In studies on
dry eye (DE) disease, an association has been identified between tear film hyperosmolarity and
inflammation severity elicited through receptor-induced increases in proinflammatory
cytokine and
chemokine release. These immune reactions might be mediated by
inflammasomes,
macromolecular complexes mounted around the
NLRP3 protein and can be activated by
reactive oxygen species (ROS) over-generation. Hence in this study we determine whether: a) ROS activated NLRP3
inflammasomes mediate hyperosmotic stress-induced
inflammation in human corneal epithelial cells (HCECs); b) the ROS-NLRP3-IL-1β axis activation is associated with environment-induced DE. Immortalized HCECs were exposed to 500 mOsm medium in the presence and absence of a ROS inhibitor,
N-acetyl-l-cysteine (NAC). HCECs transfected with NLRP3
siRNA or a negative control (NC)
siRNA. Intracellular ROS was measured by fluorometric analysis using the probe 2',7'-
dichlorofluorescin diacetate (
DCFH-DA). Real-time PCR evaluated NLRP3, ASC,
pro-caspase-1 and pro-IL-1β
mRNA levels. Western blot analysis assessed
NLRP3 protein expression whereas
caspase-1 activity was determined with a fluorometric assay. Bioactive IL-1β release was assessed by ELISA. ROS production, NLRP3
inflammasome and pro-IL-1β gene expression as well as IL-1β secretion were also evaluated in the conjunctival epithelial cells and tear fluid samples of environment-induced DE patients and normal subjects. NAC suppressed hyperosmolarity-induced rises in ROS levels, NLRP3
inflammasome formation and activation,
caspase-1 activity and IL-1β release. On the other hand, NLRP3
siRNA knockdown inhibited hyperosmotic stress-induced NLRP3 activation, which led to ASC,
pro-caspase-1 and pro-IL-1β
mRNA down-regulation followed by suppression of associated
caspase-1 activity and IL-1β secretion. In addition, in ocular surface samples of environment-induced DE patients, ROS generation, NLRP3, ASC,
pro-caspase-1 and pro-IL-1β gene expression as well as IL-1β secretion were upregulated. Taken together, NLRP3 mediated innate immune responses triggered by rises in ROS generation induce
inflammation in hyperosmotic stressed HCECs. ROS-NLRP3-IL-1β signaling pathway might play a priming role in environment-induced DE development.