Abstract
Purpose :
To determine if 24-hour exposure to desiccating stress activates NFkB and NLRP3 inflammasome pathways in the mouse cornea epithelium.
Methods :
6-to-8-week-old C57BL/6J mice were housed under normal humidity (nonstressed) or subjected to desiccating stress from a drafty, low humidity environment combined with SC scopolamine QID for 1 day to suppress tear production (DS1). Eyes were embedded and sectioned for immunofluorescent staining or the corneal epithelium was scraped for immunoassay, caspase-1 assay, western blot or real time PCR. A TransAM kit was used to detect phospho-NFkB p65 in the nuclear fraction. Western blot and real-time PCR were performed to detect NFkB and NLRP3 pathway proteins and gene transcripts, respectively.
Results :
There was significantly increased nuclear p-p-65 protein and gene expression of the NFkB inducible cytokine IL-12 (1.9-fold) and its receptors (IL-12Rb1 and IL-12Rb2, 3 and 1.9-fold, respectively) in DS1 compared to nonstressed control. NLRP3 and Caspase 1 proteins (6.4 and 3.1-fold, respectively), RNA transcripts (1.4 and 1.9 fold, respectively), as well caspase 1 enzyme activity (1.5-fold, P=0.006) increased in DS1. IL-18, the inflammasome inducible cytokine protein increased 3.1-fold, while RNA expression of its receptor IL-18Rap increased 1.5-fold (P=0.001). There was no change in IL-1b expression. Immunostaining for NLRP3, Caspase 1 and IL-18 increased in the cornea epithelium at DS1 compared to nonstressed.
Conclusions :
These findings indicate that two key innate inflammatory pathways (NFkB and NLRP3 inflammasome) are activated following short term desiccating stress. Cytokines stimulated by these pathways, IL-12 and IL-18, are required for full expression of the dry eye associated cytokine IFN-γ by T and natural killer cells.
This is a 2021 ARVO Annual Meeting abstract.