Extracellular vesicles released by microglia exposed to elevated hydrostatic pressure promote retinal neural cell loss and microglia reactivity

Abstract

Purpose : Microglia-mediated neuroinflammation plays a role in retinal degeneration in glaucoma, a leading cause of blindness. Nevertheless, how microglial cells send the inflammatory signal to other retinal cells is still poorly understood. Previous studies have demonstrated that brain microglia under pathological conditions release exosomes, small extracellular vesicles (30-150 nm) that amplify the inflammatory milieu and contribute to neurodegeneration. Herein, we hypothesise that retinal microglia exposed to elevated pressure spread the inflammatory signal through the release of exosomes orchestrating the loss of retinal ganglion cells.

Methods : Microglial cells were exposed to elevated hydrostatic pressure (EHP, 70 mmHg above atmospheric pressure) for 24 hours. Exosomes were isolated by sequential ultracentrifugation and characterized. These vesicles were incubated in naïvemicroglial cells or primary retinal neural cell cultures and the effects of exosomes on microglia reactivity and neural cell loss was determined. Exosomes were injected intravitreally in C57BL6 mice and microglia reactivity and the number of retinal ganglion cells were assessed.

Results : The exposure to EHP increased the release of exosomes by microglia. EHP-derived microglial exosomes alter naïve microglia towardsa pro-inflammatory phenotype with increased expression of markers of reactivity, pro-inflammatory mediators and augmented motility and phagocytic efficiency. In addition, exosomes derived from EHP-challenged microglia increased the production of ROS and cell death in primary retinal neural cell cultures. Preliminary results suggest that injection of EHP-derived exosomes induce microglia reactivity and decrease the number of retinal ganglion cells.

Conclusions : These results demonstrate that exosomes derived from microglia impact the inflammatory milieu and contribute to the loss of retinal ganglion cells, suggesting a role in glaucoma.
Support: Foundation for Science and Technology (FCT), Portugal (PhD fellowships SFRH/BD/115003/2016, PD/BD/127821/2016, PD/BD/114115/2015, and PD/BD/52294/2013, Grant PTDC/BIM-MEC/0913/2012, and Strategic Project UID/NEU/04539/2013), COMPETE-FEDER (FCOMP-01-0124-FEDER-028417; POCI-01-0145-FEDER-007440), Centro 2020 Regional Operational Programme (CENTRO-01-0145-FEDER-000008: BRAINHEALTH 2020), Banco Santander Totta (Grant FMUC-BST-2016-224).

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.