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Yasunari Munemasa, Yasushi Kitaoka, Kaori Kojima, Ayano Hirano, Hitoshi Takagi; Role of Toll like receptor-4 ligand after neuronal cell degeneration in the mice retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5714.
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© ARVO (1962-2015); The Authors (2016-present)
Toll like receptor-4 (TLR4) has been indicated in response to various ligands related with cell death signaling, in addition to autoimmune response. The purpose of this study is to investigate the novelty of TLR4 ligand in retinal ganglion cell (RGC) degeneration.
Eight week male TLR4 knock out mice (TLR4 -/-) and wild type (C57BL/6) were used in this study. To generate optic nerve crush (ONC), the nerve of one eye was exposed and clamped, contralateral eye was used as a control. RGC was labeled with fluoro-gold at superior colliculus and morphological analysis was performed with whole mount retina at 2 weeks after ONC. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOFMS) was used to detect TLR4 ligands after immunoprecipitation with TLR4 antibody and isolation of retinal membrane proteins at 5 days after ONC. Pull-down assay with His-tag protein and TLR4 was performed to confirm the interaction TLR4 and its ligand. Induction of RGC degeneration with TLR4 ligand was studied with cell viability assay using RGC-5 cells and intravitreal injection of TLR4 ligand on TLR4 -/-, and wild mice. Assesment of angiogenesis was studied with fluorescein angiography (FAG) after intravitreal injection of TLR4 ligand.
A significant decrease in RGC was observed in the retina of wild type mice compared to TLR4 -/- mice 2 weeks after ONC. MALDI-TOF MS identified Histone H2B type1-B as the ligand of TLR4. Histone H2B induced RGC-5 cells death in vitro. Pull down assay confirmed interaction of HistoneH2B and TLR4 with immunoblot. A decrease in RGC density was more apparent in the retina of wild type mice compared to TLR4 -/- mice at 7 days after intravitreal injection of Histone H2B. RGC-5 degeneration after treatment of Histone H2B was related with Myd88 and TRIF pathways. FAG showed that intravitreal injection of Histone H2B induced ischemia in peripheral retina of wild type mice. In contrast, retinal ischemia after Histone H2B injection was not observed in retina of TLR4 -/- mice.
Histone was identified as ligand of TLR4 after ONC. Treatment of Histone H2B induced RGC degeneration and ischemia through Myd88 and TRIF pathways.
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