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

炎症已经被认为是肝纤维化的一个重要方面，然而针对这种情况的细胞因子的治疗方法到目前为止还没有成功。这一失败的部分原因是我们对肝纤维化中复杂的炎症过程没有完全了解。白介素15(IL-15)是一种在NK和NK-T细胞生理中起关键作用的重要细胞因子。最近，有报道称IL-15受体α(IL-15RA)KO小鼠对肝纤维化的易感性增加。然而，我们的初步研究显示，IL-15KO小鼠的反应相反，与野生型小鼠相比，肝纤维化减轻。这一争议可能是由IL-15和IL-15RA基因组成的KO小鼠的先天缺陷引起的。然而，更多的初步结果支持IL-15的促纤维化作用。首先，我们观察到在TGFb刺激的原代肝细胞中IL-15的强烈诱导，并在原代巨噬细胞中检测到几种由IL-15强烈诱导的促纤维化细胞因子。其次，我们观察了IL-15在小鼠和人肝纤维化和肝硬变中的诱导作用。因此，我们的初步结果有力地表明，IL-15通过诱导巨噬细胞中的促纤维化表型来促进纤维化。要阐明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深入的了解，包括表达IL-15的细胞的鉴定，基于OMICS的肝白细胞、肝细胞和非实质细胞的分析，以及在不改变肝白细胞内平衡环境的情况下实现IL-15或IL-15RA缺失的体内实验条件。在这项应用中，我们将首先利用IL-15-CFP报告小鼠确定IL-15的主要细胞来源，并建立IL-15表达与人和鼠肝纤维化炎症微环境之间的相关性。其次，通过分析他莫昔芬对照、Cre依赖的IL-15或IL-15RA缺失成年小鼠后肝纤维化的严重程度。第三，对肝纤维化小鼠的肝细胞、星状细胞、巨噬细胞、NK细胞和NK-T细胞以及IL-15和IL-15RA缺失的小鼠进行转录转录分析。最后，我们将使用多光子活体显微镜，通过将荧光标记的细胞过继转移到纤维化小鼠(对照组和IL-15或IL-15RA缺失)，对巨噬细胞、NK和NK-T细胞进行体内功能分析(即对肌成纤维细胞的运动性和细胞毒性)。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确定介导IL-15促纤维化作用的关键因素(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在肝纤维化中影响免疫反应的贡献和机制，为专注于这种新的肝脏疾病分子的诊断和治疗应用奠定基础。

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.