Abstract
Purpose :
We have previously shown that aged C57BL/6J (B6) mice have increased cathepsin S in tears, lacrimal gland lysates, and spleen. High cathepsin levels have been linked to the generation of autoreactive T cells. This study investigated the effects of cathepsin S inhibition on age-related dry eye disease in B6 mice.
Methods :
Female B6 mice aged 15.5 to 17 months were randomized (n=16/group) to receive either a medicated diet formulated by mixing the cathepsin S inhibitor (R05461111, 262.5mg/kg chow, Roche, Switzerland) or standard diet (placebo) for 12 consecutive weeks. Eye draining lymph nodes and lacrimal glands (LGs) were excised and prepared for either histology or assayed by flow cytometry to quantify infiltrating T cells. The number of inflammatory foci (>50 cells) was counted under a 10X microscope lens. Image analysis of scanned H&E-stained LGs was used to calculate focus score/4mm2 and foci area (μm2). Eyes and adnexae were collected and processed for histology. Goblet cell density was investigated in PAS-stained sections. Expression of Tnf, Ifn, Il12, and Cxcl10 was investigated by real-time PCR in LG and conjunctiva.
Results :
Compared to the standard diet, mice subjected to cathepsin S inhibition showed a decrease in CD4+IFN-γ+ (49.6±9 vs. 35±7.5%) and an increase in CD4+Foxp3+ (9.2±4 vs. 19.4±5 %) cells in the LGs (n=5/group, P<0.05 or lower). A similar non-statistically significant trend was observed in ocular draining nodes. The frequency of total CD45+IL-12+ cells was decreased in cathepsin S-inhibitor diet LGs, accompanied by a decrease in frequency and activation (CD86 expression) in CD45+CD11c-MHC II+CD11b+F4/80+ cells. While there was no change in focus score between the treatment groups (2.2±0.7 vs. 2.62±0.7 focus score/4mm2), a significant increase in conjunctival goblet cell density was observed in the inhibitor-treated animals (45±5.5 vs. 30±7.8 goblet cells/mm, P <0.01) compared to the standard diet. Significant lower levels of Ifn, Tnf, Il12 (range 25-40% decrease) were observed in LG, while low levels of Ifn, Il12, and Cxcl10 (range 40-70% decrease) were present in the conjunctiva in the inhibitor-treated mice.
Conclusions :
Altogether our results indicate that therapies aimed at reducing cathepsin S can improve age-related dry eye disease. Further studies with a longer duration are needed to evaluate the role of cathepsin S in the formation of inflammatory foci in the aged lacrimal gland.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.