New Approaches for the Study of Lung Fibrocytes

描述（由申请方提供）：纤维细胞被认为是一种独特的骨髓源性细胞，在肺损伤期间有助于胶原蛋白积聚。纤维细胞被定义为CD 45 +/I型Col I+，强调其推定的同时造血和结缔组织表型。然而，用于识别和分离纤维细胞的方法的局限性沿着对其基本生物学的理解不足，限制了该领域的进展，并引发了对其在肺纤维化反应中作用的怀疑。迄今为止，用于研究纤维细胞的方法主要提供间接信息，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，分离涉及体外单核细胞的长期培养。作为回应，我们开发了一种直接分离活纤维细胞的策略，并正在寻求R21资金，以利用这项技术来推进该领域的发展，并为未来的工作奠定基础。为此，我们采用了在I型Col启动子（Col-GFP）控制下表达GFP的转基因小鼠。从该小鼠中，可以通过流式细胞术容易地分离CD 45 +/GFP+细胞。值得注意的是，GFP+细胞活跃地转录Col mRNA;因此，CD 45 +/GFP+细胞显示出经典定义纤维细胞的性质。在正常和5天博来霉素处理的肺中，纤维细胞占总肺CD 45+细胞的约8-9%，并表达髓样标志物。随着损伤，肺纤维细胞的绝对数量增加~2倍，伴随I型Col基因mRNA增加15倍。这些发现提出了一个基本的问题：损伤中纤维细胞的扩增和活化是局限于驻留细胞还是募集细胞？为了回答这个问题，我们还开发了一种胸部屏蔽方法，在移植过程中保护驻留的肺CD 45+细胞，因此，支持肺CD 45+和骨髓CD 45+细胞群的嵌合体的发展，每个细胞群表达不同的荧光标记物。拥有屏蔽模型和Col-GFP小鼠提供了一个独特的机会来回答这个基本问题，同时为肺纤维细胞开发一个知识库，其有效性不受技术问题的影响。因此，我们提出提供关于它们的定位和个体发生的基本信息（目的1），检验它们是稳定的常驻群体的假设（目的1），检验肺损伤期间常驻纤维细胞中发生胶原活化的假设（目的2），并产生2个比较基因组签名，其将提供关于肺纤维细胞身份、新的候选标记物、以及控制其激活的调节途径（目标2）。长期目标是为试剂的合理设计奠定基础，以明确解决肺纤维化中纤维细胞的命运和功能。 公共卫生相关性：该提案的目的是识别和理解控制身体如何对肺损伤做出反应的复杂分子途径。我们希望这项工作的发现将有助于设计有助于肺损伤愈合的治疗方法。

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.