Phagocytosis is active in retinal microglia during Experimental Autoimmune Uveitis (EAU)

Abstract

Purpose : Although activated microglia are detected in the retina during uveitis, their functions remain unknown. Recently we have demonstrated that healthy retinal pigment epithelial cells suppress the process of phagocytosis. Since, activated microglia may play a role in presenting antigen to effector T cells during EAU, we hypothesized retinal microglia exhibit any phagocytic activity.

Methods : Mice (C57BL/6) were immunized with complete Freund’s adjuvant and IRBP followed by Pertussis Toxin injections to induced EAU. The EAU was scored once a week by fundus examination. When the EAU reached a score of 3, the eyes were collected. The eyes were dissected and the neural retinas were separated into two groups. The neural retinas in Group 1 were incubated with opsonized-AlexaFluor488-E. coli bioparticles in serum free medium for 24 hours. They were fixed in 4% paraformaldehyde and immunostained for Iba-1. The density of Iba-1 positive cells containing bioparticles was counted. In Group 2, the retinal microglia were isolated from the neural retina by centrifugation through a discontinuous 35% and 70% Percoll gradient, the microglial cells were collected at the 35% and 70% interface. The microglial cells were incubated with opsonized pHrodo-S. aureus bioparticles for 24hr. Peritoneal macrophages were used as a positive control for phagocytosis. The fluorescence intensity determined as a measure of phagocytic activity.

Results : In healthy whole mount neural retina (n=3) the density of Iba-1 positive microglia cells in the nerve fiber layer (NFL) was 60.4±0.5 cells/mm², and none in the outer nuclear layer (ONL). In the EAU whole mount neural retina (n=3), the density of Iba-1 positive microglia cells in the NFL was 179.3±54.4 cells/mm², and in the ONL was 121.8±41 cells/mm². In the EAU neural retina the density of Iba-1 positive cells containing AlexaFluor488-E. coli bioparticles was 11.1±4.8 cells/mm² in NFL, and 42.8±24.5 cells/mm² in ONL. In contrast, there was no detectable phagocytized bioparticles in healthy neural retina. The isolated retinal microglia showed similar differences in that microglial cells from retina with EAU demonstrated phagocytic activity, whereas, the microglial cells from naïve mice showed none.

Conclusions : The results demonstrate little phagocytic activity by microglial cells in healthy retina, which is activated in EAU.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.