Reactive oxygen species activated NLRP3 inflammasomes prime environment-induced murine dry eye

Abstract

Tear film hyperosmolarity along with exposure to oxidant stress are factors that can induce chronic ocular surface inflammation and pain. However, there is limited information on how increases in reactive oxygen species (ROS) generated by oxidant exposure can induce inflammation. There is emerging evidence in other tissues that innate immune responses to a variety of environmental stresses stem from ROS-induced cytosolic NLRP3 inflammasome activation. Once this occurs, pro-caspase-1 is converted into its catalytic active form, which in turn cleaves pro-IL-1β thereby generating its bioactive form. We determined the role of ROS generation in mediating increases in IL-1β secretion through caspase-1 activation caused by NLRP3 inflammasome activation in an environment-induced murine dry eye (DE) model. An intelligently controlled environmental system (ICES) induced evaporative DE in female 4-6 week old C57BL/6J mice. Increases in ROS production preceded rises in corneal and conjunctival gene expression of NLRP3 inflammasome components and IL-1β that were identified using real-time PCR. Confocal microscopy evaluated concomitant increases in NLPR3, caspase-1 and IL-1β immunostaining. Increases in caspase-1 activity were used as an indicator of inflammasome activation. Rises in ROS generation occurred after 1 week of ICES exposure, which preceded increases in gene expression of three NLRP3 inflammasome components (i.e. NLRP3, ASC and caspase-1) leading to rises in bioactive IL-1β release. Increases in caspase-1 activity occurred after 2 weeks of ICES exposure. Eyedrops containing 0.3% N-acetyl-l-cysteine (NAC) were applied to quench ROS generation by mice kept in the ICES for 2 weeks. This scavenger reduced corneal fluorescein staining and decreased ROS production. NAC also down-regulated both increases in NLRP3, ASC, caspase-1 and IL-1β mRNA levels, along with their immunostaining. It robustly attenuated rises in inflammasome mediated increases in caspase-1 catalytic activity. We show in a dessicating DE disease murine model that rises in ROS generation trigger NLRP3 inflammasome complexation and activation leading to increases in bioactive IL-1β secretion. These results prompt us to suggest that the ROS-NLRP3-IL-1β signaling pathway might play a priming role in environment-induced DE progression. Finally, our findings provide a basis for developing novel strategies that may improve the management of patients requiring treatment for environment-induced dry eye disease.

Keywords: ICES; IL-1β; NLRP3; caspase-1; dry eye; inflammasomes; inflammation; reactive oxygen species.