Lipopolysaccharide-induced inflammatory modulation of the apoptotic pathways in human primary aniridia limbal stromal cells, in vitro

PURPOSE: This study investigated apoptosis-related responses to a standardized 24-hour lipopolysaccharide (LPS) challenge in primary limbal stromal cells (LSCs) derived from patients with congenital aniridia (AN-LSCs) and control donors. METHODS: LSCs were isolated from limbal tissue obtained from patients with congenital aniridia and control donors. Cells were treated with 0, 2.5, 10, and 17.5 µg/mL LPS for 24 hours. Apoptosis was assessed using Annexin V-APC/PI staining and flow cytometry. Apoptosis-related mRNA levels were quantified by qPCR, cell-associated protein fluorescence was assessed by flow cytometry, and secreted TNF-α was measured by ELISA. RESULTS: The mean basal apoptosis rate was numerically higher in AN-LSCs than in control LSCs (2.10% vs. 0.52%), although the donor-level difference was not statistically significant. The exploratory linear mixed-effects model showed a significant main effect of LPS condition and a significant Group × LPS interaction. Apoptosis increased significantly at 10 and 17.5 µg/mL LPS compared with the corresponding untreated condition in both groups, and the increase from the untreated condition to 17.5 µg/mL LPS was significantly greater in AN-LSCs than in control LSCs (adjusted P = 0.003). qPCR and flow-cytometry analyses identified marker-specific LPS-associated changes, but no molecular Group × LPS interaction reached statistical significance. Cell-associated TNF-α fluorescence was distinguished from secreted TNF-α measured by ELISA. CONCLUSIONS: Acute LPS exposure increased apoptosis in both groups, with a greater increase in AN-LSCs at the highest concentration. Molecular changes were marker- and concentration-specific without evidence of differential molecular response patterns between groups. Given the small number of independent donors, the marked donor-age imbalance, and the acute nature of the model, these findings should be considered exploratory and hypothesis-generating and require confirmation in a larger independent cohort of appropriately age-matched donors.