The role of TLR4 in photoreceptor {alpha}a crystallin upregulation during early experimental autoimmune uveitis.
Purpose. Previous studies indicate that the upregulation of alphaA crystallin prevents photoreceptor mitochondrial oxidative stress-mediated apoptosis in experimental autoimmune uveitis (EAU). In this study, the role of TLR4 was investigated in the upregulation of alphaA crystallin in the retinas of animals with EAU. Methods. TLR4(-/-), iNOS(-/-), TNF-alpha(-/-), MyD88(-/-), wild-type (WT) control (C57BL/6), and nude mice (B6.Cg-Foxn1(nu)) were immunized with IRBP mixed with complete Freund's adjuvant; eyes were enucleated on day 7 after immunization. Real-time polymerase chain reaction was first used to detect upregulated inflammatory cytokines and alphaA crystallin in retinas with EAU; confirmed with Western blot analysis, and the site of upregulation was localized by immunohistochemistry. Oxidative stress was localized using 8-OHdG, and TUNEL staining was used to detect apoptosis. Results. In early EAU, increased expression of TNF-alpha, iNOS, and alphaA crystallin genes were detected in the retinas of WT mice, whereas such upregulation was absent in TLR4-deficient mice (P < 0.001). alphaA Crystallin was not elevated in MyD88(-/-), TNF-alpha(-/-), and iNOS(-/-) mice with EAU. Immunostaining revealed TNF-alpha, iNOS, and alphaA crystallin localization in the photoreceptor inner segments and outer plexiform layer in the WT controls with EAU; but such staining was absent in TLR4-deficient mice with EAU. 8-OHdG staining showed oxidative stress in the photoreceptors in WT mice with EAU and there was no apoptosis. Conclusions. TLR4 plays an important role in the upregulation of alphaA crystallin through the interaction of MyD88 and the subsequent generation of TNF-alpha and iNOS in the EAU retina. Such crystallin upregulation may prevent oxidative stress-mediated apoptosis of photoreceptors in uveitis.
Saraswathy
Sindhu
S
Doheny Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.
Nguyen
Angeline M
AM
Rao
Narsing A
NA
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D017209:Apoptosis; D001327:Autoimmune Diseases / Q000378:metabolism*; D015153:Blotting, Western; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004195:Disease Models, Animal; D005136:Eye Proteins; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D018384:Oxidative Stress; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D012177:Retinol-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation; D014605:Uveitis / Q000378:metabolism*; D038202:alpha-Crystallin A Chain / Q000378:metabolism*; D000099214:Retinol-Binding Proteins, Interstitial
D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine; D005136:Eye Proteins; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D052247:Nitric Oxide Synthase Type II; D012177:Retinol-Binding Proteins; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D038202:alpha-Crystallin A Chain; D000099214:Retinol-Binding Proteins, Interstitial; C507780:Myd88 protein, mouse; C493487:Tlr4 protein, mouse; C496317:Nos2 protein, mouse