CD45+/ Col I+ Fibrocytes: Major source of collagen in the fibrotic lung, but not in passaged 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 by flow cytometry a large increase in fibrocyte number and Col I expression associated with fibrosis. In contrast, fibroblast number was not significantly increased. A large increase (>50-fold) in fibrocyte number associated with fibrosis was also observed by single cell RNAseq. In this case, fibroblasts increased 5-fold. Single cell RNAseq also revealed that myofibroblast markers in fibrotic tissue are associated with a cluster containing a similar number of fibrocytes and fibroblasts, not with a resident fibroblast cluster. Some investigators claim that fibrocytes are not present among primary fibroblasts. However, we found that fibrocytes were the predominant cell type present in these cultures prior to passage. Fewer fibrocytes were present after one passage, and almost none after two passages. Our 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 by flow cytometry that in mice treated with bleomycin and WCSD compared to bleomycin alone, there was a large decrease in the number of fibrocytes present but not in the number of fibroblasts. In summary, fibrocytes are a major collagen-producing cell type that is increased in number in association with fibrosis as well as a major source of myofibroblasts. The common observation that collagen-producing spindle-shaped cells associated with fibrosis are CD45- may be an artifact of passage in cell culture.