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

Abstract

Autoimmune dacryoadenitis and altered lacrimal gland (LG) secretion are features of Sjögren's syndrome (SS). Activity of cathepsin S (CTSS), a cysteine protease, is significantly and specifically increased in SS patient tears. The soluble chemokine, CX3CL1 (fractalkine), is cleaved from membranebound CX3CL1 by proteases including CTSS. We show that CX3CL1 is significantly elevated by 2.5fold in tears (p = 0.0116) and 1.4-fold in LG acinar cells (LGAC)(p = 0.0026) from male NOD mice, a model of autoimmune dacryoadenitis in SS, relative to BALB/c controls. Primary mouse LGAC and human corneal epithelial cells (HCE-T cells) exposed to interferon-gamma, 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 5.1fold (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), including increased T-cells and macrophages, in LG from NOD mice relative to BALB/c. CTSS-mediated induction/cleavage of CX3CL1 may contribute to ocular surface and LG inflammation in SS. Sjögren's syndrome (SS) is a systemic autoimmune disease associated with lymphocytic infiltration of lacrimal glands (LG) and salivary glands (SG), associated with dacryoadenitis and sialoadenitis, respectively 1. SS patients develop associated complications including reduced tear and saliva production, blurred vision, corneal damage, dental cavities and oral thrush 1. Inflammation in the LG promotes release of pro-inflammatory cytokines to the ocular surface, which can further compromise tear secretion by disruption of corneal sensory and efferent nerve responses 2,3. This further reduces tear flow and alters tear composition, resulting in pro-inflammatory and proteolytic tears 4 which may elicit apoptosis and autophagy to further damage the ocular surface 5. Reduced tear volume, tear film instability and ocular surface inflammation all contribute to the reduced visual acuity and increased patient discomfort associated with dry eye symptoms in SS 2,6. The male non-obese diabetic mouse (NOD) is commonly used as a model of the autoimmune dacryoadenitis and ocular surface inflammation characteristic of SS. While the ocular manifestations of SS spontaneously develop in the males from 8-10 weeks of age, the female mouse develops a later autoimmune sialoadenitis from 14-16 weeks of age 7. The male NOD mice share many ocular surface system manifestations with SS patients including lymphocytic infiltration of the LG 7,8 , reduced tear flow 7-10 , generation of a proteolytic tear film 7,9,10 , altered distribution and expression of Rab3D 9,11 , reduced myoepithelial cells 12 , loss of extracel...

Integrative Identification of Hub Genes Associated With Immune Cells in Atrial Fibrillation Using Weighted Gene Correlation Network Analysis

“…CTSS participated in the abdominal aortic aneurysm (AAA) formation, and inhibition of CTSS suppressed AAA formation in mice ( 29 ). A previous study showed that CTSS -mediated induction of CX3CL1 might contribute to the ocular surface and lacrimal glands inflammation in Sjögren's syndrome with a 4.5-fold increase in CX3CR1 -expressing macrophages ( 30 ). In chronic obstructive pulmonary disease, reduction in CTSS expression prevents loss of lung function, reduces inflammation, and slows the lung tissue remodeling ( 31 ).…”

Rab27a Contributes to Cathepsin S Secretion in Lacrimal Gland Acinar Cells

“…Proinflammatory cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor (TNF-α) are highly expressed in NOD mouse LG [ 27 ]. LGAC treated in vitro with IFN-γ exhibit characteristics of NOD mouse LGAC including reduced Rab3D [ 27 ], increased MHC II [ 27 ] and increased CX3CL1 expression [ 17 ]. Cultured LGAC exposed to IFN-γ also exhibit enhanced CTSS secretion with CCh stimulation, while β-hex release is unaffected [ 27 ].…”

“…Cathepsin S (CTSS) is a lysosomal cysteine protease associated with many physiological processes, including extracellular matrix degradation [ 14 ], major histocompatibility complex II (MHC II) processing in antigen presentation [ 15 ] and protein catabolism [ 15 , 16 , 17 , 18 ]. Dysregulation of CTSS is implicated in multiple autoimmune diseases [ 14 ], including rheumatoid arthritis [ 19 ], systemic lupus erythematosus [ 20 ], multiple sclerosis [ 21 ] and SS [ 19 , 20 , 22 , 23 ].…”

“…CTSS activity is significantly elevated in tears of SS patients compared to tears from patients with non-SS dry eye or other autoimmune diseases [ 23 ]. CTSS activity is also significantly increased in the LG and tears of an SS murine model, the male NOD mouse [ 17 , 24 , 25 ]. Male NOD mice share many characteristics of ocular manifestation with SS patients including reduced tear flow [ 17 , 23 , 24 , 25 , 26 ], lymphocytic infiltration of the LG [ 17 , 24 , 25 ], development of a proteolytic and inflammatory tear film [ 17 , 27 ], loss of extracellular matrix [ 24 ], and altered acinar Rab3D distribution [ 26 , 28 ].…”

“…In vitro studies have shown that CTSS, at concentrations equal to those found in SS patient tears, induces release of pro-inflammatory cytokines from cultured corneal epithelial cells [ 29 ]. CTSS is also involved in activation and release of the chemokine, CX3CL1, from LG acinar cells (LGAC) and into tears in male NOD mice [ 17 ]. In vivo inhibition of CTSS activity in the male NOD mouse significantly reduced lymphatic infiltration of the LG and increased tear flow [ 29 ].…”