Abstract
Purpose :
We reported that aged C57BL/6 (B6) had increased lacrimal gland (LG) lymphoid infiltrates that are suggestive of ectopic lymphoid structures. However, B6 mice are T helper 1 prone mice. This study investigates the presence and composition of LG infiltrates in aged mice. We also analyse the diversity of mice with different genetic background.
Methods :
Female B6 mice were used at 2-4, 12, and 24months (M) of age (n=10/age). A separate group of diversity outbred mice (DO, Jackson strain 009376) were used at 2-4 and 24M of age (n=9-10/age/sex). LGs were excised and used for histology, bulk RNA sequencing, or gene expression analysis using qPCR. Flow cytometry investigated B cell frequency. Eyes were excised for goblet cell density evaluation using PAS staining. The number of inflammatory foci (>50 cells) was counted under a 10X microscope lens. H&E-stained LGs were scanned, photographed, and image analysis calculated the focus score. The expression of B-cell-related genes was investigated by real-time PCR.
Results :
Bulk RNA seq of aged B6 LGs showed upregulation of many inflammatory pathways. B-cell-related genes were among the top hits. A significant increase in B220+ cells was seen with aging from 8 to 12 to 24M in the LG (3, 20, and 38% among live cells, P<0.05). qPCR validation showed a significant increase in Cd19 (70 and 151-fold), Cd22 (46 and 138-fold) Cxcl13 (5 and 11-fold), Ltb (9 and 7-fold), Lta (4 and 5-fold), Glycam-1 (915 and 3095-fold), Ccl19 (11 and 15-fold) at 12 and 24M compared to young (all P<0.05 or lower). T helper follicular genes were also upregulated with aging: Cxcr5 (3 and 3-fold), Ccr7 (8 and 4-fold), Il21 (27 and 24-fold) (all P<0.05 or lower). Aged female DO mice have increased immune infiltrates in aged LGs (3.2±3.5 vs 0 focus score/4mm2, P<0.01), but male DO mice do not (1.6±2 vs 0 focus score/4mm2, P>0.05). Interestingly, female and male DO mice have decreased goblet cell density with aging (~35% loss compared to young DO, P<0.01 for both sexes).
Conclusions :
Many pathways are altered in the aged LG in B6 mice, including B cell infiltration. There is an upregulation of B and T helper follicular-related genes as early as 12M of age. In addition, LG immune infiltrates preferentially accumulate in female mice, while goblet cell loss is observed in both sexes. These results show that age-related dry eye and LG infiltration is not exclusive of a mouse genotype.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.