IL-36α/IL-36RA/IL-38 signaling mediates inflammation and barrier disruption in human corneal epithelial cells under hyperosmotic stress

Abstract

To explore the distinct expression and diverse roles of IL-36 cytokines in dry eye disease using an in vitro hyperosmolarity model of human corneal epithelial cells (HCECs). Primary HCECs were cultured from fresh donor limbal explants. Hyperosmolarity model was established by switching HCECs from isosmotic (312mOsM) to hyperosmotic medium (350-500 mOsM) alone or with addition of rhIL-36RA or rhIL-38 for 2-48h. Some cultures were treated with IL-36α (1-10ng/ml) with or without rhIL-36RA or rhIL-38. Gene expression was detected by RT-qPCR; and protein production and barrier disruption were evaluated by ELISA and/or immunofluorescent staining. IL-36 cytokines were differential expressed in primary HCECs. Among 3 pro-inflammatory agonists, IL-36α, but not IL-36β and IL-36γ, was distinctly induced at osmolarity-dependent manner while two antagonist IL-36RA and IL-38 were significantly suppressed in HCECs exposed to hyperosmotic stress. IL-36α increased to 4.4-fold in mRNA and 6.9-fold at protein levels (116.0±36.33pg/ml vs 16.79±6.51pg/ml in controls) by 450 mOsM, but dramatically inhibited by addition of rhIL-36RA or rhIL-38. Exogenous rhIL-36α stimulated expression of TNF-α and IL-1β at mRNA and protein levels and disrupted tight junction proteins ZO-1 and occludin. However, rhIL-36RA or rhIL-38 suppressed TNF-α and IL-1β production and protected HCECs from barrier disruption in response to IL-36α or hyperosmolarity. Our findings demonstrate that the stimulated pro-inflammatory IL-36α with the suppressed antagonists IL-36RA and IL-38 is a novel mechanism by which hyperosmolarity induces inflammation in dry eye. IL-36RA and IL-38 may have a therapeutic potential in dry eye.