Ectoine protects corneal epithelial survival and barrier from hyperosmotic stress by promoting anti-inflammatory cytokine IL-37

To explore novel role and molecular mechanism of a natural osmoprotectant ectoine in protecting corneal epithelial cell survival and barrier from hyperosmotic stress. Primary human corneal epithelial cells (HCECs) were established from donor limbus. The confluent cultures in isosmolar medium were switched to hyperosmotic media (400–500 mOsM), with or without ectoine or rhIL-37 for different time periods. Cell viability and proliferation were evaluated by MTT or WST assay. The integrity of barrier proteins and the expression of cytokines and cathepsin S were evaluated by RT-qPCR, ELISA, and immunostaining with confocal microscopy. HCECs survived well in 450mOsM but partially damaged in 500mOsM medium. Ectoine well protected HCEC survival and proliferation at 500mOsM. The integrity of epithelial barrier was significantly disrupted in HCECs exposed to 450mOsM, as shown by 2D and 3D confocal immunofluorescent images of tight junction proteins ZO-1 and occludin. Ectoine at 5–20 mM well protected these barrier proteins under hyperosmotic stress. The expression of TNF-α, IL-1β, IL-6 and IL-8 were dramatically stimulated by hyperosmolarity but significantly suppressed by Ectoine at 5–40 mM. Cathepsin S, which was stimulated by hyperosmolarity, directly disrupted epithelial barrier. Interestingly, anti-inflammatory cytokine IL-37 was suppressed by hyperosmolarity, but restored by ectoine at mRNA and protein levels. Furthermore, rhIL-37 suppressed cathepsin S and rescued cell survival and barrier in HCECs exposed to hyperosmolarity. Our findings demonstrate that ectoine protects HCEC survival and barrier from hyperosmotic stress by promoting IL-37. This provides new insight into pathogenesis and therapeutic potential for dry eye disease.