Role of the interleukin-15/macrophage axis in the immune response of liver fibrosis

白细胞介素15/巨噬细胞轴在肝纤维化免疫反应中的作用

• 批准号: 329715688
• 负责人: Professor Dr. Jan G. Hengstler, since 6/2018
• 金额: --
• 依托单位: Forschungsbereich Toxikologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/329715688?language=en
• 关键词: Role; interleukin; 15; macrophage; axis

Inflammation has been recognized as a crucial aspect of liver fibrosis, however therapeutic approaches targeting cytokines for this condition have been so far unsuccessful. This failure is in part due to our incomplete understanding of the complex process of inflammation in liver fibrosis. Interleukin-15 (IL-15) is a potent cytokine with key functions in NK and NK-T cell physiology. Recently, it was reported that IL-15 receptor alpha (IL-15RA) KO mice showed enhanced susceptibility to liver fibrosis. However, our preliminary studies reveal an opposite response in IL-15 KO mice, showing reduced liver fibrosis compared to wild type mice. This controversy could be caused by the congenital defects in both IL-15 and IL-15RA constitutive KO mice. However, additional preliminary results support a pro-fibrotic role of IL-15. First, we observed a strong induction of IL-15 in primary hepatocytes stimulated with TGFb and detected several pro-fibrotic cytokines strongly induced by IL-15 in primary macrophages. Second, we observed induction of IL-15 in mouse and human fibrotic and cirrhotic liver. Therefore, our preliminary results strongly suggest IL-15 promotes fibrosis by inducing a pro-fibrotic phenotype in macrophages. Elucidating the complex role of the IL-15/macrophage axis in liver fibrosis requires a more refined knowledge of inflammation during fibrosis, including identification of cells expressing IL-15, Omics-based analysis of liver leukocytes, hepatocytes and non-parenchymal cells, and experimental in vivo conditions whereby deletion of IL-15 or IL-15RA is achieved, without altering the homeostatic milieu of liver leukocytes. In this application, we will approach this by first identifying the main cell source of IL-15 using IL-15-CFP reporter mice, and establishing correlations between IL-15 expression and the inflammatory microenvironment in liver fibrosis in mouse and human. Second, by analyzing the severity of liver fibrosis after tamoxifen-controlled, Cre-dependent deletion of IL-15 or IL-15RA in adult mice. Third, by transcriptomics analyses of hepatocytes, stellate cells, macrophages, NK and NK-T cells from fibrotic mice of control and IL-15 and IL-15RA-deleted. Finally, we will perform functional in vivo analyses of macrophages, NK and NK-T cells, using multiphoton intravital microscopy (i.e. motility, cytotoxicity towards myofibroblasts) by adoptive cell transfer of fluorescently-labeled cells into fibrotic mice (control and IL-15 or IL-15RA-deleted). In addition, we will use in vitro co-culture systems of macrophages, NK/NK-T cells and stellate cells to identify the key factors (i.e. secreted cytokines, surface molecules) mediating the pro-fibrotic effects of IL-15. We expect that our project will unveil the contribution and the mechanisms by which IL-15 influences immune responses in liver fibrosis, setting the basis for diagnostic and therapeutic applications focused on this novel molecular player in liver disease.

炎症已被认为是肝纤维化的一个重要方面，然而针对这种病症的细胞因子的治疗方法迄今为止尚未成功。这种失败部分是由于我们对肝纤维化中炎症的复杂过程的不完全理解。白细胞介素-15（IL-15）是一种有效的细胞因子，在NK和NK-T细胞生理学中具有关键功能。最近，据报道，IL-15受体α（IL-15 RA）KO小鼠显示出对肝纤维化的易感性增强。然而，我们的初步研究揭示了IL-15 KO小鼠的相反反应，与野生型小鼠相比，显示出肝纤维化减少。这种争议可能是由IL-15和IL-15 RA组成型KO小鼠的先天性缺陷引起的。然而，额外的初步结果支持IL-15的促纤维化作用。首先，我们在用TGF β刺激的原代肝细胞中观察到IL-15的强烈诱导，并在原代巨噬细胞中检测到IL-15强烈诱导的几种促纤维化细胞因子。第二，我们观察到IL-15在小鼠和人纤维化和硬化肝脏中的诱导。因此，我们的初步结果强烈表明IL-15通过在巨噬细胞中诱导促纤维化表型来促进纤维化。 阐明IL-15/巨噬细胞轴在肝纤维化中的复杂作用需要对纤维化过程中的炎症有更精确的了解，包括鉴定表达IL-15的细胞，基于Omics的肝白细胞、肝细胞和非实质细胞分析，以及在不改变肝白细胞的稳态环境的情况下实现IL-15或IL-15 RA缺失的体内实验条件。在本申请中，我们将首先使用IL-15-CFP报告小鼠鉴定IL-15的主要细胞来源，并建立小鼠和人类肝纤维化中IL-15表达与炎症微环境之间的相关性。第二，通过分析成年小鼠中他莫昔芬控制的、Cre依赖的IL-15或IL-15 RA缺失后肝纤维化的严重程度。第三，通过转录组学分析来自对照和IL-15和IL-15 RA缺失的纤维化小鼠的肝细胞、星状细胞、巨噬细胞、NK和NK-T细胞。最后，我们将通过将荧光标记的细胞过继细胞转移到纤维化小鼠（对照和IL-15或IL-15 RA缺失）中，使用多光子活体显微镜（即运动性、对肌成纤维细胞的细胞毒性）对巨噬细胞、NK和NK-T细胞进行体内功能分析。此外，我们将使用巨噬细胞、NK/NK-T细胞和星状细胞的体外共培养系统来鉴定介导IL-15促纤维化作用的关键因子（即分泌的细胞因子、表面分子）。我们希望我们的项目将揭示IL-15影响肝纤维化免疫反应的贡献和机制，为专注于肝脏疾病中这种新型分子的诊断和治疗应用奠定基础。