CD45+/Col I+ Fibrocytes: Major Source of Collagen in the Fibrotic Lung, but not in Fibroblast Cultures

The role of cells of the hematopoietic lineage in fibrosis is controversial. Here we evaluate the contribution of Col I+/CD45+ cells (fibrocytes) to lung fibrosis. Systemic bleomycin treatment was used to induce fibrosis in a bone marrow transplant and two transgenic mouse models. Lung cells from these mice were analyzed by flow cytometry, both immediately upon release from the tissue or following growth on tissue-culture plastic. Fibrotic and control human lung tissue were also used. Fibroblasts and fibrocytes derived from a transgenic mouse model were compared in terms of their morphology, growth, and adhesion to fibronectin. Single cell RNAseq was performed with the analysis focusing on CD45-/Col I+ “fibroblasts” and CD45+/Col I+ “fibrocytes” in control and fibrotic mouse lung tissue. Finally, we inhibited fibrosis in mice using a novel, water-soluble version of caveolin scaffolding domain (CSD) called WCSD. In both mouse and human lung tissue, we observed a large increase in fibrocyte number and Col I expression associated with fibrosis. In contrast, fibroblast number is not significantly increased. These results on increased fibrocyte number in fibrotic mouse lung tissue were strongly supported by single cell RNAseq. RNAseq also revealed that myfibroblast markers are associated with a fibrocyte cluster, not with a resident fibroblast cluster. Some investigators claim that fibrocytes are not present among primary fibroblasts. However, we find that fibrocytes are the predominant cell type present in these cultures prior to passage. Fewer fibrocytes are present after one passage, and almost none after two passages. Like the classic description of fibroblasts, passage 0 fibrocytes are spindle-shaped (i.e. not the fried-egg shape of macrophages). Experiments suggest that fibrocytes are crowded out of cultures during passage because fibroblasts have a larger footprint than fibrocytes, even though fibrocytes bind more efficiently to fibronectin. Finally, we observed that in mice treated with bleomycin and WCSD, there was a large decrease in the number of fibrocytes present, but not in the number of fibroblasts, compared to bleomycin alone. In summary, fibrocytes are the major collagen-producing cell type that is increased in number in association with fibrosis as well as the major source of myofibroblasts. The common observation that collagen-producing spindle-shaped cells associated with fibrosis are CD45- appears to be an artifact of passage in cell culture.