Immunoglobulin laden exosomes induce complement activation and inflammation in retinal endothelial cells in diabetes.

Abstract

Purpose : Low-grade chronic inflammation is known to induce vascular damage during diabetic retinopathy (DR). Activation of the complement system was shown to lead to pro-inflammatory changes in the retina. However, the role and activation of the complement system in DR are not well understood. Exosomes, membrane vesicles (40-200nm) that are secreted into extracellular environment, were shown to contribute to the pro-inflammatory changes seen in several tissues. The present study aims to elucidate the role of plasma exosomes in activation of the complement pathway during DR pathogenesis.

Methods : Plasma exosomes were isolated from control or STZ-induced diabetic C57/B6 mice, Wistar control rats, and control and type II diabetic patients via ultracentrifugation or the ExoQucik method, and further purified via density gradient separation. Static Light Scattering (SLS) was used to quantify the exosomes. Classical complement activation was measured in vitro by C1 activation assay and C1s activity was determined by Western blot.

Results : In this study we found that most immunoglobulin binds to exosomes in plasma. SLS quantification of plasma exosomes showed significantly higher number of vesicles in diabetic mice (1.14 x10¹¹/mL) when compared to control (0.735x10¹¹/mL) (n=9, p<0.05). Exosome makers, CD63 and ALIX, were used to confirm results and were analyzed via western blot (n=9 p<0.05). Importantly, diabetic mouse plasma had higher exosome-bound IgG levels when compared to control (n=9, p<0.05). Immunoglobulin bound to exosomes caused activation of the classic complement complex, C1, which is the first step in downstream complement protein recruitment (C4 and C2), formation of C3 and subsequent C5 convertase, and release of pro-inflammatory anaphylatoxins. Stimulation with pro-inflammatory anaphylatoxins (C3a/C5a) for 3 and 6 hours induced significant VEGFA (C5a: 30% increase relative to control) and IL1β (C3a: 30% increase relative to control) upregulation in human retinal endothelial cells via their receptors (n=6, p<0.05).

Conclusions : Diabetes-induced increase in immunoglobulin-laden exosomes in circulation can activate classical complement pathway and induce a pro-inflammatory response in retinal endothelial cells. This finding suggests that exosomes can contribute to the development of DR.

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