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Mirja Ramke, Xiaohong Zhou, Ashish Chintakuntlawar, Jaya Rajaiya, James Chodosh; MyD88 Adaptor Protein is Required for Adenovirus Keratitis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4841.
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© ARVO (1962-2015); The Authors (2016-present)
The innate immune response is robust in the development of epidemic keratoconjunctivitis (EKC). EKC is a hyperacute and highly contagious ocular surface infection by human adenovirus species D (HAdV-D) types 8, 37, 53, 54, 56, and 64. Toll-like receptors (TLRs) are important contributors to innate immunity, and recruit intracellular adaptor protein myeloid differentiation factor 88 (MyD88) to form active signaling complexes. Initiation of TLR and MyD88 downstream signaling cascades leads to activation of transcription factors and secretion of proinflammatory and antiviral cytokines. The role of TLR-dependent pathways in adenovirus keratitis is unknown.
Stromal keratitis was induced by intracorneal injection of 105 infectious units of HAdV-D37 or dialysis buffer control in C57Bl/6j (WT), TLR2-/-, TLR9-/-, MyD88-/-, and TLR2/9-/- mice. Flow cytometry, ELISA, real-time RT-PCR, confocal microscopy, and Western blot analysis were performed on the cornea 4 days post infection (dpi). Cultured human corneal fibroblasts (HCF) at 2nd passage were infected with HAdV-D37 or dialysis buffer control, and treated with MyD88 inhibitory peptide prior to Western blot analysis and real-time RT-PCR.
Clinically evident keratitis developed in TLR2-/-, and TLR9-/-mice within 1 dpi, whereas keratitis was not evident or was significantly reduced in MyD88-/- and TLR2/9-/- mice, respectively. Flow cytometric analysis showed reduction in CD45+ leukocytes in infected MyD88-/- corneas as compared to WT. ELISA showed significantly higher CXCL1, IL-6, CCL2, and CXCL2 expression in infected WT corneas than in MyD88-/- or TLR2/9-/-. By confocal microscopy using Cy3-labeled HAdV-D37, viral entry appeared similar between WT and MyD88-/-, but E1A gene expression was reduced in MyD88-/-corneas. Src kinase phosphorylation and CXCL1 induction were also decreased in infected MyD88-/- corneas as compared to WT. Pretreatment with MyD88 inhibitory peptide in HCF in vitro reduced the phosphorylation of Src kinase and decreased CXCL8 and CCL2 gene expression.
Cytokine expression in adenovirus keratitis is MyD88 and Src dependent, and relies on the synergistic activation of TLR2 and 9. The innate immune response is critical to the development of stromal keratitis in EKC.
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