Caspase-8 Mediated Activation of NLRP3 and NLRC4 inflammasomes in Experimental Dry Eye Mouse Model and Human Corneal Epithelial Cells Exposed to Hyperosmolarity

Abstract

Purpose: To explore the underlying mechanisms of innate immunity in response to desiccating stress using an experimental dry eye mouse model and human corneal epithelial cells (HCECs) exposed to hyperosmolarity, an in vitro model mimicking dry eye environment.

Methods: The experimental dry eye was induce by desiccating stress in C57BL/6 mice exposed to an air draft in <40% ambient humidity with subcutaneous injection of scopolamine hydrobromide for 5 days. Primary HCECs were established from donor limbal explants. The cultures in iso-osmolar medium (312 mOsM) were switched to hyperosmotic media (400, 450 and 500 mOsM), with or without prior incubation of NLRP3 inhibitor (glybenclamide), caspase-1 inhibitor (z-YVAD fmk), or caspase-8 inhibitor (z-IETD fmk). Reverse transcription and quantitative real time PCR (RT-qPCR), immunofluorescent staining, Western blotting, caspase activity assays and ELISA were performed to evaluate the concerned molecules.

Results: NLRP3 and NLRC4 inflammasomes were found to be up-regulated with increased production and activity of ASC, caspase-1, caspase-8, IL-1β and IL-18 in corneal and conjunctival epithelia of the dry eye mice, as well as in HCECs exposed to hyperosmotic media, as evaluated by RT-qPCR, immunofluorescent staining, Western blotting, ELISA and caspase activity assays. Glybenclamide inhibited NLRP3 activation, and also blocked the activation of caspase-1. Both inhibitors to NLRP3 or caspase-1 suppressed maturation of IL-1β and IL-18. Interestingly, blockage of caspase-8 by z-IETD fmk down-regulated the NLRP3 and NLRC4, and further suppressed the caspase-1 dependent activation of IL-1β and IL-18.

Conclusions: These findings uncovered a novel mechanism and signaling pathway of innate immunity by ocular surface mucosal epithelium in response to desiccating environment stress, which activates NLRP3 and NLRC4 inflammasomes through caspase-8 mediated pathway to promote the casapase-1 dependent activation of IL-1β and IL-18 during dry eye development. These results provide new insight in the innate immunity that contributes to pathogenesis of dry eye.