Epidermal Growth Factor Protects Lung Epithelial Cells After Injury

Abstract Rationale: Thrombocytopenia is independently associated with worse outcomes among individuals with acute lung injury, but the mechanism(s) by which platelets protect patients after lung insult remains uncertain. We used a discovery-driven approach to determine which platelet-derived protein(s) contributes to the observed cytoprotective phenotype and validated potential candidate proteins in vitro. Methods: We first performed single nucleus RNA sequencing (sn-RNA seq) on isolated nuclei from flash-frozen lungs from thrombocytopenic (Mpl-/-) mice treated with either vehicle control or wild-type mouse platelets after intratracheal P. aeruginosa supernatant (SN) insult. Next, we performed bulk RNA sequencing on cultured mouse lung epithelial cells (MLE-12) treated with either vehicle, platelet releasate (PR), SN to induce injury, or SN followed by PR rescue. We then performed in vitro cytotoxicity assays using MLE-12 cells treated with SN with or without epidermal growth factor (EGF) rescue. Lastly, we repeated cytotoxicity assays with or without pre-incubation with the small molecule EGF receptor (EGFR) inhibitor erlotinib. Results: In our sn-RNA seq data, network analysis identified an upregulation of the EGFR pathway in lung epithelial cells after P. aeruginosa SN insult in mice that received platelet transfusion. These results suggested EGF, a platelet protein involved in cell survival that binds EGFR, as a candidate protein for investigation. We next performed network analysis on bulk RNA sequencing data and confirmed upregulation of the EGFR pathway in both the PR alone and the SN with PR groups. When comparing SN with PR rescue versus SN alone, EGFR is the most significantly upregulated pathway. In cultured murine lung epithelial cells, treatment with 10μM EGF produced a significant reduction in cytotoxicity compared to SN alone (EGF normalized median fold change 0.66; SN alone normalized median fold change 1; p-value = 0.02). Pre-treatment with 10μM erlotinib abrogated the cytoprotective effect of EGF when compared to vehicle pre-treatment (EGF with erlotinib normalized median fold change 1.12; EGF with vehicle normalized median fold change 0.68; p-value = 0.03). Conclusions: EGF, a conserved protein present in platelet releasate, is an important factor in lung epithelial response following injury. We show that EGF attenuates cytotoxicity after insult in cultured lung epithelial cells, an effect which is abrogated with inhibition of EGFR. These results suggest that the platelet-epithelial EGF-EGFR axis may play an integral role in cytoprotection after insult.