶细胞和相应机制，为改善心梗预后提供新的治疗位点。