Abstract
Purpose:
To investigate the response to polyinosinic:polycytidylic acid [Poly(I:C)], an analog of viral double-stranded RNA produced during viral replication in the barrier function of immortalized conjunctival epithelium.
Methods:
Immortalized conjunctival epithelial cells were cultured on 12-mm Transwell filters at a density of 4x105 cells/cm2. The cultured cells were then stimulated with 2.5µg/ml, 25µg/ml, and 250µg/ml of Poly(I:C). Transepithelial electrical resistance (TER) was then measured using endohm electrodes (World Precision Instruments, LTD., Hertfordshire, UK). After 3 hours of exposure to Poly(I:C), the expressions of tight junction-related protein ZO-1, occludin, and claudin-1,-4, and -7 were analyzed by Western blotting. Immunoreactive bands were visualized by chemiluminescence, and densitometry analysis was then performed.
Results:
After 3 hours of exposure to Poly(I:C), TER was increased in a dose-dependent manner (control: 51.6±6.9Ω･cm2, 2.5µg/ml stimulation: 64.4±2.9Ω･cm2 , 25µg/ml stimulation: 114.9±9.6Ω･cm2, and 250µg/ml stimulation: 309.2±34.7Ω･cm2). Poly(I:C) challenge also increased the TER in a time-dependent manner (0hour: 53.9±1.3Ω･cm2, 1hour: 119.4±7.4Ω･cm2 ,: 2hours 194.4±14.7Ω･cm2, and 3hours: 241.8±19.9Ω･cm2 by 25µg/ml stimulation). Claudin-4 expression was decreased, yet no change was observed in the other tight junction-related proteins.
Conclusions:
The findings of this study show that Poly(I:C) challenge, which mimics a viral infection, increased the barrier function of ocular surface epithelia, and decreased claudin-4 expression due to a strengthening of the barrier. Thus, we theorize that the increased barrier function must be a kind of defense reaction to viral infection.