Figure 3

CYLD negatively regulates S. pneumoniae -induced TLR2 up-regulation. (A) Human alveolar epithelial cell A549 and human middle ear epithelial cell HMEEC-1 were treated with S.p., and CYLD mRNA expression was measured 5 hours after treatment by Q-PCR analysis. (B) WT mice were intratracheally or transtympanically inoculated with S.p. for pneumonia and otitis media model, respectively, and CYLD mRNA expression was measured from the lungs and middle ear tissues of inoculated mice 3 hours after inoculation and 9 hours after inoculation, respectively. Data in A & B are means ± S.D. (n = 3). *, p < 0.05 compared with CON, CON, control treated with vehicle. (C) Cells were transfected with NF-κB reporter gene with CYLD siRNA or empty vector. S.p.- or PLY-induced NF-κB promoter activity was measured as relative luciferase activity. (D) Cells were transfected either with CYLD siRNA or empty vector, and S.p.-induced TLR2 mRNA expression was measured 5 hours after treatment by Q-PCR analysis. (E) WT and CYLD KO mice were intratracheally inoculated with S.p., and TLR2 mRNA expression was measured from the lungs of inoculated mice 6 hours after inoculation by Q-PCR analysis. Data are means ± S.D. (n = 3). *, p < 0.05 compared with CON, **, p < 0.05 compared with S.p. in MOCK (A-C) or in WT mice (D), CON, control inoculated with vehicle, MOCK, transfected with empty vector.