Expression Of A Pulmonary Fibrosis Associated Surfactant Protein C Mutant, SP‐CI73T, In Alveolar Type 2 Cells Induces Lung Inflammation and Aberrant Parenchymal Remodeling
Authors: Venosa Alessandro , Tomer Yaniv , Jamil Sarita , Beers Michael 
Published: November 2023

Epithelial cell dysfunction has been postulated as an important driver in the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF). Mutations in the Surfactant Protein C [SP‐C] gene [SFTPC], an alveolar type 2 cell [AT2] restricted protein, have been linked to sporadic and familial forms of IPF. The missense isoleucine to threonine substitution at position 73 [I73T] is the most common disease‐associated SFTPC mutation. In order to temporally model early phenotypic changes following induction of expression of SP‐CI73T, a hemagglutinin (HA)‐tagged mSP‐CI73T sequence was knocked into the mouse sftpc locus. The founder line, whose alleles retained an intronic FRT flanked PGK‐neomycin (PGK‐neo) selection cassette demonstrated hypomorphic expression of sftpcI73T mRNA (“Hypo‐SP‐CI73T/I73T”) and developed age‐dependent cellular and mesenchymal expansion of alveolar septae with CD45(+) and AT2 cell hyperplasia. To test the effects of more physiological (full allelic) expression of SP‐CI73T, we generated an “inducible” SP‐CI73T expressing line (“IER‐SP‐CI73T”) by crossing Hypo‐SP‐CI73T/I73T mice to a R26FlpOER line to promote tamoxifen mediated FLPo recombinase removal of the inhibitory PGK‐neo cassette from sftpc alleles. In the absence of tamoxifen, adult IER‐SP‐CI73T mice retained a phenotype identical to Hypo‐SP‐CI73T/I73T founders. Intraperitoneal tamoxifen (iTAM) administration to IER‐SP‐CI73T mice resulted in rapid increases in proSP‐CI73T protein expression accompanied by allele and iTAM dose dependent weight loss and mortality. During this same period, treated IER‐SP‐CI73T mice developed a substantial increase in total BAL cell counts and in absolute numbers of macrophages, PMNs, and eosinophils. Histological sections revealed widespread alveolar infiltrates and diffuse alveolar damage. FACS analysis of whole lung digests revealed a reduction in the number of SiglecFhiCD11bintCD64hiCD11chiCD206hi alveolar macrophages in IER‐SP‐CI73T mice at 2 weeks. In contrast, there was an increase in a subset of macrophages (SiglecFhiCD11bintCD64hiCD11chi) expressing lower levels of CD206, as well as SSChiCD11b+SiglecFhiCCR3+ eosinophils and Ly6GhiCD11b+ neutrophils in BAL and lung digest. BAL from the treated cohorts also showed increases in IL5, Eotaxin, IL6, and MCP‐1 at 1–2 weeks with sustained elevation of KC out to 6 weeks. Transcriptomic profiling of AT2 cells from IER‐SP‐CI73T mice 2 weeks after iTAM showed upregulation in MCP‐1, IL‐5, Eotaxin, and CCL17 expression. IER‐SP‐CI73T mice surviving to 6 weeks went on to develop a novel fibrotic lung phenotype marked by alveolar septal thickening, diffuse collagen deposition, and foci of a‐SMA (+) cells adjacent to dilated airspaces lined by hyperplastic AT2 cells. We conclude that the AT2 epithelial cell dysfunction induced by mutant proSP‐CI73T expression in vivo is sufficient to drive a cascade of profibrotic events including acute spontaneous alveolitis and ultimately fibrotic remodeling supporting the concept of epithelial dysfunction as a key component of IPF.