Investigation On The Mechanisms Of IL-17A And Macrophage Polarization On Experimental Ocular Neovascularization

Purpose: Neovascularization(NV),as a cardinal complication of several ocular diseases,has been intensively studied,and increasing researches have shown its close association with inflammation and immune cells.The purpose of the present study is to investigate the mechanisms and the role of IL-17 A in macrophage activation and polarization shift as well as angiogenesis in the process of ocular neovascularization(NV)both in vivo and in vitro.Methods: 1.C57BL/6J mice were prepared for retinopathy of prematurity(ROP)and laser-induced choroidal neovascularization(CNV).VEGF over-expressed C57BL/6J mice were also employed.Retina or choroid whole mount staining was performed to analyze the NV area of several mice models of ocular NV with IL-17 A neutralizing antibody(NAb)or recombinant IL-17A(rIL-17A)and PBS administrated.2.Wild type or IL-17 A knockout(IL-17A-/-)C57BL/6J mice at postnatal day 7(P7)were randomized to control and ROP model groups.Inflammation and macrophage phenotype associated cytokines in retinas were evaluated by realtime RT-PCR.The polarization pattern of macrophages in IL-17A-/-and wild type ROP mice was analyzed by flow cytometry after sorting with CD11 b beads at different time points.3.Mouse macrophage cell line RAW264.7 cells were cultivated in vitro with different concentrations of rIL-17A(0?10?50?100ng/ml)for 24 h.Western blots were performed to detect the phosphorylation state of signal pathway proteins of MAPK(p38,ERK and SAPK/JNK)and AKT,through which macrophages might be modulated by IL-17 A,and protein expression of Notch1.Macrophage polarization associated cytokines were evaluated by realtime RT-PCR.Quantibody cytokine array kit was applied to examine cytokine concentrations in culture supernatant of RAW264.7 treated with rIL-17 A.Supernatant was also collected for measurement of the accumulation of nitrite according to the Griess reaction.4.Human umbilical vascular endothelial cells(HUVECs)were cultured in vitro with different concentrations of rIL-17A(0?10?50?100ng/ml)or supernatants of RAW264.7 cells treated with rIL-17 A.Realtime RT-PCR was applied to examine the expression of VEGFR1 and VEGFR2.Cell proliferation was measured with CCK-8 and tube formation assay were performed to study the effects of IL-17 A on endothelial cells in vitro.Results: Mice intravitreally administrated with IL-17 A NAb developed significantly reduced choroidal NV(CNV)and RNV compared to age-matched controls.IL-17 A deficiency shifted macrophage polarization towards an M2 phenotype during RNV with significant reduced M1 cytokine expressions compared to wild type controls.In vitro assays revealed that IL-17 A polarized macrophages to pro-inflammatory and pro-angiogenic M1 phenotype,which probably gave rise to elevated HUVECs proliferation and tube formation as well as VEGFR1 and VEGFR2 expression.Conclusions: IL-17 A neutralization alleviated ocular NV by promoting macrophage polarization shift towards M2 phenotype and reducing VEGF expression from M1 macrophages,indicating a paracrine role of IL-17 A in endothelial cells and macrophage cross talk in angiogenesis.