Role Of IL-17A In Diabetic Retinopathy And Involved Mechanism

Background and objectives:Diabetic retinopathy(DR),which represents one of the most serious complications of diabetes,is a leading cause of blindness in working age individuals worldwide.The pathogenesis of DR is complex,and several vascular,inflammatory,and neuronal mechanisms are involved.The involvement of inflammatory processes in the induction of structural and functional alterations associated with DR is gaining increasing attention,and it has been particularly associated with the early stages of DR.Since the retina does not have well-developed lymphatic drainage but it has the blood-retinal barrier(BRB),it is generally deemed that lymphocytes do not appear in the retinal parenchyma to produce specific immune inflammatory response.Therefore,the present evidence regarding inflammatory events in DR is restricted to the involvement of innate immunity driven by retinal macrophages and microglia.The involvement of adaptive immune cells,particularly T cells,in DR has not been elucidated.Interleukin-17A(IL-17A)is a proinflammatory cytokine released from T lymphocytes and has been implicated in the pathogenesis of several autoimmune and inflammatory diseases.We have recently reported that plasma IL-17 level is increased in type 2 diabetic patients,suggesting that IL-17 may be involved in DR.Thus,in the present study,we performed in vitro and in vivo experiments and employed several DR animal models to demonstrate the role of IL-17A in DR as well as the target cells and the signaling transduction pathway involved in the IL-17A action on DR.The current study provides a new insight into the inflammatory pathogenesis of DR and also the basic experimental data for potential use of anti-IL-17A-neutralizing antibody in therapeutic strategy for DR.Methods:(1)Ins2Akita(Akita)mutation mice and streptozotocin(STZ)-induced diabetic mice both used as DR models at the 13-14 weeks after the onset of diabetes.(2)IL-17A-deficient Akita mice were obtained by the hybridization of IL-17A-knockout mice and Akita mice and used as IL-17A-deficient DR model at the 13-14 weeks after the mice initiated diabetes.(3)Akita DR mice were injected with IL-17A(8 ng/2 ?l/eye or 40 ng/2 ?l/eye)or Ad-Actl-shRNA(Ad-GFP as a control)in the vitreous cavity.At the 3rd day after IL-17A injection or at the 3rd week after Ad-Actl-shRNA injection,related measurements in the following 8-15 items were performed,but retinal angiography was performed at the 3rd week despite the injections.(4)STZ-induced DR mice were injected with anti-IL-17A-neutralizing monoclonal antibody(mAb)(2 ?g/2 ?l/eye or 10 ?g/2 ?l/eye)or anti-IL-17 receptor(R)A-neutralizing mAb(0.4 ?g/2 ?l/eye or 2 ?g/2 ?l/eye)in the vitreous cavity.Simultaneously,intravitreal injection of PBS(2?l/eye)was used as a control.At the 3rd day after the injections,related measurements in the following 8-15 items were performed,but retinal angiography was performed at the 3rd week after the injections.(5)Rat Müller cell line(rMC-1)and rat primary Müller cells were incubated with high glucose(HG,15 mM or 25 mM)for 48 h,and they were used as DR cell models.(6)The rMC-1 in the HG medium was treated with IL-17A(10 ng/ml,25 ng/ml or 50 ng/ml),anti-IL-17A-nutralizating mAb(1 ?g/ml or 2 ?g/ml),or anti-IL-17RA-neutralizating mAb(1 ?g/ml)for 24 h.The relevant measurements in the following 8-15 items were performed.(7)The primary Müller cells were treated with IL-17A(25 ng/ml),the IKK inhibitor Wedelolactone(Wedel,10 ?M),or Ad-Act1-shRNA(Ad-GFP as a control)for 24 h.The related measurements in the following 8-15 items were performed.(8)Western blot was used to examine expression levels of the molecules in the retinas and Müller cells,IL-17A,IL-17RA,GFAP,VEGF,GS,EAAT1,Actl,TRAF6,occludin,and ZO-1,as well as phosphorylation levels of NF-?B and caspase-3.(9)Enzyme-linked immunosorbent assay(ELISA)was used to determine contents or concentrations of IL-17A,GFAP and VEGF in retinal and Müller cell lysates and in Müller cell culture supernatants.(10)Glutamate content in the retina and Müller cells was detected by high performance liquid chromatography(HPLC).(11)Fluorescent immunohistochemistry was employed to observe the co-expression of GS,a marker of Müller cells,and IL-17A,IL-17RA or VEGF in the retina,as well as the co-expression of NeuN,a marker of neurons,and IL-17A or IL-17RA in the retina.(12)Fluorescein isothiocyanate(FITC)-conjugated concanavalin A(FITC-Con A)labeled adherent leukocytes in the retinal vessels,and then the labeled adherent leukocytes were counted in each retina.(13)Retinal angiography was performed by perfusion of FITC-dextran into the blood circulation.The flat mounted retina was viewed for FITC-dextran exudation under a confocal laser scanning microscope.(14)Apoptosis of retinal ganglion cells was assessed by immunohistochemistry and TUNEL staining.(15)The ultra-structure of the retinal ganglion cell apoptosis was observed under a transmission electron microscope.Results:(1)At three months after the onset of diabetes,the Akita mice exhibited upregulated IL-17A and IL-17RA expression as well as an increased IL-17A content in the retina compared with the wild-type(WT)mice.Similarly,at three months after the onset of diabetes,STZ-treated mice manifested upregulated IL-17A and IL-17RA expression as well as an increased IL-17A content in the retina,with respect to vehicle-treated control mice.(2)In the retinas of Akita mice,cells that co-expressed GS and IL-17A or GS and IL-17RA were identified;however,the co-localization of NeuN and IL-17A or NeuN and IL-17RA was not identified.(3)In HG-treated rMC-1,the numbers of both IL-17A-and IL-17RA-stained cells,the expression of both IL-17A and IL-17RA and the secretion of IL-17A were increased.Similarly,the expression and secretion of IL-17A and the expression of IL-17RA by primary Müller cells were increased in HG condition.(4)IL-17A enhanced HG-induced GFAP expression and production,VEGF expression and secretion,and glutamate production,but downregulated GS and EAAT1 expression in rMC-1.In contrast,anti-IL-17A-neutralizing mAb or anti-I L-17RA-neutralizing mAb reduced all the changes induced by HG,and anti-IL-17RA-neutralizing mAb blocked the IL-17A effects.(5)HG induced Act1 and TRAF6 expression as well as p65 phosphorylation.IL-17A enhanced HG-induced Act1 and TRAF6 expression and p65 phosphorylation,but Ad-Actl-shRNA reduced the HG-induced Actl-TRAF6-p65 activation,and also inhibited the effect of IL-17A promoting this signaling pathway activation.Wedel abolished the p65 phosphorylation by either HG or IL-17A.(6)IL-17A enhanced HG-induced GFAP expression and production,VEGF expression and secretion,and glutamate production,but downregulated GS and EAAT1 expression in primary Müller cells.Both Ad-Actl-shRNA and Wedel reduced all the changes induced by HG,and also inhibited IL-17A effects of exacerbating Müller cell activation and dysfunction.(7)At the three months after the onset of diabetes of Akita mice,the expression of Actl and TRAF6 and the phosphorylation of p65 in the retinas were increased relative to WT mice.Intravitreal injection of IL-17A in Akita DR mice further enhanced the expression of Actl and TRAF6 and the phosphorylation of p65 in the retinas.In contrast,intravitreal injection of Ad-Actl-shRNA in Akita DR mice reduced retinal Actl-TRAF6-p65 activation.(8)Intravitreal injection of IL-17A in Akita DR mice resulted in upregulation of GFAP and VEGF expression,downregulation of GS and EAAT1 expression,enhancement of GS and VEGF co-expression,elevation of glutamate content,increase in vascular adherent leukocyte number,aggravation of vascular leakage,and increases in the numbers of NeuN/TUNEL double-positive cells and NeuN/Caspase-3 double-positive cells in the ganglion cell layer in the retinas.In contrast,intravitreal injection of Ad-Actl-shRNA in Akita DR mice resulted in the contrary effects to those of IL-17A.(9)Intravitreal injection of anti-IL-17A-neutralizing mAb or anti-IL-17RA-neutralizing mAb in STZ-induced DR mice led to downregulated GFAP and VEGF expression,upregulated GS and EAAT1 expression,attenuated GS and VEGF co-expression,decreased glutamate content,diminished vascular adherent leukocyte number,upregulated occludin and ZO-1 expression,alleviated vascular leakage,reduced NeuN/TUNEL double-positive cell number in the ganglion cell layer,and ameliorated ultrastructural changes of ganglion cell apoptosis in the retinas,with respect to intravitreal injection of PBS.(10)Compared with Akita DR mice,IL-17A-deficient Akita DR mice manifested a downregulation of GFAP and VEGF expression,an upregulation of GS and EAAT1 expression,an attenuation of glutamate content,a decrease in vascular adherent leukocyte number,an upregulation of ZO-1 expression,an alleviation of vascular leakage,a reduction of NeuN/TUNEL double-positive cell number in the ganglion cell layer,and a decrease in caspase-3 activity in the retinas.Conclusions:(1)DR induces retinal production of IL-17A and activates the IL-17A-related inflammatory signaling pathway,IL-17RA-Actl-TRAF6-NFKB.Retinal Müller cells play a critical role in the changes induced by DR.(2)IL-17A exacerbates DR via activation of IL-17RA-Actl-TRAF6-NF?B inflammatory signaling pathway,including Müller cell activation and dysfunction,BRB breakdown,retinal vascular leakage,and retinal ganglion cell apoptosis.Müller cells are the crucial target cells in the effects of IL-17A on DR.(3)Both the treatment with anti-IL-17A-neutralizing mAb and the IL-17A-deficient Akita DR mice alleviate DR,demonstrating that IL-17A plays an important role in DR inflammatory pathogenesis.These findings provide a new clue for potential use of anti-IL-17A-neutralizing mAb to treat DR.