New Approaches for the Study of Lung Fibrocytes

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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.