Epithelial Membrane Protein 2 Deletion Protects from Lung Injury and Fibrosis

Abstract Rationale: Epithelial membrane protein 2 (EMP2) is a tetraspan protein highly expressed by alveolar epithelial type 1 (AT1) cells. We have previously reported that EMP2 regulates expression of adhesion molecules in AT1 cells and is required for transepithelial migration of neutrophils into the airspace after inhaled lipopolysaccharide. Here, we investigated roles for EMP2 in lung fibrosis. Methods: Emp2-/-, Emp2Sftpc (lung epithelial-specific Emp2-deficient), and Emp2Sftpc-ER (AT2-specific Emp2-deficient) mice were exposed to intratracheal bleomycin and inflammatory and fibrotic (hydroxyproline assay, trichrome staining) responses profiled. Roles for the alveolar epithelium were further explored using inhaled silica and adenoviral-TGFβ, alternate models that induce less primary epithelial injury, and butylated hydroxytoluene (BHT), which induces severe AT1-predominant necrotic injury.Results: Compared to controls, Emp2-/- and Emp2Sftpc mice had reduced lung fibrosis after bleomycin, whereas there was no reduction in fibrosis in Emp2Sftpc-ER mice. No attenuation in fibrosis was observed in any of the Emp2-deficient strains after silica or adenoviral TGFβ. Consistent with attenuated early AT1 injury, BALF RAGE and Pdpn levels were reduced after bleomycin, as were weight loss; airway recruitment of monocytes and monocyte-derived macrophages; airway pro-inflammatory cytokines and TGFβ; and airway albumin. Emp2-/- and Emp2Sftpc mice exhibited reduced γH2AX (DNA double-strand break marker) and attenuated induction of p53 target genes in both AT1 and AT2 cells as early as 24 hours post-bleomycin, and reduced cellular senescence in AT1, AT2, and pre-AT1 transitional state (PATS) cells at later timepoints. Pro-fibrotic Krt8+B6+ PATS cells were reduced in number. Single cell RNA-sequencing analysis of bleomycin-challenged Emp2Sftpc mice corroborated reduced PATS cells and identified reduced p53 activation and dysregulated complement gene expression in PATS cells, together pointing to attenuation of quantitative and qualitative acquisition of the pro-fibrotic transitional cell state. Emp2Sftpc mice had attenuated weight loss and mortality after BHT, further suggesting that Emp2-null AT1 cells may be broadly protected against toxic insults. AT1-specific Emp2Ager mice and Emp2-null Sftpc-CreER-TdTomato AT2-lineage tracing mice are currently under study to confirm whether Emp2 deletion in AT1 cells is specifically protective and reduces AT2-PATS-AT1 flux. Conclusion: Emp2 deletion is protective against lung injury and fibrosis in the bleomycin model and from mortality in the BHT model. Our findings suggest that Emp2 deletion protects alveolar epithelial cells from early, acute DNA damage. Our finding of reduced damage in both AT1 and AT2 cells as early as 24 hours suggests that AT1 EMP2 may co-regulate AT2 damage via either AT1-AT2 contact-dependent or paracrine mechanisms.