Figure 3
S. pneumoniae mediates TNF-α mRNA stability independent of TLRs. (A) RAW-TNF-AU+ cells were treated with vehicle, S. pneumoniae (5 × 107 bacteria/ml), LPS (100 ng/ml), Pam3Csk4 (200 ng/ml), or ODN1826 (1 μM). Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (B) RAW-TNF-AU+ cells were treated with the TLR9 antagonist ODN2088 (3 μM) 15 min prior to addition of vehicle or S. pneumoniae (5 × 107 bacteria/ml) to the cell cultures. Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (C and D) RAW-TNF-AU+ cells were treated with a MyD88 inhibitor peptide or a control peptide for 24 h before addition of (C) vehicle or S. pneumoniae (5 × 107 bacteria/ml) or (D) LPS (100 ng/ml) or TNF-α (25 ng/ml) plus IFN-γ (10 ng/ml). (C) Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (D) Supernatants were harvested 36 h post stimulation, and nitrite was measured by Griess assay. Similar results were obtained in 2–3 independent experiments. The data are shown as means +/- SEM.