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

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 membrane-bound CX3CL1 by proteases including CTSS. We show that CX3CL1 is significantly elevated by 2.5-fold 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 5.1-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), 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.

. Gating strategy to identify immune cell populations. As reported 1 , the dot plot shows selected windows and the gating strategy applied to identify immune cell populations in the LG, shown with solid arrows. The gating strategy begins on the top row, R1 to R3 gate for single live immune cells, which were then gated with CD11c and CD11b to identify CD11c+ dendritic cells. CD11b+ CD11ccells in R5 were further identified as macrophages (MΦ) and monocytes (Mo) with F4/80 and Ly6C. CD11b-CD11c-cells in R6 were distinguished as T-cells and nature killer (NK) cells by CD3 and CD335 markers, respectively. Broken arrows show additional gating steps to identify immune cell population within CX3CR1+ cells in R4. Supplementary Fig. S4. Representative images and quantitative of lymphocytic infiltration in LG of female NOD. 2 to 7 month (M) old female NOD mice (N = 4 mice per group) LGs were sectioned and stained with H&E. A. Image quantification from B. showed little to no sign of lymphocyte infiltration in female mouse LG. One-way ANOVA with Tukey's multiple comparison test was used to for statistical comparison. No significant difference were detected among age groups. Data represents Mean ± SD. LG were determined by qPCR. One-way ANOVA with Tukey's multiple comparison test was used to for statistical comparison. No significant difference were detected among age groups. Data represents Mean ± SD.
Supplementary Fig. S6. Blood glucose levels in 1 to 6 month (M) old male NOD mice. The levels of glucose were measured in blood to check for diabetes in 1 to 6 M old NOD mice (N=6-8 mice per group). None of the mice developed diabetes (>250 mg/dL) through the experimental endpoint. Data represents Mean ± SD and each point represents a value from one mouse. One-way ANOVA with Tukey's multiple comparison test was used to evaluate significance. No significant changes were observed between the different groups.

Supplementary methods
Cell and tissue processing and confocal fluorescence microscopy: Cells, LG and cornea were processed for immunofluorescence labeling as described previously. 2 The LG and cornea were fixed in 4% paraformaldehyde and 4% sucrose solution in PBS for 3 hr at room temperature. After fixation, tissues were washed with PBS and transferred to a 30% sucrose/PBS solution and left at 4°C overnight. Tissues were embedded in O.C.T compound and frozen on dry ice. O.C.T blocks were sectioned at 5 µm thickness and mounted on glass micro slides (VWR, Radnor, PA). LG cryosections were quenched with NH4Cl (50 mM) in PBS for 5 min and permeabilised with 0.1% Triton X-100 for 10 min. Sections were blocked with 1% BSA for 1 hr at room temperature, followed by primary antibody and secondary antibody incubations, each for 1 hr at 37°C. PBS washes were applied after each antibody incubation. Corneal cross sections were quenched with NH4Cl (50 mM) in PBS for 5 min and permeabilised with 0.3% Triton X-100 for 30 min.
Corneal sections were blocked with 5% BSA in 0.3% Triton X-100 for 1 hr at room temperature and incubated in primary antibody at 4°C overnight. The sections were washed three times with PBS and incubated with secondary antibody for 1 hr at 37°C. Mouse LGAC were washed twice with PBS after gently removing the culture medium. Cells were then fixed and permeabilised with methanol and acetone (1:1) at -20°C for 10 min, followed by two 5-min PBS washes, and then blocked with 1% BSA at room temperature for 1 hr. The cells were then incubated sequentially with primary antibody and secondary antibody, with three 5-min PBS washes following each incubation. After the final wash, all samples were fixed with ProLong anti-fade mounting medium and imaged the next day. Images were acquired with a Zeiss LSM 800 with Airyscan. Image quantification was performed using a reported image processing pipeline using python and ImageJ. 3,4 Blood glucose measurements: As previously described, 5 mice were anesthetised briefly with isoflurane through a nose cone. Peripheral blood was collected by tail nick and measured with Free Style Lite test strips. Mice with blood glucose >250 mg/dl 6 were considered diabetic.
Histology analysis of lymphocytic infiltration in female NOD LG: As previously described, 5,7 LG from female NOD mouse were fixed in 10% neutral buffered formalin solution, embedded in paraffin and cut and Gapdh (Mm99999915_g1) and were from Applied Biosystems (Grand Island, NY).