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YU SU, Franco Aparecido Rossato, Yin Shan Eric Ng; Bacterial infection promotes spontaneous choroidal neovascularization (sCNV) development in the JR5558 mouse. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4902. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We hypothesized that bacterial infection induces sub-clinical inflammation of the retinal pigment epithelium (RPE) via the activation of the Toll-like receptor 2 (TLR2) and contributes to sCNV pathogenesis. We aim at determining the effect of short-term systemic antibiotic on sCNV development and the mechanism of TLR2-mediated RPE inflammation.
Two different breeding groups of JR5558 mice were set up from the same litter (F0), with and without Ditrim (0.96 mg/ml) in drinking (F1 mice), and for different duration (F2-A and F2-B mice) (Fig 1A). FITC-conjugated isolectin B4 and anti-CD45 staining were used to quantify sCNV and immune cells, respectively. NF-κB expression was determined by staining. RPE/choroid expression of VEGFA, TLR2, VE-cadherin, CD45, MCP-1, IL-6 was determined by qPCR. RPE/choroid/sclera explants from C57B6J mice were cultured with and without Pam2+CEP (carboxyethyl-pyrrole-dipeptide) on the transwell, and expression of VEGFA, TLR2, VE-cadherin, CD45, IL-6 was determined by qPCR.
In the F1 eyecups both the number of sCNV (13±9 vs. 32±15, P<0.0001) and CNV area per eye (21076±14687 vs. 70210±49389 pixel area, P<0.0001) were significantly reduced in the Ditrim group compared to control. In the F2-A group, in which Ditrim was stopped at P0, the number of CNV (9 ± 2 vs. 32±15, P<0.0001) and CNV area per eye (12048 ± 3161 vs. 70210±49389 pixel area, P<0.0001) were significantly reduced, and the numbers of CD45+ leukocytes in both the F2-A and F2-B groups were significantly reduced compared to the control (93±37, 62±20, vs. 284±53, P<0.001 for both) (Fig 1B). NF-κB staining was lower in all the F2 groups compared to control. Expression of TLR2, VEGFA, IL-6 by RPE/choroid was significantly decreased in all the Ditrim treated F1 mice compared to control (P<0.05 for all). For the explants, Pam2+CEP treatment significantly increased the expression of VE-cadherin, VEGF,TLR2 and MCP-1 compared to controls (P<0.03 for all).
Systemic Ditrim inhibits sCNV and suppresses inflammation of the RPE/choroid indirectly, which is likely mediated by the anti-bacterial activity of Ditrim. Furthermore, activation of TLR2 by bacterial ligand induces robust pro-inflammatory cytokines and angiogenic marker expression by RPE/choroid/sclera explants and drives sCNV development.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
Fig1 (A) Animal experiment design.(B) Quantification of sCNV and inflammation.
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