Hyperosmotic Stress-Induced TRPM2 Channel Activation Stimulates NLRP3 Inflammasome Activity in Primary Human Corneal Epithelial Cells

PURPOSE. The purpose of this study was to determine whether either a hyperosmotic or oxidative stress induces NLRP3 inflammasome activation and increases in bioactive IL-1 beta secretion through transient receptor potential melastatin 2 (TRPM2) activation in primary human corneal epithelial cells (PHCECs). METHODS. Real-time PCR, Western blots, and immunofluorescent staining were used to evaluate TRPM2 and NLRP3, ASC, caspase-1, and IL-1 beta mRNA and protein expression levels, respectively. A CCK-8 assay evaluated cell viability. Hyperosmotic 500 mOsm and oxidative 0.5 mM H2O2 stresses were imposed. TRPM2 expression was inhibited with a TRPM2 inhibitor, 20 lM N-(p-amylcinnamoyl) anthranilic acid (ACA), or TRPM2 siRNA knockdown. RESULTS. In the hypertonic medium, TRPM2, NLRP3, ASC, caspase-1, and IL-1 beta gene and protein expression levels rose after 4 hours (P <= 0.043), whereas ACA preincubation suppressed these rises (P <= 0.044). Similarly, H2O2 upregulated TRPM2 protein expression by 80%, and induced both NLRP3 inflammasome activation and increased bioactive IL-1 beta secretion (P <= 0.036), whereas ACA pretreatment suppressed these effects (P <= 0.029). TRPM2 siRNA transfection reduced TRPM2 gene expression by 70% (P = 0.018) in this hyperosmotic medium and inhibited the increases in NLRP3, caspase-1, and IL-1 beta gene (P <= 0.028) and protein expression (P <= 0.037). CONCLUSIONS. TRPM2 activation by either a hyperosmotic or oxidative stress contributes to mediating increases in NLRP3 inflammasome activity and bioactive IL-1 beta expression because inhibiting TRPM2 activation or its expression blunted both of these responses in PHCECs. This association points to the possibility that TRPM2 is a viable target to suppress hyperosmotic-induced corneal epithelial inflammation.