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M. Zheng, H. Li, H. Qian, M.A. Fields, F. Yu, S.S. Atherton; Involvement of Toll–Like Receptor 7 (TLR7) in the Pathogenesis of HSV–1 Induced Acute Retinal Necrosis . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1649.
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Purpose:To investigate the role of TLR7 in the pathogenesis of HSV–1–induced acute retinal necrosis (ARN) in murine model. Methods: The anterior chamber of the right eye of BALB/c mice was inoculated with HSV–1 (KOS, 2×104 pfu/eye) or with an equivalent volume of tissue culture medium. On day 3, 5, 6, 7, 9 and 14 p.i., 5 uninoculated eyes were enucleated from experimental and control mice (day 3 only) and pooled; total mRNA was isolated or the eyes were sonicated for Western blot. Frozen sections were made for immunohistochemistry. RPE cells were isolated from BALB/c mice and their identity was confirmed by RPE65 staining. Cultured RPE cells were infected with HSV–1 at an MOI of 5, harvested at 1, 2, 4, 8 and 18h p.i., and RT–PCR for TLR7 and immunohistochemical staining for TLR7 and HSV–1 were performed. Results:In control eyes, TLR7 was expressed in the inner and outer plexiform layers of the retina. TLR7 was upregulated in the HSV–1 infected contralateral eyes at the peak of acute retinitis (day 9–10 p.i.). A few TLR7+ cells were also HSV–1+. Some TLR7+ cells were F4/80+ and some TLR7+ cells were RPE65+. Rare TLR7+, F4/80+ cells or TLR7+, RPE65+ cells were also HSV–1 positive. RT–PCR for TLR7 revealed a biphasic upregulation of TLR7 mRNA during the pre–clinical phase (day 6 p.i.) and at the beginning of resolution phase (day 14 p.i.) in the HSV–1 infected contralateral eyes. Western blot for TLR7 showed gradual intensification of TLR7 from day 7 to 14 p.i. in the HSV–1 infected contralateral eye. In HSV–1 infected cultured RPE cells, TLR7 mRNA was upregulated gradually from 2h to 8h p.i. and decreased by 18h p.i. Immunohistochemical staining of HSV–1 infected cultured RPE cells for TLR7 confirmed the RT–PCR results. Interestingly, the majority of TLR7+ cells were also HSV–1 infected. Conclusions:Upregulation of TLR7 in RPE cells and in the uninoculated eye was closely associated with HSV–1 antigen expression suggesting that TLR7 expressed by activated migrating RPE cells and by infiltrating macrophages may play a role in the pathogenesis of HSV–1 induced acute retinal necrosis.
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