miR146b调节M1型巨噬细胞激活而抑制结肠炎进展的分子机制

Interleukin-10 (IL-10) is a multi-cell sourced and multifunctional cytokine in regulating growth and differentiation of immune cells involved in inflammatory responses, and is regarded as an important antiinflammatory and immunosuppressive factor. Accumulated evidence revealed that IL-10 has critical functions in many cell types including macrophages. However, the exact molecular mechanims of IL-10 in control of M1 macrophage activation and how IL-10 affects the development of inflammatory diseases is incompletely understood. Recently, we demonstrate that IL-10 induces microRNA 146b (miR-146b), which targets IRF5 resulting in the control of M1 macrophage activation by RNA immunoprecipitation assay and 3`UTR luciferase reporter assay. However, the molecular mechanisms involved in the regulation of miR-146b-IRF5 axis in macrophages, the contribution of miR-146b in the innate immune, the development of murine colitis and their roles in inflammatory bowel diseases (IBD) need to be further addressed. In this project, we will further investigate how miR-146b regulates the release of proinflammatory cytokines and anti-inflammatory cytokine, and affects the T helper cell activation. In addition, we will evaluate if miR-146b can severe as a small molecular drug to treat the endoxin shock, Listeria monocytogenes infection and ameliorates colitis development in IL-10 deficient mice. In IBD patients, we will screen the IL-10R2 mutation in IBD patient samples to analyze miR-146b expression. Furthermore, we also will detect the differenation of monocytes derived from peripheral blood with or without IL-10R2 mutation by transfecting miR-146b mimic or inhibitor. Our hypothesis is that miR-146b negatively controls M1 macrophage differentiation in inflammation by regulating IRF5 activation and targeting miR-146b would lead to the development of novel therapy for autoimmune/inflammatory diseases.

白介素10(interleukin 10, IL-10)是一种多细胞源、多功能的细胞因子, 是目前公认的炎症与免疫抑制因子。近年来对IL-10的功能和调节机制有了更深入研究。但是目前为止,IL-10负反馈调节M1巨噬细胞激活的机制以及控制炎症性肠病发展过程的机制尚不清楚。我们的前期研究发现miR-146b在小鼠M1巨噬细胞激活过程起到重要作用,并且通过其靶基因IRF5调控 M1巨噬细胞相关特征性基因的表达。在本项目中,我们将在原有工作基础上,进一步深入研究小鼠巨噬细胞中IL-10诱导miR-146b表达的分子机制,阐明miR-146b对M1巨噬细胞生物学特性影响,包含对自身细胞因子和对辅助性T细胞的影响,以及评估miR-146b模拟物作为小分子药物在治疗小鼠李斯特菌感染、内毒素休克以及自发性肠炎中的治疗效果,为以miR-146b模拟物小分子药物治疗相关疾病提供实验依据和理论支持。

白介素10(interleukin 10, IL-10)是目前公认的炎症与免疫抑制因子，我们的前期研究结果显示IL-10缺失可促进M1巨噬细胞的分化，并易于激活Th1细胞。但IL-10负反馈调节M1巨噬细胞激活的机制以及控制炎症性肠病发展过程的机制仍有待于进一步研究。我们的前期研究发现miR-146b在小鼠M1巨噬细胞激活过程起到重要作用，并且通过其靶基因IRF5调控 M1 巨噬细胞相关特征性基因的表达。在本项目从细胞水平、动物水平并结合临床样本开展研究，进一步深入探讨了小鼠巨噬细胞中IL-10诱导miR-146b表达的分子机制,，研究了miR-146b对M1巨噬细胞生物学特性影响, 包含对自身细胞因子和对辅助性T细胞的影响，明确了miR-146b模拟物作为小分子药物在治疗小鼠李斯特菌感染、内毒素休克以及自发性肠炎中的治疗效果。.主要研究结果包括：我们发现IL-10和Rag1双重敲除小鼠（IL-10−/−/Rag1−/−）自发地发生结肠炎并伴有M1巨噬细胞相关信号分子表达升高，提示IL-10可调控M1巨噬细胞参与结肠炎的发展。同时，用IL-10或LPS刺激可诱导巨噬细胞中表达miR-146b，而在IL-10缺失的巨噬细胞中miR-146b表达受损。此外，RNA免疫沉淀实验表明，miR-146b和IRF5位于同一miRNA诱导沉默复合物（miRNA-induced silencing complex，miRISC）中。此外，miR-146b模拟物显著降低IRF5 3′UTR活性及LPS诱导的IRF5蛋白表达。我们构建了miR-146b缺陷小鼠，发现miR-146b缺陷小鼠体内M1巨噬细胞极化增强，而且miR-146b−/−巨噬细胞更能促进Th1细胞激活。最后，用miR146b模拟物治疗可显著抑制M1巨噬细胞的活化，并改善结肠炎和内毒素休克的发生发展。以上结果表明IL-10-miR-146b-IRF5轴在调控巨噬细胞活化过程中具有重要作用，并揭示了miR-146b在免疫反应调节中的重要作用,为以miR-146b模拟物作为小分子药物治疗相关疾病提供实验依据和理论支持。