Abstract
Purpose :
Cathepsin S (CTSS) has increased activity in tears of male NOD mice, a murine model of Sjögren’s Syndrome (SS), and is also increased in tears of patients with SS. However, the contribution of its increased activity in tears to the ocular surface inflammation that is a part of SS has not been investigated. We hypothesized that elevated CTSS in tears might drive ocular surface inflammation by altering gene and protein expression of factors linked to corneal inflammation
Methods :
The human corneal epithelial cell line (HCE-T) was cultured and treated with recombinant human CTSS at activity levels seen in SS patient tears. Gene expression of pro-inflammatory cytokines, proteases, and protease-activated receptor-2 (PAR-2) were measured after 2-,4-,8-,and 24-hours of treatment by RT-and q-PCR. CTSS protein expression and its activity in lysates were determined using Western Blotting and CTSS activity assay kits, respectively. PAR-2 protein expression was measured by immunofluorescence.
Results :
Recombinant human CTSS induced gene expression of IL-6, IL-8, TNF-α and IL-1β at 2 and 4 hours. Increased IL-1β and TNF-α reached a peak at 2 hours (Relative Quantity (RQ)=2, p≤0.001 and ≤0.05 respectively). Increased IL-8 and IL-6 reached a peak at 4 hours (RQ=4, p≤0.001). CTSS, MMP-9, and PAR-2 gene expression was increased after 24-hour treatment (RQ= 4, 2, and 1.5 respectively, p≤0.001). Exposure of HCE-T cells to heat-inactivated CTSS was not as effective as in elevating IL-6 and IL-8 gene expression, suggesting that CTSS protein activity was important in eliciting the effect. Recombinant human CTSS exposure also increased expression of endogenous CTSS protein levels and endogenous CTSS activity (fold change=3.5, p≤0.05) after 24 hrs. Finally, recombinant human CTSS increased PAR-2 immunofluorescence after 24 hours.
Conclusions :
These findings suggest that elevated CTSS levels in tears of SS patients may induce inflammatory cytokines, proteases, and PAR-2 expression in corneal epithelial cells, possibly contributing to ocular surface inflammation in SS patients.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.