Assessing the role of metabolism in monocyte to macrophage differentiation in pulmonary fibrosis

项目摘要 特发性肺纤维化(IPF)的预后比大多数癌症更差，只有20%-30%的患者 确诊后存活5年。IPF的病理生理学与损伤后的修复失调有关。 通过肺泡毛细血管膜损伤与成纤维细胞激活的联系机制 和无序的修复是不完全理解的。在最近的一份出版物中，我们提供了明确的遗传证据 肺泡巨噬细胞是小鼠纤维化发展过程中的关键效应细胞，这一点在 人肺泡巨噬细胞取自肺纤维化患者。我们报告说单核细胞招募了 在肺损伤和纤维化过程中表达独特的标志物，并改变其形态，使其类似于肺泡 巨噬细胞。使用组合的遗传谱系追踪，有针对性的遗传策略和转录分析 (RNA-Seq)我们发现“单核细胞来源的肺泡巨噬细胞”和 “组织驻留的肺泡巨噬细胞”在肺纤维化的发展中起着不同的作用。关键是， 不依赖组织驻留肺泡的单核细胞来源的肺泡巨噬细胞的基因删除 巨噬细胞可减轻博莱霉素诱导的肝纤维化程度。我们的结果与以下结论一致 其他人则认为，单核细胞向组织驻留巨噬细胞的分化是 通过对局部组织微环境的提示做出反应的表观遗传变化。因为在这个过程中 单核细胞向肺泡巨噬细胞的分化是肺特有的，针对这一过程的治疗是在 单核细胞已经被招募到肺内，很可能避免与全身毒性相关的全身毒性 单核细胞耗尽。博莱霉素肺纤维化模型的一个特征是纤维化的自发消退。 超过2-3个月，这不是IPF的总结，在IPF中肺纤维化的持续进展是常态。 然而，我们和其他人已经证明，石棉小鼠的肺纤维化模型确实证明 进行性纤维化，因此可以更准确地概括人类IPF。为了提供一个更有说服力的 靶向单核-巨噬细胞分化作为IPF治疗靶点的理论基础，我们计划解决 我们的数据在续订申请中提出了三个重要问题。首先，单核细胞来源的肺泡 巨噬细胞在石棉小鼠肺纤维化模型中扮演着类似的角色？我们已经这么做了 生成了支持这一假设的初步数据。作为这些实验的一部分，我们将进行单细胞 转录组学(Drop-Seq)确定单核细胞中促纤维化基因的表达是否源于 肺泡巨噬细胞属于一种细胞亚群。第二，单核细胞来源的缺失可以吗 肺泡巨噬细胞促进肺纤维化的消退？第三，我们可以用我们的 转录数据为靶向单核细胞来源的肺泡巨噬细胞以改善纤维化的策略提供依据？ 我们的数据有力地表明，脂质代谢对单核细胞来源的肺泡的分化是必不可少的。 巨噬细胞。我们将通过肺泡巨噬细胞靶向缺失脂肪酸来阻断这一途径 合成酶基因(FASN)与药物对脂肪酸合成的抑制作用 肺纤维化过程中单核细胞来源的肺泡巨噬细胞分化和基因表达。为了 建立与我们活体工作的生物学相关性，我们还将收集IPF患者的肺泡巨噬细胞和 比较对照的AM和AM的转录差异。具体目标1：确定是否 单核细胞来源的巨噬细胞在非消退性肺纤维化模型中驱动肺纤维化。具体目标2： 确定单核细胞来源的巨噬细胞缺失是否能缓解未溶解肺中的肺纤维化 纤维化模型。特定目标3：确定抑制单核细胞来源的脂质生物合成 巨噬细胞在博莱霉素和未消退肺纤维化模型中预防肺纤维化。

Project Summary Idiopathic Pulmonary Fibrosis (IPF) has a prognosis worse than most cancers, with only 20-30 percent of patients surviving 5 years after diagnosis. The pathophysiology in IPF relates to dysregulated repair after injury to the alveolo-capillary though the mechanisms that link alveolar capillary membrane injury with fibroblast activation and disordered repair are incompletely understood. In a recent publication we provide clear genetic evidence that alveolar macrophages are key effector cells in the development of fibrosis in mice which was validated in human alveolar macrophages obtained from pulmonary fibrosis patients. We reported that monocytes recruited to the lung during injury and fibrosis express unique markers and change their morphology to resemble alveolar macrophages. Using combined genetic lineage tracing, a targeted genetic strategy and transcriptomic analysis (RNA-Seq) of flow-sorted myeloid populations we showed that “monocyte-derived alveolar macrophages” and “tissue-resident alveolar macrophages” play distinct roles in the development of lung fibrosis. Critically, genetically deleting monocyte derived alveolar macrophages independent of tissue resident alveolar macrophages reduced the severity of bleomycin induced fibrosis. Our results are consistent with findings from others who have suggested that the differentiation from monocytes into tissue-resident macrophages is driven by epigenetic changes in response to cues from the local tissue microenvironment. Because the process of monocyte to alveolar macrophage differentiation is specific to the lung, therapies that target this process after a monocyte has been recruited into the lung are likely to avoid the systemic toxicity associated with systemic monocyte depletion. One feature of the bleomycin lung fibrosis model is the spontaneous resolution of fibrosis over 2-3 months, which does not recapitulate IPF in which continuous progression of lung fibrosis is the norm. However, we have and others have shown that the asbestos mouse model of lung fibrosis does demonstrate progressive fibrosis and thus may more accurately recapitulate human IPF. In order to provide a more compelling rationale for targeting monocyte-macrophage differentiation as a therapeutic target for IPF, we plan to address three important questions raised by our data in our renewal application. First, do monocyte-derived alveolar macrophages play a similar role in asbestos mouse models of non-resolving lung fibrosis? We have already generated preliminary data supporting this hypothesis. As part of these experiments, we will perform single cell transcriptomics (DROP-Seq) to determine whether the expression of pro-fibrotic genes in monocyte-derived alveolar macrophages is attributable to a subpopulation of cells. Second, can the deletion of monocyte-derived alveolar macrophages promote the resolution of fibrosis after it is established? Third, can we use our transcriptomic data to inform strategies to target monocyte-derived alveolar macrophages to ameliorate fibrosis? Our data strongly suggest that lipid metabolism is essential for the differentiation of monocyte-derived alveolar macrophages. We will interrupt this pathway through alveolar macrophage-targeted deletion of the fatty acid synthetase gene (FASN) and pharmacologic inhibition of fatty acid synthesis to examine the effects on monocyte-derived alveolar macrophage differentiation and gene expression during lung fibrosis. In order to establish biological relevance to our in vivo work we will also collect alveolar macrophages from IPF patients and examine transcriptomic differences compared to AM from controls. Specific Aim 1: To determine whether monocyte derived macrophages drive lung fibrosis in non-resolving lung fibrosis models. Specific Aim 2: To determine whether deletion of monocyte-derived macrophages resolves lung fibrosis in the non-resolving lung fibrosis models. Specific Aim 3: To determine whether inhibiting lipid biosynthesis in monocyte-derived macrophages prevents lung fibrosis in the bleomycin and non-resolving lung fibrosis models.