Reciprocal effects of conditioned medium on gene and protein expression of limbal epithelial cells and limbal fibroblasts in congenital aniridia

Abstract

Congenital aniridia is marked by substantial inflammatory changes to the ocular surface. However the exact mechanisms of epithelial-stromal interaction are not fully understood. The purpose of this study was to investigate inflammatory cytokine expression in limbal epithelial cells and fibroblasts following exposure to each other’s conditioned medium (CM). Healthy primary limbal epithelial cells (pLECs) and healthy (LFC) or aniridia primary limbal fibroblasts (AN-LFC) were isolated. A PAX6-deficient limbal epithelial cell line (mut-LSCs) modeled aniridia. pLECs underwent siRNA-mediated PAX6 knockdown (siPAX6 pLECs) with control cells transfected with non-specific siRNA (siCtrl pLECs). siCtrl and siPAX6 pLECs were treated with LFC-CM and AN-LFC-CM for 24 hours, while LFC and AN-LFC were treated with pLECs-CM and mut-LSCs-CM for 48 hours. Gene and protein expression of IL-1β, IL-6, IL-8, TNF-α and VEGF-A were measured using qPCR and ELISA. Except for an increased IL-8 protein expression in siPAX6 pLECs treated with LFC-CM, gene and protein levels of inflammatory biomarkers remained unchanged in siCtrl and siPAX6 pLECs, regardless of treatment with LFC-CM or AN-LFC-CM. In LFCs, pLECs-CM decreased TNF-α mRNA and IL-8 protein, while increasing IL-1β, IL-6, IL-8 mRNA and IL-6 protein. LFCs treated with mut-LSCs-CM showed decreased TNF-α mRNA and increased IL-6 protein. AN-LFCs treated with pLECs-CM showed increased IL-6, IL-8 and VEGF-A mRNA and IL-6 protein. mut-LSCs-CM did not alter AN-LFC expression. Limbal fibroblasts’ secretome minimally inflames limbal epithelial cells, suggesting a supportive niche role. In contrast, pLECs-CM induces a stronger fibroblast response, indicating abnormal interactions in congenital aniridia.