Abstract
Purpose :
Toll-like receptor 3 (TLR3) and the retinoic acid-inducible gene I (RIG-I) like RNA helicase gene family are pattern recognition receptors in viral infection which recognize double-stranded (ds) RNA viruses and activate innate immunity. Both polyinosinic-polycytidylic acid [poly(I:C)] and polyadenylic-polyuridylic acid [poly(A:U)] are synthetic analogs of dsRNA that interact with TLR3, however, only poly(I:C) triggers the RIG-I like RNA helicase gene family. The purpose of this present study was to investigate the barrier function in immortalized human corneal epithelial (HCLE) cells and human conjunctival epithelial (HCjE) cells via stimulation of TLR3 by synthetic dsRNA.
Methods :
HCLE and HCjE cells were first cultured on 12-mm Transwell® inserts (Corning®), and then stimulated with 25µg/ml poly(I:C) or 25µg/ml poly(A:U). To block TLR3, 40μM TLR3/dsRNA Complex Inhibitor (Sigma-Aldrich) was then added to the medium. After stimulation for 24 hours, transepithelial electrical resistance (TER) was measured. The flow mechanism was evaluated on poly(I:C) stimulation by dividing into the following four groups: 1) control group, 2) poly(I:C) in both apical and basal chambers group, 3) poly(I:C) in an apical chamber group, and 4) poly(I:C) in a basal chamber group. After stimulation for 1 hour, all poly(I:C) was changed into the culture medium, and measurement of TER was performed.
Results :
Poly(I:C) and poly(A:U) stimulation increased TER after 24-hours (p<0.01). In comparison to the control group, the TER increase in the HCLE cells and HCjE cells in the poly(I:C) group was 73.6% and 94.4%, respectively, while the TER increase in the HCLE cells and HCjE cells in the poly(A:U) group was 49.2% and 12.3%, respectively. In all cells in both groups, the TER increase was blocked by TLR3 inhibitor (p<0.01). Evaluation of the poly(I:C) flow mechanism revealed that TER increased in all groups stimulated with poly(I:C) (p<0.05) at 3 hours, and that the increase remained until 24 hours.
Conclusions :
The barrier function of ocular surface epithelium increased, specifically through TLR3. The reaction between poly(I:C) and TLR3 might possibly occur inside the cells. This increased barrier function acts as one of the host defense mechanisms against viral infection.
This is a 2021 ARVO Annual Meeting abstract.