Adeno-associated Virus (AAV8) Vector Mediated Gene Delivery Of Angiotensin-converting Enzyme 2 (ACE2) Ameliorates Experimental Autoimmune Uveitis

Background: Uveitis is a sight-threatening inflammatory ocular disease caused by autoimmune or infection.It is one of the main causes of visual impairment,even blindness within the working population worldwide.Experimental autoimmune uveitis(EAU)is a classic animal model that represents human autoimmune uveitis.It is a T cell mediated autoimmune disease model that closely resembles several types of uveitic disorders,such as Beh?et’s disease(BD)and Vogt-Koyanagi-Harada disease(VKH).It is now well recognized that two different effector T cell subsets: Th1 and Th17 cells play important roles in the pathogenesis of uveitis.The rennin angiotensin system(RAS)is a hormone system which plays essential roles in modulating the physiologic functions of the cardiovascular and renal systems.Dysfunction of RAS also attributes to the pathogenesis of inflammation and autoimmune diseases.The newly found axis of RAS:ACE2/Ang-(1–7)/Mas axis has not only showed counterbalanced effects of the deleterious ACE/Ang II/AT1 R axis,but also revealed a variety of beneficial effects including anti-fibrosis and anti-inflammation effects.Activating of ACE2/Ang-(1–7)/Mas axis played anti-inflammatory effects in diabetic retinopathy and LPS induced ocular inflammation.However,the effect and the underlying mechanisms by which over-expression of ACE2 in response to ocular autoimmune inflammation remain poorly understood.Based on the aforementioned background,the following aspects are investigated in our present study:(1)Whether AAV8-mediated ACE2 gene delivery plays protective effects in EAU?(2)Whether ACE2 over-expression can regulate the local immune responses in the eyes of EAU?(3)The involvement of ACE2/Ang-(1-7)/Mas axis in the protective effect of ACE2 over-expression.(4)The possible underlying signaling mechanisms of ACE2’s protective effects on EAU.We aim to provide new evidences for discovering new therapeutic targets to protect against uveitis.Purpose: The purpose of this study is to investigate the effect and the possible mechanism of AAV8-mediated gene delivery of ACE2 in protecting against EAU in mice.Methods: AAV8-ACE2 or control vector were subretinally injected to B10.RIII mice 3 weeks prior to interphotoreceptor retinoid binding protein(IRBP161-180)subcutaneous injection to induce EAU.The anterior segment of the mice was examined at different time points after IRBP injection by slit lamp and clinical scores were determined at the same time.Morphology and histological scores were evaluated by HE staining.The retinal function was assessed by electroretinogram(ERG).The m RNA levels of retinal inflammatory cytokines and the markers of M1/M2 macrophages were analyzed by real-time PCR.The involvement of the possible signaling pathways were investigated by using corresponding inhibitors and detected by ELISA.The protein expressions of the pathways were evaluated by Western blotting.Results: The inflammation of anterior segment and the clinical scores was significantly lower in the AAV8-ACE2+EAU group than the AAV8-e GFP+EAU group.Histological examination showed less retinal folds and inflammatory cells in AAV8-ACE2 treated eyes.The amplitude of a-,b-wave in both light-and dark-adapted ERG was higher in the AAV8-ACE2+EAU group compared with the AAV8-e GFP+EAU group and EAU group.The m RNA levels of inflammatory cytokines were significantly reduced in AAV8-ACE2 treated group.The transduction of AAV8-ACE2 in EAU mice resulted in the inhibition of ocular Th1/Th17 response and the polarization from M1 macrophages to M2 macrophages.The pathways involvement study showed that MAPK,NF-κB and STAT3 were all involved in the protective role play by ACE2 over-expression.An Ang-(1-7)antagonist,A779 abrogated the protective effects of AAV8-ACE2.Conclusions: AAV8-mediated ACE2 over-expression exerts protective effect on ocular inflammation in EAU mouse model by activating ACE2/Ang-(1-7)/Mas axis.This protective axis may regulate the differentiation of Th1/Th17 cells and the polarization of M1/M2 macrophages in the eye.These results support the notion that activating local ACE2/Ang-(1-7)/Mas axis plays a potent role in modulating ocular immune response and that enhancing ACE2 provides a novel therapeutic strategy for uveitis.