Novel Mechanisms of Pulmonary Fibrosis

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This proposal explores the role of a pulmonary osteoclast-like cell (POLC) in silicosis. We first discovered POLCs while studying pulmonary alveolar microlithiasis (PAM), a rare, autosomal recessive disorder caused by mutations in the epithelial sodium phosphate co-transporter, Npt2b. Phosphate accumulates in the alveolar lining fluid and complexes with calcium to form spherical hydroxyapatite microliths containing bone matrix proteins and surfactant components. Contact of microliths with tissue resident alveolar macrophages (TR-AM) and recruited monocytes (Mo-AM) induces osteoclastic transformation in the Npt2b-/- mouse, with expression of the full repertoire of osteoclast signature gene and protein expression in multinucleated giant cells (MNGC) including tartrate resistant acid phosphatase (TRAP), cathepsin K (CTSK), calcitonin receptor (CALCR) and the proton pump, ATP6V0D2. Single cell RNA sequencing (scRNAseq) of human PAM lung also confirmed a robust osteoclast signature in AM, and IHC confirmed the presence of TRAP and CTSK positive MNGC. Like humans, Npt2b-/- animals develop modest pulmonary fibrosis and a marked restrictive physiologic defect. We found that microliths induce alveolar expression of the requisite osteoclastogenic cytokines for POLC differentiation (i.e. M- CSF, RANKL) and expression of hydrochloric acid and CTSK that both dissolve stones and damage tissues. We were surprised to find that the Npt2b-/- mice also develop pulmonary alveolar proteinosis, which has not been reported in PAM, but is known to be associated with another particulate exposure syndrome, silicoproteinosis. This led us to consider that osteoclastic transformation of TR-AM and Mo-AM is a stereotypic response to inhaled particles, resulting in the concomitant loss of AM surfactant catabolic and host defense functions (in the form of susceptibility to Tb). Indeed, snRNAseq, rtPCR measures of gene expression and IHC assessments revealed that silica challenge is also associated with RANKL-dependent osteoclastic transformation of BAL and parenchymal cells, and TRAP, CTSK and hydrochloric acid production, culminating in destructive remodeling and pulmonary fibrosis, and associated with a decrease in proteins required for lipid metabolism. Anti- RANKL treatment of mice attenuates the silicoproteinosis, POLC formation, fibrosis and the restrictive physiologic defect that occurs in silica-challenged mice. The differential tissue responses to dissolvable vs. persistent particulates forms the basis for our hypothesis that persistent acid and matrix degrading enzymes produced by POLC may be primary drivers of particulate-induced fibrosis. To test this hypothesis in three aims, we will determine; 1) Cell lineages and alveolar factors that give rise to POLC; 2) The role of osteoclastic functions and products in silica- induced fibrosis; and 3) The role of POLC in the pathogenesis of pulmonary fibrosis, in vivo. This proposal directly addresses mechanisms relevant to the alarming rise in the prevalence of progressive massive fibrosis in black lung claimants, and successful completion of the aims will provide preclinical evidence supporting anti- osteoclastic strategies such as bisphosphonates and anti-RANKL in exposed subjects.