Interleukin-17 neutralization ameliorates retinal degeneration in Cx3cr1-/-/Ccl2-/-/Crb1rd8 mice

Abstract

Purpose: Interleukin-17A (IL17A) is a driving force in chronic inflammation and its overexpression has been linked to age-related macular degeneration (AMD). IL17A reduces ARPE-19 cell viability in vitro. This study explores the contribution of murine Il17a to retinal degeneration in Cx3cr1-/-/Ccl2-/-/Crb1rd8 (DKO/rd8) mice.

Methods: DKO/rd8 mice were administered intravitreal adeno-associated viral vector serotype 2 encoding soluble IL17 Receptor A (sIL17R) in right eyes and control vector (EV) in left eyes. Pathology was assessed by fundoscope, histology, and biochemically. Mice were sacrificed 2 months after injection. Eye sections were analyzed by H&E staining and transmission electron microscopy (TEM). Expression of the sIL17R vector was verified by ELISA and by analysis of mRNA expression of Il6 and Il17a. Molecular changes due to the injection were assessed by quantification of retinal A2E and mRNA expression of Il17rc, and by Western blotting of p38, Erk1/2 and Akt signaling pathways. For western blots, neuroretinas were dissected from sIL17R- and EV-injected eyes and cultured ex vivo for 2 h in serum free media prior to 0, 5, or 15 min stimulation with 50 ng/ml recombinant mouse Il17a.

Results: Two independent trials were performed with reproducible results. sIL17R was detected in the retina by ELISA and we measured a reduction in Il6 mRNA but no change in Il17a mRNA. By fundoscopy, 26/40 (65%) of sIL17R-treated eyes showed improvement, 12/40 (30%) were similar to EV-treated eyes, and 2/40 (5%) were worse. By histology, 12/15 (80%) of sIL17R-treated eyes showed improvement whereas just 3/15 (20%) worsened. A2E was significantly lower in sIL17R-treated eyes compared to EV eyes, which was confirmed by TEM. Treated eyes had healthier inner and outer photoreceptor segments as well as healthier RPE cells. The sIL17R treatment reduced expression of Il17rc in the retina relative to EV controls. Treatment also resulted in inhibition of signal transduction via the MAPKs p38 and Erk1/2 but had no effect on Akt activity.

Conclusions: Local neutralization of Il17a via intravitreal injection of AAV2.sIL17R significantly ameliorated retinal degeneration in DKO/rd8 mice. Protection was likely conferred from a decrease in MAPK activity. Based on similarities in disease pathogenesis between these mice and AMD, we believe this pathway should be considered in the treatment of AMD.

Keywords: 490 cytokines/chemokines • 412 age-related macular degeneration • 557 inflammation