Abstract
Purpose :
Retinopathy of unknown etiology involving central serous retinal detachment (CSRD) can be associated with occult bacterial infection. A putative role for bacterial peptidoglycan in development of CSRD has not been elucidated. We investigate the presence, activity, and induction of N-acetylmuramyl-L-alanine amidase [NAMAA; also known as peptidoglycan recognition protein (PGRP-2)] as well as NOD ligand-induced stress in human retinal pigmented epithelium (HRPE) cells as an in vitro model of bacteria-induced retinopathy.
Methods :
HRPE NAMAA activity was measured densitometrically using cleavage of FITC-labeled MDP by HRPE cell lysates followed by agarose gel electrophoresis. The effects of NOD1, NOD2, and NOD1/2 ligand stimulation on the RPE amidase activity, as well as on other biochemical markers [caspase-3, gamma-glutamyl transpeptidase (GGT), and glutathione (GSH)] were tested. Specific inhibition of the NAMAA activity was determined by anti-PGRP specific antibodies and was visualize in ethanol fixed HRPE by immunofluorescent antibody labelling.
Results :
The agarose gel method to detect NAMAA cleaved FITC-MDP (NOD2 ligand) was sensitive down to 5 ng. HRPE cell lysates contained enzymatic activity that cleaves FITC tagged MDP in a dose and time-dependent manner. Multiple PGRP (PGN receptor proteins) were detected in HRPE cells by immunofluorescent antibody (IFA). Antibodies to PGRP 1 and 2 were shown to inhibit the HRPE’s NAMAA activity by inhibiting its cleavage of FITC-MDP. NOD1/2 ligand stimulation of RPE cells did not stimulate increased NAMAA or caspase activity, but NOD ligand incubation for 24-72 h decreased glutathione (GSH) and increased gamma-glutamyltranspeptidase (GGT) activity.
Conclusions :
The results suggest that HRPE NAMAA activity may contribute to retinal innate immunity; potentially acting against the effects of bacterial infection. That is, the NAMAA may cleave L-alanyl containing peptidoglycans and inhibit pro-inflammatory activity. Concomitantly, the results suggest that NOD ligand stimulation of HRPE cells leads to oxidative stress suggested by loss of anti-oxidant potential (reduced GSH) and increased GGT activity (GSH recapture).
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.