S. pneumoniae induces TNF-α production and stabilizes TNF-α mRNA through AREs in the 3'-UTR. (A) C57BL/6 macrophages were stimulated with vehicle, S. pneumoniae, N. meningitidis (5 × 107 bacteria/ml), LPS (100 ng/ml), Pam3Csk4 (200 ng/ml), or ODN1826 (1 μM). Supernatants were harvested 18 h later, and TNF-α was measured by ELISA. (B) C57BL/6 macrophages were treated with 1 μM actinomycin D 15 min prior to treatment with vehicle, S. pneumoniae, N. meningitidis (both 5 × 107 bacteria/ml), or LPS (100 ng/ml). The cells were lysed at the indicated time points and Total RNA was harvested. TNF-α and β-actin mRNAs were detected by qPCR, and normalized ratios were calculated. The data is shown as percent remaining TNF-α mRNA compared to untreated control. (C) RAW-TNF-AU+ and RAW-TNF-AU÷ cells were seeded and treated with vehicle, S. pneumoniae, N. meningitidis (both 5 × 107 bacteria/ml), or LPS (100 ng/ml). Total cell lysates were harvested 20 h later and CAT was measured by ELISA. Similar results were obtained in 2–4 independent experiments. The data are shown as means +/- SEM.