Deciphering the age-dependent changes of pulmonary fibroblasts in mice by single-cell transcriptomics.

Background and objectives: The heterogeneity of pulmonary fibroblasts, a critical aspect of both murine and human models under physiological and pathological conditions, is well-documented. Yet, consensus remains elusive on the subtypes, lineage, biological attributes, signal transduction pathways, and plasticity of these fibroblasts. This ambiguity significantly impedes our understanding of the fibrotic processes that transpire in lung tissue during aging. This study aims to elucidate the transcriptional profiles, differentiation pathways, and potential roles of fibroblasts within aging pulmonary tissue. Methods: We employed single-cell transcriptomic sequencing via the 10x Genomics platform. The downstream data were processed and analyzed using R packages, including Seurat. Trajectory and stemness of differentiation analyses were conducted using the Monocle2 and CytoTRACE R packages, respectively. Cell interactions were deciphered using the CellChat R package, and the formation of collagen and muscle fibers was identified through Masson and Van Geison staining techniques. Results: Our analysis captured a total of 22,826 cells, leading to the identification of fibroblasts and various immune cells. We observed a shift in fibroblasts from lipogenic and immune-competent to fibrotic and myofibroblast-like phenotype during the aging process. In the aged stage, fibroblasts exhibited a diminished capacity to express chemokines for immune cells. Experimental validation confirmed an increase of collagen and muscle fiber in the aged compared to young lung tissues. Furthermore, we showed that TGFß treatment induced a fibrotic, immunodeficient and lipodystrophic transcriptional phenotype in young pulmonary fibroblasts. Conclusion: We present a comprehensive single-cell transcriptomic landscape of lung tissue from aging mice at various stages, revealing the differentiation trajectory of fibroblasts during aging. Our findings underscore the pivotal role of fibroblasts in the regulation of immune cells, and provide insights into why age increases the risk of pulmonary fibrosis.