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A6209 - A Novel Murine Model of Expressing a Surfactant Protein C BRICHOS Mutation Charts a Path from Epithelial Endoplasmic Reticulum Stress to Spontaneous Acute Lung Injury and Fibrotic Remodeling
Author Block: J. Katzen1, Y. Tomer1, M. Kopp1, A. Venosa1, S. Mulugeta2, M. F. Beers2; 1Department of Medicine Division of Pulmonary and Critical Care Medicine, University of Pennsylvania, Philadelphia, PA, United States, 2Department of Medicine Division of Pulmonary and Critical Care Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, United States.
Rationale: Mutations in the BRICHOS region of the alveolar type 2 (AT2) cell specific Surfactant Protein C [SP-C] gene [SFTPC], have been shown to produce misfolded conformers inducing an AT2 ER stress response in vitro. Similar molecular signatures have been detected in familial forms of interstitial lung disease (ILD) associated with BRICHOS SP-C mutations and sporadic ILD/idiopathic pulmonary fibrosis (IPF). A robust SFTPC BRICHOS mutation mouse model can provide a platform for proof of concept studies linking SFTPC mutations with ILD and permit broader insights into the pathway from aberrant AT2 quality control to the development of ILD/IPF.
Approach: To model previously reported ILD associated SFTPC BRICHOS mutations, a cysteine to glycine substitution at position 121 [C121G] was knocked into the mouse sftpc locus using a targeted ES cell approach. The founder line had hypomoprhic sftpc gene expression due to retention of an intronic LoxP flanked PGK-neomycin [PGK-neo] selection cassette. PGK-neo excision and full allelic expression was achieved by crossing the founder line with a CMV-Cre constitutive deleter line or tamoxifen inducible Rosa26-ERT2-Cre or SFTPC-ERT2-Cre lines.
Results: In all three in vivo models, AT2 cells expressing the SP-CC121G mutation showed evidence of ER retention of pro-SP-C characterized by its aberrant processing on Western blotting and a reticular pattern with peri-nuclear aggregates on immunofluorescent staining of histological sections. Constitutive embryonic PGK-neo removal in heterozygous CMV-Cre/SP-CC121G mice produced grossly normal day P0.5 lung histology, but highly penetrant lethal early postnatal (P2-3) respiratory failure. Tamoxifen mediated excision from adult Rosa26-ERT2-Cre/SP-CC121G homozygous mice resulted in a fatal acute alveolitis at 7-9 days. AT2 cells isolated from these mice revealed evidence of ER stress (increased GRP78), and JNK activation by Western blotting. Increased AT2 cell apoptosis was also evident by caspase 3 staining of lung sections. qRT-PCR of AT2 mRNA showed significant up-regulation of chemokines eotaxin, IL-5, and MCP-1 with commensurate increases in BAL IL-5, eotaxin, and MCP-1. Tamoxifen treated heterozygous SFTPC-ERT2-Cre/SP-CC121G mice, survived the early alveolitis stage, but subsequently developed fibrotic remodeling with sub-pleural fibroblastic foci of SMA(+) fibroblasts and hyperplastic AT2 cells.
Conclusions: This in vivo SFTPC BRICHOS mutation model provides direct evidence for the role of a dysfunctional AT2 cell phenotype in the pathogenesis of pulmonary fibrosis. The model will permit further elucidation of the pathway(s) from ER stress to subsequent immune cell recruitment, dysregulated inflammation, and aberrant fibrotic remodeling, and will provide insights into potential therapeutic targets for clinical ILDs.