Effects of age, TNF-α and calorie restriction on cathepsin S and other inflammatory markers in the lacrimal gland

Abstract

Purpose : Calorie restriction reduces the expression of inflammation and increases life span in many organisms. Aged C57BL/6 (B6) lacrimal glands (LGs) have increased inflammation and Th1 infiltration. In this study, we investigate the effects of calorie restriction (CR) or loss of Tnf-α. on the expression of Cathepsin S and markers of inflammation in the LGs of mice during aging.

Methods : B6 mice were used at 2-4, 12, and 24M of age. A separate group of 6M B6 mice received 40% CR or had food ad libitum (AL) for 4M. Young (2-4M) and aged (19-24M) TNF-α^-/- were compared to WT mice. LGs were excised and used for histology or gene expression analysis using qPCR. The number of inflammatory foci (>50 cells) was counted under a 10X microscope lens. H&E-stained LGs were scanned, photographed, and image analysis was used to calculate focus score/4mm² and foci area of foci (μm²). Expression of TNF-α, IL-1β, IFN-γ, MHC II, IL12, and Ctss was investigated by real-time PCR.

Results : A progressive increase in TNF-α (1.7 and 1.9-fold) and IFN-γ (2.8 and 6.2-fold) was observed in 12 and 24M B6 LGs (P<0.001). Expression of Ctss, MHC II, and IL-12 peaked at 12M (7, 5.8, and 122-fold, respectively, P<0.001) and remained significantly elevated at 24M (3.6,3.6, and 56-fold, respectively, P<0.001). IL-1β levels were increased at 24M (2.3-fold, P<0.001). 40% CR significant decreased TNF-α (46%), IFN-γ (60%), MHC II (64%), Ctss (46%), and IL12 (81%) (all P<0.05) but not IL-1β mRNAs. Aged TNF-α^-/- LGs had a 50% decrease in lymphocytic infiltration compared with aged WT (1±0.7 vs. 2±0.8 focus score/4mm², P<0.01) and smaller foci area (47046±34745 vs. 95730±49794 um², P=0.008). A significant increase in TNF-α (3-fold), IFN-γ (9.7-fold), MHC II (10-fold), IL-12 (260-fold), Ctss (7.2-fold), and IL-1β (3.4-fold) mRNA was observed in aged WT compared with young WT LG (P<0.01). However, IFN-γ (1.3-fold), MHC II (4.4-fold), IL-12 (53-fold), and Ctss (2-fold) mRNA levels were decreased in aged TNF-α^-/- compared to WT LGs (P<0.01), but not IL-1β (1.7 fold).

Conclusions : Taken together, our results indicate that alti-inflammatory therapies (such as CR) or inhibition of TNF-α can ameliorate the age-related increase in cytokine production, and in particular, Ctss, and can decrease LG inflammation. Further studies are needed to evaluate the role of Ctss during aging-related inflammation.

This is a 2021 ARVO Annual Meeting abstract.