Ocular inflammatory cell infiltrates and angiogenesis are augmented by administration of the toll-like receptor-2 (TLR-2) ligand in a mouse model of choroidal neovascularization (CNV)

摘要

Purpose : Modified lipids and proteins present in AMD tissues might activate innate immune system including toll-like receptors. Our study investigates the effect of TLR-2 activation on ocular inflammation and angiogenesis in a murine laser-induced CNV model. Methods : PAM3CSK4 (PAM) is a synthetic lipopeptide mimicking bacterial cell walls, activating TLR-2. Single i.p. injections of 50 ?μg of PAM (a dose previously identified to optimally induce retinal cytokine production) were administered at either day -1, 0, 1, 2, 3, or 4 relative to laser application. Laser pulses were applied to mice eyes and CNV area measured 7 days later by i.v. injection of a vascular label, flat mount imaging, and quantification of CNV area by morphometric analysis. There were 3 laser pulses per eye and 10 mice per group, yielding 60 data points per condition. Mice challenged with PAM or saline were also administered systemic VEGF antibodies (a??4G3a?? at 3 mg/kg dosed 3x per week i.p.).To study cellular infiltration, labeled antibodies were used to identify neutrophils and microglia (GR-1 and Iba-1) at day 3 and 7 after laser in the RPE-choroid complex. Images were captured with a single fluorescent microscope with consistent exposure times on the same day for each experiment. Intensity of CNV label was assessed in MatLab. To confirm PAMa??s mechanism of action, CNV area was assessed in sex matched TLR-2 KO and wild-type litter-mate controls. Results : Single i.p. injections of PAM increased CNV area with the largest effect at 2 days after laser increasing CNV area by an average of 67% (+/- 18%, p a?¤ 0.003, n = 4 independent studies) compared to mice injected with saline. . I.p. injections of PAM increased neutrophil and microglia cell intensity in the CNV lesion by 480% and 89% on day 3 compared to mice injected with PBS. Minimal differences in antibody label intensity were observed in day 7 tissues. CNV area was similar in both WT and TLR-2 KO mice injected with PBS. PAM increased CNV area in WT mice, but not in the TLR-2 KO mice. Anti-VEGF Ab administration reduces laser-induced CNV area 62% (p 0.0001 vs vehicle) in mice challenged with PAM i.p. injections 2 days after laser. Conclusions : PAM injection increases ocular inflammation and exacerbates ocular angiogenesis by a TLR-2 dependent mechanism that is also sensitive to VEGF inhibition. This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.