Single-Cell Transcriptomic Analysis Reveals Age-Associated Cellular and Transcriptional Changes in Murine Lacrimal Glands

Purpose: To characterize age-associated changes in lacrimal gland cellular composition, transcriptional states, and intercellular communication and to explore their relationship with lacrimal gland dysfunction and age-related dry eye disease (DED). Methods: Single-cell RNA sequencing (scRNA-seq) was performed on extraorbital lacrimal glands from young (12-week-old) and aged (20-month-old) male C57BL/6J mice. A total of 47,537 high-quality cells were analyzed using the Seurat pipeline. Differential expression, pathway enrichment (gene set enrichment analysis), and cell-cell communication (CellChat) analyses were used to assess aging-associated changes in immune composition and signaling dynamics. Results: Aging was associated with pronounced immune-cell infiltration accompanied by epithelial and stromal remodeling, with higher observed representation of T cells, B cells, and plasma cells and broad transcriptional alterations across immune populations. Regulatory T cells (Tregs) increased in frequency but exhibited transcriptional profiles suggestive of reduced suppressive capacity. Aged T cells showed enrichment for Th1, Th2, and Th17 differentiation pathways. Also, γδ T cells formed a distinct Th17-skewed subset, and type 2 innate lymphoid cells (ILC2s) expanded with stress-adaptive reprogramming. Migratory dendritic cells and macrophages exhibited activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT), tumor necrosis factor (TNF), and FoxO signaling. CellChat analysis revealed enhanced programmed death ligand 1 (PD-L1) signaling and T-cell-centric communication, alongside diminished homeostatic interactions. Conclusions: Aging disrupts lacrimal gland immune homeostasis through coordinated remodeling of lymphoid, myeloid, and stromal compartments. These changes are associated with chronic inflammation and fibrosis and parallel mucosal immune aging observed in other mucosa-associated lymphoid tissues such as the gut and lung. This high-resolution, single-cell atlas highlights IL-6/transforming growth factor-beta (TGF-β), JAK-STAT, PD-L1, and FoxO signaling as candidate pathways for further investigation, providing a framework for future mechanistic studies and therapeutic exploration in aging-associated DED.