NHE1 exacerbates profibrotic activity in idiopathic pulmonary fibrosis and post‐COVID‐19 infection

Idiopathic pulmonary fibrosis (IPF) is a destructive, aging-related pulmonary disease characterized by formation of lung scar tissue. Like other coronaviruses, SARS-COV-2 leads to long term lung damage in some patients in the form of lung fibrosis. In either disease, aggregation of fibroblasts, cell differentiation and excessive tissue remodeling causes the lung tissue to lose lung compliance and oxygen exchange.Sodium hydrogen exchanger isoform 1 (NHE1) is a membrane bound transport protein that functions to support cell movement, regulate cell activity, and balance intracellular pH. A multitude of signaling pathways involved in the regulating of NHE1 function are also involved in the profibrotic activity of fibroblasts and myofibroblasts; therefore we investigated if NHE1 is involved in profibrotic activity and tissue damage associated with IPF and COVID-19 infection. Because NHE1 is key in the remodeling of actin stress fibers and is involved in stiffness of both disorders, we first investigated the impact of NHE1 on stress fiber formation using agonists involved with both fibrosis and COVID response. Lung fibroblasts were treated with three agonists: transforming growth factor-β (TGF-β), serotonin (5HT), and lysophosphatidic acid (LPA) in the presence and absence of an NHE1 inhibitor, ethyl-isopropyl amiloride (EIPA). LPA induced stress fiber formation 65.40+/-3.3 percent over the control, and both 5HT and TGF-β also stimulated the formation of stress fibers by 50.27+/−5.7 and 50.91+/−1.4 percent over the untreated cells. In each case, inhibiting NHE1 with EIPA blocked agonist-activated stress fiber formation, delineating that all three profibrotic factors need NHE1 activity for cytoskeletal remodeling. Another indicator of fibrosis is the fibroblast to myofibroblast transdifferentiation. Myofibroblasts are apoptosis-resistant cells that excessively deposit extracellular matrix proteins; the build up of these cells further compromises the structural integrity of the alveoli and contributes to lung dysfunction. TGF- β, a proinflammatory cytokine, significantly contributes to profibrotic activity by stimulating both fibroblast-myofibroblast transition and excess collagen production. Cells treated with TGF-β to induce cell differentiation showed delayed or significantly reduced myofibroblast transformation in the presence of pharmacological inhibitors of NHE1. Specifically, expression of α-smooth muscle actin (α-SMA), the marker for myofibroblast differentiation, was diminished in each CCL39, LL29, and WI38 cell lines by at least 30 percent and a several day delay of the marker's expression. This study demonstrates the potential for NHE1 as a novel target to fight IPF and lung fibrosis in post-COVID-19 infection.