Assessing the Independent and Combined Effects of Hyperosmolarity and Inflammatory Stress on Human Corneal Epithelial Cells

Purpose: Inflammation and hyperosmolarity drive dry eye disease (DED) by inducing oxidative stress, apoptosis, and immune activation, disrupting tear film homeostasis and epithelial integrity. This study aimed to evaluate the independent and combined effects of hyperosmolarity and inflammatory stress on human corneal epithelial (HCE) cells. Methods: HCE cells were exposed to hyperosmolar conditions using sodium chloride, lipopolysaccharide, or a combination of both for 12, 24, and 48 hours. The effects on cell adhesion, cell proliferation, cytokine secretion, and gene expression were analyzed. Expression of genes and inflammatory cytokines was evaluated for stress response pathways and antioxidant marker regulation. Results: LPS exposure alone significantly increased inflammatory cytokine secretion at 24 hours (e.g. IL-8 = 2481.4 ± 32.5, P < 0.001; IL-17 = 1095 ± 32, P < 0.01) but did not affect adhesion. The combination of LPS and hyperosmolar stress exacerbated cellular damage, with increased levels of IL-6, TNF-α, IL-1β, and TLR2 and 4. Gene expression analysis revealed upregulation of stress response genes (HMOX-1 = 13 ± 1.5, P < 0.001; COX2 = 2.2 ± 0.1, P < 0.001; and TonEBP = 4.7 ± 1.7, P < 0.001) and downregulation of antioxidant markers (SOD1 = 0.5 ± 0.0, P < 0.001). Cytokine and oxidative stress marker secretion (IL-2, IL-8, IL-12, IL-17, IL-23, HMOX-1, COX2, and SOD1) peaked at 48 hours under hyperosmolar and combined stress conditions. Conclusions: This study demonstrates for the first time the combined effect of hyperosmolarity and inflammation that disrupts corneal epithelial homeostasis in vitro, leading to increased oxidative stress by expressing HMOX1, COX2, TonEBP, HIF1, and inflammatory cytokine production. These findings provide further insights into the pathophysiology of DED and support the development of an in vitro model that mitigates hyperosmolar and inflammatory stress-induced epithelial defects.