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H. Yi, N. Kothari, C. P. Sodhi, D. J. Hackam, A. S. Hackam; Cross-Talk Between the Neuroprotective Wnt Signaling Pathway and the Innate Immunity Toll-Like Receptor 4 (tlr4) in Glia. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6169.
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Chronic activation of the innate immune response in the macula has been implicated in age-related macular degeneration (AMD). Several lines of evidence suggest that the innate immunity mediator Toll-like receptor 4 (TLR4) may be involved in photoreceptor death in AMD. Another critical signaling pathway in the retina is the Wnt pathway. We previously demonstrated that Wnt signaling is upregulated in Muller glia and microglia during photoreceptor death in the rd1 mouse, and that Wnt activation protected cultured photoreceptors from oxidative stress. The relationship between Wnt signaling and innate immunity/TLR4 in retinal glia has not been examined. In this study, we investigated the functional interaction between the Wnt and TLR4 pathways.
The Muller glia cell line MIO-MI and the microglia cell line BV-2 were used in this study. Activation of Wnt signaling by the Wnt3a ligand was measured using luciferase reporter assays with the TOP-FLASH and Renilla plasmids. TLR4 signaling was activated by 0.05-50 ug/ml lipopolysaccharide (LPS) and was measured by IL-6 gene expression with sybr green quantitative PCR and by immunodetection of p65. Nerve growth factor (NGF) expression was measured by quantitative PCR.
Muller glia MIO-M1 cells and microglia BV-2 cells express high levels of TLR4. Both cell types responded to the TLR4 ligand LPS, as shown by dose-dependent nuclear translocation of p65 and upregulation of the TLR4 target gene IL-6. Activation of TLR4 suppressed Wnt3a-mediated Wnt signaling by up to 42% in MIO-M1 cells (SEM=7%, n=3, p<0.05). Furthermore, LPS reduced expression of the Wnt target gene NGF by up to 90% in MIO-M1 cells, whereas Wnt3a increased expression of NGF by 45%. Finally, Wnt3a upregulated TLR4 expression in microglia BV-2 cells (3.25-fold, SEM=0.849, compared with control, n=3, p<0.05) but did not regulate TLR4 expression in MIO-M1 cells (n=4).
Our results indicate that the innate immunity receptor TLR4 negatively regulates the neuroprotective Wnt pathway, suggesting potential cross-talk between these pathways in the retina. These data raise the hypothesis that TLR4 may regulate photoreceptor death during retinal injury by inhibiting Wnt signaling, thereby identifying a potential therapeutic target for AMD.
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