New Approaches for the Study of Lung Fibrocytes

描述(申请人提供)：纤维细胞被认为是一种独特的骨髓来源的细胞，在肺损伤过程中有助于胶原蛋白的积累。纤维细胞被定义为CD45+/I型Col I+，强调它们可能同时具有造血型和结缔组织表型。然而，用于鉴定和分离纤维细胞的方法的局限性以及对其基本生物学的了解不足，限制了该领域的进展，并加剧了对其在肺纤维化反应中所起作用的怀疑。到目前为止，用于研究纤维细胞的方法主要是提供间接信息，因为纤维细胞鉴定和分离技术不能直接产生活细胞；在这方面，分离涉及在体外长期培养单核细胞。作为回应，我们开发了一种直接分离可存活的纤维细胞的策略，并正在寻求R21资金，以利用这项技术来推动该领域的发展，并为未来的工作奠定基础。为此，我们采用了一种在I型Col启动子(Col-GFP)控制下表达GFP的转基因小鼠。用流式细胞仪可以很容易地从小鼠体内分离出CD45+/GFP+细胞。值得注意的是，GFP+细胞正在积极转录Col mRNA；因此，CD45+/GFP+细胞表现出经典定义为纤维细胞的特性。在正常肺和博莱霉素处理5d的肺组织中，纤维细胞占肺CD45+细胞总数的8~9%，并表达髓系标志。随着损伤程度的增加，肺纤维细胞绝对数增加~2倍，并伴有I型Col基因mRNA增加15倍。这些发现提出了一个基本问题：损伤中纤维细胞的扩张和激活是否局限于驻留细胞或招募细胞？为了回答这个问题，我们还开发了一种胸壁屏蔽方法，在移植期间保护常驻的肺CD45+细胞，结果是支持肺CD45+和骨髓CD45+细胞群体的嵌合体的发展，这些细胞群体各自表达不同的荧光标记。拥有屏蔽模型和Col-GFP小鼠为回答这一基本问题提供了一个独特的机会，同时也为肺纤维细胞开发了一个知识库，其有效性不受技术问题的影响。因此，我们建议提供关于它们的定位和个体发育的基本信息(目标1)，检验它们是稳定的常驻群体的假设(目标1)，检验在肺损伤期间常驻纤维细胞中发生胶原激活的假设(目标2)，并产生2个比较基因组签名，它们将提供关于肺纤维细胞身份的关键信息，新的候选标记，以及控制其激活的调控途径(目标2)。长期目标是为试剂的合理设计奠定基础，以确定纤维细胞在肺纤维化中的命运和功能。 与公共卫生相关：这项建议的目标是识别和理解控制身体如何对肺损伤做出反应的复杂的分子途径。我们希望这项工作的发现将有助于设计治疗方法，这将有助于肺损伤的愈合。

DESCRIPTION (provided by applicant): Fibrocytes are thought to be a distinct marrow-derived cell that contributes to collagen accumulation during lung injury. Fibrocytes are defined as CD45+/type I Col I+, underscoring their putative, simultaneous hematopoietic and connective tissue phenotype. Limitations in methods used to identify and isolate fibrocytes along with deficiencies in understanding their basic biology, however, restrict progress in the field, and fuel skepticism regarding their role in lung fibrogenic reactions. To date, methods used to study fibrocytes have primarily provided indirect information due to the fact that fibrocyte identification and isolation techniques do not yield viable cells directly; in this regard isolation involves prolonged culturing of monocytic cells in vitro. In response, we developed a strategy to directly isolate viable fibrocytes, and are seeking R21 funding to use this technology to advance the field and establish a foundation for future work. For this, we employ a transgenic mouse that expresses GFP under control of the type I Col promoter (Col-GFP). From this mouse, CD45+/GFP+ cells can be readily isolated by flow cytometry. Of note, GFP+ cells are actively transcribing Col mRNA; thus CD45+/GFP+ cells display properties that classically define fibrocytes. In the normal and 5 d bleomycin-treated lung, fibrocytes comprise ~8-9% of total lung CD45+ cells and express myeloid markers. With injury there is a ~2-fold increase in the absolute number of lung fibrocytes accompanied by a 15-fold increase in type I Col gene mRNA. These findings raise the basic question: is the expansion and activation of fibrocytes in injury localized to resident or recruited cells? To answer this question, we also developed a thoracic shielding method that protects resident lung CD45+ cells during transplantation, and as a result, supports development of chimeras with lung CD45+ and marrow CD45+ cell populations that each expresses a different fluorescent marker. Having the shielding model and Col-GFP mice in hand provides a unique opportunity to answer this basic question, and at the same time to develop a knowledge base for lung fibrocytes whose validity is not undermined by technical concerns. We, thus, propose to provide basic information regarding their localization and ontogeny (Aim 1), to test the hypothesis that they are a stable resident population (Aim 1), to test the hypothesis that collagen activation occurs in resident fibrocytes during lung injury (Aim 2), and to generate 2 comparative genomic signatures that will provide key information about lung fibrocyte identity, new candidate markers, and regulatory pathways controlling their activation (Aim 2). The long-term objective is to lay the groundwork for the rationale design of reagents to definitively resolve fibrocyte fate and function in lung fibrosis. PUBLIC HEALTH RELEVANCE: This objective of this proposal is to identify and understand the complex molecular pathways that control how the body responds to a lung injury. It is our hope that findings from this work will facilitate the design of treatments for that will help with the healing from lung injury.