Cathepsin S activation contributes to elevated CX3CL1 (fractalkine) levels in tears of a Sjögren’s syndrome murine model

Abstract

Purpose : Autoimmune dacryoadenitis and altered lacrimal gland (LG) secretion are features of Sjögren’s syndrome (SS). The activity of the cysteine protease, cathepsin S (CTSS), is significantly and specifically increased in SS patient tears. The soluble chemokine, CX3CL1 (fractalkine), is cleaved from membrane-bound CX3CL1 by proteases including CTSS. Here we investigate the association of CX3CL1 with elevated CTSS in tears, cornea and LG of a murine model of SS, the male NOD mouse.

Methods : Age-matched male NOD ShiLtJ (NOD) and BALB/cJ (BALB/c) mice were used from 16-17 weeks. CX3CL1 and CTSS activity in mouse reflex tears, cornea and LG were measured by ELISA and using a commercial CTSS activity kit, respectively. In vitro culture of primary mouse lacrimal gland acinar cells (LGAC) from BALB/c mice and human corneal epithelial cells (HCE-T) were used to elucidate further mechanisms of CX3CL1 induction and shedding. Flow cytometry analysis was used to quantify CX3CR1+ immune cells, which bind CX3CL1, in mouse LGs.

Results : CX3CL1 was significantly elevated by 2.5-fold in tears (p=0.0116) and 1.4-fold in LG (p=0.0026) from NOD mice relative to BALB/c controls. Primary mouse LGAC and HCE-T cells exposed to IFN-γ, a cytokine elevated in SS, showed up to 9.6-fold (p≦0.0001) and 25-fold (p≦0.0001) increases in CX3CL1 gene expression, and 1.9-fold (p=0.0005) and 196-fold (p≦0.0001) increases in CX3CL1 protein expression, respectively. Moreover, exposure of HCE-T cells to recombinant human CTSS at activity equivalent to that in SS patient tears increased cellular CX3CL1 gene and protein expression by 2.8-fold (p=0.0021) and 5.1-fold (p≦0.0001), while increasing CX3CL1 in culture medium by 5.8-fold (p≦0.0001). Flow cytometry demonstrated a 4.5-fold increase in CX3CR1-expressing immune cells (p≦0.0001 in LG from NOD mice relative to BALB/c.

Conclusions : CX3CL1 is elevated in tears, cornea and LG of a murine model of the ocular symptoms of SS, correlated with increased CTSS. CTSS-mediated induction/cleavage of CX3CL1 may contribute to the ocular surface and LG inflammation in SS.

This is a 2020 ARVO Annual Meeting abstract.

 

Proposed mechanism for CX3CL1 cleavage/induction by CTSS and IFN-γ.