Single cell analysis of mutant Surfactant Protein-C injury and remodeling

Mutation in the alveolar epithelial type 2 cell Surfactant Protein-C gene [SP-CI73T] is associated with pulmonary fibrosis (PF). We have previously demonstrated that inducible expression of the most common PF-linked mutation in the SP-C gene triggers inflammatory exacerbations of lung injury, progressing to fibrosis. We used single-cell sequencing to define the phenotype of epithelial, endothelial, immune and stromal cells accumulating in the lung at a time coordinated with peak injury (14 d post induction) and fibrotic remodeling (42 d post induction). The analysis showed at least 7 phenotypically distinct clusters of monocytes/macrophages during injury. Of these clusters, three exhibited distinctive pro-fibrotic signatures, based on expression of the prototypical markers TGFb1, osteopontin/SPP1 and fibronectin1. Notably SPP1+ cells expressed the highest levels of complement (C1qa, b, and c) and lipid associated genes (apoliporproteinE/ApoE, Cd36, Marco, Ldlr, Lrp1). We then set aim to examine the role of ApoE in inflammatory cell activation by crossing the SP-CI73T line with ApoE knock out mice. Mice heterozygous and homozygous for mutant ApoE were included to this analysis (both 14 and 42 da post injury). Survival analysis demonstrate unchanged inflammatory cell composition, but flow cytometric analysis noted a shift in myeloid (neutrophil and eosinophil) and lymphoid (B cells) cell recruitment in the ApoE mutant mice. Together, these results demonstrate time related changes in monocyte/macrophage dynamics and supports the notion that lipid homeostasis may be involved in their pro-fibrotic activation. Overall design: Single cell sequentinc of collagenase digested lung tissue from SP-C mutant mice at steady state and 14 d post injury