Regulation of Lung Fibrosis by Alternatively Activated Macrophages and Arginase-1

描述(由申请者提供)：本计划研究的目标是提供支持和持续的指导，以促进申请者发展成为间质性肺疾病和肺免疫学的独立科学研究者。这将会实现的 通过旨在获得坚实的基础科学背景的课程，包括实验室实践经验、定期教学、频繁的演示和成功的内科科学家的密切指导。该提案将为申请者提供至少75%的受保护研究时间。以研究提案为平台，申请者将获得一些基本科学技术方面的专业知识，包括：小鼠纤维化肺病的分析、细胞特异性基因敲除小鼠的产生以及巨噬细胞和成纤维细胞功能的评估。这一努力将涉及一个充满活力的研究小组，该小组在一个培养尖端研究的整体大学环境中发挥作用。研究项目：纤维性肺病是导致发病率和死亡率的重要原因，目前尚缺乏有效的治疗方法。调节顽固性肺纤维化的机制尚不完全清楚。我们的实验室最近描述了一种严重的进行性纤维化模型，以应对非感染性肺损伤。CXCR3是IFNG诱导的趋化因子CXCL9(Mig)、CXCL10(IP-10)和CXCL11(ITAC)的受体。这些趋化因子被认为是Th1免疫的重要介质。与野生型对照小鼠相比，CXCR3基因缺失的小鼠在气管内注射博莱霉素治疗后出现严重的进行性纤维化。微阵列分析已经确定了与Th2免疫相关的基因在缺乏CXCR3的情况下表达的优势。此外，我们还鉴定了一些与交替激活的巨噬细胞表型相关的基因。这些受体包括甘露糖受体、YM1和清道夫受体。与交替激活的巨噬细胞表型相关的最显著的基因是精氨酸酶-1的36倍诱导。虽然另一种激活的巨噬细胞参与了免疫介导的组织纤维化，但它们在非免疫调节过程中的作用尚不清楚。精氨酸酶-1是由交替激活的巨噬细胞和成纤维细胞产生的，以应对非感染性肺损伤。它有可能直接介导纤维化的发生。精氨酸酶-1负责将L-精氨酸转化为脯氨酸和多胺。多胺参与细胞增殖，而脯氨酸是胶原蛋白的直接前体。巨噬细胞和成纤维细胞表达精氨酸酶-1在进行性纤维化中的作用尚不清楚。根据我们的初步数据，我们假设巨噬细胞和成纤维细胞中精氨酸酶-1的表达在非感染性纤维肺损伤后纤维化的发展中起关键作用。我们将在以下特定目标中验证这一假设：目标1：使用区分招募和常驻人群的新的流式细胞术方法，确定CXCR3基因缺失小鼠在非感染性肺损伤后招募到肺中的特定巨噬细胞表型。目的：用Arg-1等位基因与靶向巨噬细胞或成纤维细胞的重组酶菌株杂交，确定非感染性损伤后巨噬细胞和成纤维细胞中精氨酸酶-1靶向缺失的效果。总而言之，这种方法将使我们能够剖析巨噬细胞和成纤维细胞产生精氨酸酶-1在进行性肺纤维化病理生物学中的作用。

DESCRIPTION (provided by applicant): The goal of this proposal research is to provide support and continued mentorship to facilitate the applicant's development into an independent scientific investigator in interstitial lung disease and lung immunology. This will be accomplished through a curriculum designed to obtain a solid basic science background involving hands on laboratory experience, regular didactic teaching, frequent presentations, and close mentoring by successful physician-scientists. The proposal will allow at least 75% of protected research time for the applicant. Using the research proposal as a platform, the applicant will gain expertise in number of fundamental scientific techniques including: the analysis of murine fibrotic lung disease, the generation of cell specific knockout mice and the assessment of macrophage and fibroblast function. This effort will involve a dynamic research group which functions in an overal university environment fostering cutting edge research. Research Project: Fibrotic lung diseases are a significant cause of morbidity and mortality for which effective therapies are lacking. The mechanisms that regulate unremitting pulmonary fibrosis are incompletely understood. Our laboratory has recently described a model of severe and progressive fibrosis in response to non-infectious lung injury. CXCR3 is the receptor for the IFNg-inducible chemokines CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (ITAC). These chemokines have been suggested to be important mediators of Th1 immunity. CXCR3 null mice develop severe and progressive fibrosis following treatment with intratracheal bleomycin relative to wild type control mice. Microarray analysis has identified a preponderance of genes associated with Th2 immunity expressed in the absence of CXCR3. In addition, we identified a number of genes that have been associated with the alternatively activated macrophage phenotype. These include Mannose Receptor, YM1 and Scavenger Receptor. The most prominent of the genes associated with an alternatively activated macrophage phenotype was a 36-fold induction in arginase-1. While alternatively activated macrophages have been implicated in mediating immunologically mediated tissue fibrosis, their roles in non-immune regulated processes are less clear. Arginase-1 is produced both by alternatively activated macrophages and fibroblasts in response to non-infectious lung injury. It has the potential to directly mediate fibrogenesis. Arginase-1 is responsible for the conversion of L-arginine to proline and polyamines. Polyamines are involved in cell proliferation while proline is a direct precursor of collagen. The role of arginase-1 expression by macrophages and fibroblasts in progressive fibrosis has not been explored. Based on our preliminary data, we hypothesize that arginase-1 expression in macrophages and fibroblasts is critical to the development of fibrosis after non-infectious fibrotic lung injury. We will test thi hypothesis in the following specific aims: AIM 1: Define the specific macrophage phenotypes recruited to the lung in CXCR3 null mice after non-infectious lung injury using a novel flow cytometric approach that distinguishes recruited and resident populations. AIM2: Determine the effects of targeted deletion of arginase-1 in macrophages and fibroblasts after non-infectious injury using a floxed arg-1 allele crossed with either a cre-recombinase strain that targets macrophages or fibroblasts. Collectively, this approach will allow us to dissect the role of arginase-1 production by macrophages and fibroblasts in the pathobiology of progressive pulmonary fibrosis.