Role of pathologic fibroblast subsets unique to silicosis

Abstract Silicosis is a chronic and potentially fatal fibrotic disease caused by inhalation of crystalline silica. Recent worldwide outbreaks of silicosis among workers who cut engineered stones for countertops underscore the necessity of understanding the cellular and molecular mechanisms underlying silicosis, for which there is no effective therapy. In silicosis lungs, activated fibroblasts generate granulomas called silicotic nodules and persistently cause fibrosis, which deteriorates lung function over time. However, the mechanisms underlying fibroblast activation and progressive fibrosis in silicosis have not been well studied. In previous work, we used lineage tracing and single-cell RNA-sequencing (scRNA-seq) to show that alveolar fibroblasts are the major origin of fibroblasts induced by bleomycin injury and in silicotic nodules in a murine model of silicosis. In preliminary work that serves as the basis for this proposal, we revealed that, in response to silica, alveolar fibroblasts differentiate into two unique populations that are not present in bleomycin-induced fibrosis or idiopathic pulmonary fibrosis. One population is localized inside silicotic nodules, expresses high levels of ECM genes, and is characterized by the up-regulation of ectopic bone regulatory pathways, which have recently been shown to be important in driving silicosis. The other population is restricted to the periphery of silicotic nodules and is characterized by hedgehog activation. This study takes advantage of our original mouse tools that allow us to target each unique fibroblast subset specifically and aims to elucidate the lineage trajectories of these fibroblast subsets and how they contribute to persistent fibrosis in silicosis. We hypothesize that pathologic fibroblasts originating from alveolar fibroblasts in silicotic nodules utilize ectopic bone regulatory pathways to cause a malicious feedback loop resulting in persistent fibrosis, while hedgehog-activated fibroblasts restrict the expansion of silicotic nodules. In Aim 1, we will use our original mouse tools to lineage- trace pathologic fibroblast subsets to elucidate the lineage trajectories and how pathologic fibroblasts in silicotic nodules are maintained for chronic fibrogenesis. In Aim 2, we will conditionally delete bone regulatory genes and the principal hedgehog receptor in alveolar fibroblasts to determine the roles of the pathways and fibroblast subsets we found to be unique to silicosis. In Aim 3, we will build a fibroblast-centric cell atlas of human silicosis with our specialized scRNA-seq strategy and high-throughput in situ hybridization to further assess the translational potential of our murine studies. These studies should provide insights into the cellular and molecular mechanisms driving silicosis, which could lead to novel therapeutic strategies for silicosis.