Investigating the anti-inflammatory mechanisms of calorie restriction in the aged lacrimal gland - Dry eye is a prevalent condition that significantly impacts the lives of millions of elderly adults worldwide, causing ocular irritation and blurred vision, thereby severely affecting their overall quality of life. The lacrimal gland (LG), responsible for producing the aqueous layer of the tear film, undergoes age-related changes characterized by immune cell infiltration and impaired regenerative capacity. While calorie restriction (CR) has been shown to slow down functional decline in various tissues, its specific effects on LG inflammation remain to be fully understood. In our preliminary studies, we successfully generated the first single-cell RNA sequencing (scRNAseq) atlas of the LG from both young and old mice fed ad libitum (AL) and those subjected to a 40% calorie-restricted (CR) diet for a duration of six months. Our preliminary findings indicate that the aging process is associated with a decline in mitochondrial and lipid metabolism, a process regulated by peroxisome proliferator-activated receptor (PPAR)-α. This metabolic decline contributes to the accumulation of lipids within the epithelial cells of the lacrimal gland. Additionally, we observed an upregulation of the inflammasome pathway, leading to inflammasome activation in the LG epithelial cells, production of inflammatory cytokines such as IL-1β and IL-18, and Gasdermin-D cleavage. Notably, our research revealed a compelling observation: CR effectively reversed the primary dry eye phenotype associated with aging. In the LG, CR led to an elevation in PPAR-α and retinoid X receptor (RXR) levels, thereby enhancing lipid and mitochondrial metabolism. Importantly, CR also resulted in a significant enhancement of the expression of the epithelial progenitor cell markers. Our data suggests that the molecular pro-resolving mechanism underlying CR involves the activation of PPAR-α signaling, that in turn, leads to a reduction in lipid accumulation within the epithelial cells, thereby mitigating cell damage, inflammasome formation and activation of immune cells. We hypothesize that the increase in PPAR-α is a key mechanism by which CR protects epithelial cells from damage and enhances the function of lacrimal gland (LG) progenitor cells. To test these hypotheses, our research will employ several approaches: 1) We will utilize Nanostring GeoMX spatial transcriptomics combined with immunostaining and flow cytometry to elucidate the specific effects of CR on epithelial cells and macrophages within the LG. 2) We will evaluate the impact of modulating PPAR-α on the function of aging LG epithelial cells and the inflammatory responses of immune cells. 3) Furthermore, we aim to determine the effect of PPAR-α modulation on LG regeneration, as well as the growth, proliferation, and differentiation of progenitor cells in LG organoids. By investigating the mechanisms underlying CR, we hold the potential to develop interventions for age-associated LG inflammation and dry eye. This research offers promising prospects for addressing these conditions and advancing the field of ocular health.
