Abstract
Purpose :
To explore the pathological role and molecular mechanism by which hyperosmolarity disrupts corneal epithelial barrier through TNF-α-induced protease cathepsin S with suppressed anti-inflammatory cytokine IL-37 using an in vitro dry eye model with human corneal epithelial cells (HCECs).
Methods :
Primary HCECs were established from fresh donor limbal tissue explants. The cultures in iso-osmolar medium were switched to medium with increasing osmolarity (350, 400, 450 and 500 mOsM), without or with prior incubation of IL-37 for different time periods (2-48 hours). The integrity of apical barrier junction proteins was evaluated by immunostaining and confocal laser scanning microscopy. The expression of cytokines and cathepsin S by HCECs and the condition medium was determined by RT-qPCR, immunofluorescent staining and ELISA.
Results :
The integrity of corneal epithelial barrier was observed to be largely disrupted as shown by 3D confocal laser scanning images of immunofluorescent staining for major junction proteins ZO-1, occludin, claudin 1, and E-cadherin, in HCECs exposed to 450-500 mOsM, when compared with iso-osmolar medium at 312 mOsM. The mRNA expression and protein production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were dramatically stimulated by hyperosmotic stress as evaluated by RT-qPCR and ELISA. The protease cathepsin S also increased significantly at both mRNA and protein levels, as evaluated by RT-qPCR and immunostaining. However, anti-inflammatory cytokine IL-37 was observed to decrease significantly as early as 4 hour and last 24 hours in HCECs exposed to hyperosmotic medium. Interestingly, recombinant human IL-37 was observed to suppress the production of pro-inflammatory cytokines and inhibit TNF-α-induced cathepsin S in HCECs exposed to hyperosmotic medium. Further investigations on molecular pathway are currently underway.
Conclusions :
Our findings demonstrate that the hyperosmotic stress disrupts the corneal epithelial barrier through stimulating pro-inflammatory cytokines and protease cathepsin S while suppressing anti-inflammatory cytokine IL-37. The results may provide a novel insight on molecular pathogenesis and potential therapeutic targets for dry eye patients.
This is a 2020 ARVO Annual Meeting abstract.