Deciphering the role of p16INK4A+ fibroblasts in lung fibrosis

Project Summary/Abstract Aging is one of the biggest risk factors for many chronic lung diseases, including idiopathic pulmonary fibrosis (IPF). However, uncertainties remain as to how age-related processes contribute to the pathogenesis of IPF, particularly in deciphering the cell types that might play a role in both aging and fibrotic transformation. Senescence, a cellular state characterized by an irreversible cell cycle arrest and other morphologic transformations, is thought to be a cellular phenotype that becomes increasingly prevalent in aged organs, contributing to the decline in function over time. p16INK4A, a tumor suppressor, is one of the best-studied biomarkers of senescent cells in vitro and in vivo. Numerous genetic models have been built to remove p16INK4A+ cells from tissues, demonstrating a functional role for these cells in promoting age-related pathologies such as lung fibrosis. Despite the knowledge gained from strategies that remove p16INK4A+ cells, deletion of these cells precludes the identification and functional characterization of p16INK4A+ cells in vivo, leaving a large gap in our understanding of how senescent cells might behave in tissues rather than in a culture dish. To address this, we constructed a novel genetic reporter of senescence by engineering an amplified fluorescent tag driven by expression of p16INK4A (mouse referred to as INK4A H2B-GFP Reporter-In-Tandem, or INKBRITE) to isolate and characterize p16INK4A+ cells in vivo and ex vivo. To our surprise, we were able to identify p16INK4A+ fibroblasts in young and healthy lungs that contribute to scar-forming cells in areas of fibrotic remodeling. Furthermore, p16INK4A+ cells are able to alter the epithelial progenitor microenvironment and alter stem cell regenerative outcomes. This led us to the hypothesis that p16INK4A+ fibroblasts are capable of contributing to fibrosis by forming scars and inducing an pathologic epithelial response, and the INKBRITE reporter could be leveraged as a screening platform to identify compounds that more precisely target p16INK4A+ fibroblasts in vivo. Through the use of single cell RNA sequencing, adoptive cell transfers, lineage tracing, high-content imaging, and various methodologies outlined in this proposal, we aim to better define the cellular contribution of p16INK4A+ fibroblasts to fibrotic transformation, and define agents that might better remove it from tissues.