Increased inflammatory mediators in the ocular surface tissue in keratoconus

Abstract

Purpose: This study aimed to characterize the inflammatory mediators present in the tear film of patients with keratoconus (KC). It also aimed to investigate the gene expression of these mediators in corneal epithelial cells and their immune activity in conjunctival epithelial cells in patients with KC compared to a control group.

Methods: This transversal study included 30 patients with KC and 23 control group participants. Tear samples were collected by washing the ocular surface with 60 μL of sterile buffered saline solution. The levels of interleukin IL-5, IL-13, IL-2, IL-6, IL-10, interferon-gamma, tumor necrosis factor-alpha, and IL-4 were measured using a LEGEND plex HU Th1/Th2 panel kit and analyzed using flow cytometry. Corneal epithelial samples were obtained via manual keratectomy from KC patients scheduled for corneal crosslinking and from individuals scheduled for photorefractive keratectomy (control group). These samples were immediately stored at -70 °C for mRNA extraction and subsequent reverse transcription polymerase chain reaction analysis to measure IL-5 and IL-6 gene expression. Conjunctival epithelium samples were collected using impression cytology and analyzed using immunohistochemistry and confocal microscopy to detect IL-5 and IL-6 immunoreactions.

Results: Our study found no statistically significant differences in the tear film cytokine concentrations between the two groups. In addition, the gene expression of IL-5 and IL-6 in the corneal epithelium was higher in the KC group than in the control group, with IL-5 showing a 50% increase and IL-6 showing a 20% increase. Immunohistochemical analysis revealed a greater immunostaining of IL-5 and IL-6 in the conjunctival epithelium of patients with KC compared to the control group.

Conclusions: In this study, despite higher levels of IL-5 and IL-6 in the tear film of patients with KC, there was no statistically significant difference compared to the control group. However, there was heightened immune activity in the corneal and conjunctival epithelial cells of patients with KC based on IL-5 and IL-6 gene expression and their immunodetection, respectively.

Figure 1

Bar graph demonstrating IL-5 and IL-6 gene expression in corneal epithelial cells from the keratoconus and control groups.

Figure 2

Immunofluorescence of conjunctival epithelial cells showing higher levels of IL-5 and IL-6 in patients with keratoconus compared to the control group. A. Immunofluorescence of control conjunctival epithelial cells labeled for 4′,6-diamidino-2-phenylindole (DAPI, blue), IL-6 (green), IL-5 (red), and merge. B. Immunofluorescence of keratoconus conjunctival epithelial cells labeled for DAPI (blue), IL-6 (green), IL-5 (red), and merge (bars=20 um). C. Intracellular fluorescence intensity of IL-6. D. Intracellular fluorescence intensity of IL 5. ****p<0.0001.