Figure 4

S. pneumoniae synergizes with NTHi to induce inflammatory response by up-regulating TLR2 expression. (A) HeLa cells were treated with S.p. with or without NTHi, and IL-1β mRNA expression was measured 5 hours after treatment by Q-PCR analysis. (B) WT mice were intratracheally inoculated with S.p. with or without NTHi, and IL-1β mRNA expression was measured from the lungs of inoculated mice 6 hours after inoculation by Q-PCR analysis. (C) Cells were transfected with TLR2 siRNA or control siRNA, treated with S.p. with or without NTHi, and IL-1β mRNA expression was measured 5 hours after infection by Q-PCR analysis. (D) NF-κB luciferase activity following S.p., NTHi, or S.p. with NTHi in MEF cells from WT and TLR2 KO mice. MEF cells from WT and TLR2 KO mice were transfected with NF-κB reporter gene, inoculated with S.p., NTHi, or S.p. with NTHi, and relative luciferase activity was measured 5 hours after inoculation. (E) NF-κB luciferase activity following S.p., NTHi, or S.p. with NTHi in MEF cells from WT and TLR4 KO mice. MEF cells from WT and TLR4 KO mice were transfected with NF-κB reporter gene, inoculated with S.p., NTHi, or S.p. with NTHi, and relative luciferase activity was measured 5 hours after inoculation. (F & G) TLR2 and IL-1β up-regulation in the lungs of WT and TLR4 KO mice following S.p., NTHi, or S.p. with NTHi in vivo. WT and TLR4 KO mice were intratracheally inoculated with S.p., NTHi, or S.p. with NTHi, and TLR2 (F) and IL-1β (G) mRNA expression was measured from the lung of inoculated mice 6 hours after inoculation. Data are means ± SD (n = 3). *, p < 0.05 compared with control, **, p < 0.05 compared with NTHi treatment, ***, p < 0.05 compared with S.p. with NTHi treatment in MOCK or WT mice.