A109-21 Loss of P16ink4a in Fibrotic Fibroblasts Causes Persistent Pulmonary Fibrosis

Abstract Background Pulmonary fibrosis is characterized by the persistence of profibrotic fibroblasts. Recent studies revealed that CTHRC1+ profibrotic fibroblasts represent a major subset localized at the leading edge of fibrogenesis. However, the fate control mechanism of profibrotic fibroblasts remains poorly understood. Our previous time-course bulk RNA-seq analysis in the single-dose bleomycin model revealed that Cthrc1-CreER-labelled fibroblasts downregulated profibrotic markers after the peak of fibrosis within residual scars without restoring the normal alveolar fibroblast signatures. This indicates that there is a program to inactivate profibrotic fibroblasts in the remaining scars. During the resolution phase, these profibrotic fibroblasts exhibited increased expression of senescence-related genes, including p16Ink4a. p16Ink4a is also reported to be upregulated in fibroblastic foci in idiopathic pulmonary fibrosis. Although cellular senescence is generally thought to limit fibroblast proliferation and promote fibrosis resolution, the specific role of p16Ink4a in regulating the fate and function of fibrotic fibroblasts during the resolution phase remains unclear. Methods To investigate the function of p16Ink4a in fibrotic fibroblasts, we generated Cthrc1-CreER; Rosa26-tdTomato; p16Ink4afl/fl mice to lineage-trace Cthrc1+ profibrotic fibroblasts and conditionally delete p16Ink4a. Pulmonary fibrosis was induced by the single-dose bleomycin administration via an oropharyngeal route. Tamoxifen was injected during the fibrotic phase. Lungs were collected on day 28 (fibrotic phase) and day 56 (resolution phase). Lung fibrosis, fibroblast abundance, and gene expression were analyzed. Results On day 28 after bleomycin, fibrosis did not differ significantly between p16Ink4awt/wt and p16Ink4afl/fl mice. On day 56, we observed worse fibrosis in p16Ink4afl/fl mice. The number of Cthrc1-CreER-labelled fibroblasts decreased on day 56 compared to the peak of fibrosis without a significant difference between p16Ink4awt/wt and p16Ink4afl/fl, suggesting that p16Ink4a did not regulate fibroblast number in the resolution phase. We observed that p16Ink4a deletion increased fibrotic markers (Col1a1 and Cthrc1) in Cthrc1-CreER-labelled fibroblasts. These data suggest that p16Ink4a deletion increased fibroblast activation and caused persistent matrix deposition during the resolution phase. Conclusion Our findings suggest that loss of p16Ink4a in fibrotic fibroblasts exacerbates lung fibrosis during the resolution phase, indicating that p16Ink4a suppresses fibroblast activation and promotes fibrosis resolution. Upregulation of p16Ink4a thus appears to be a homeostatic mechanism to suppress the persistent activation of profibrotic fibroblasts and facilitate fibrosis resolution. This abstract is funded by: None