Upon stimulation with CpG, TPL-2 negatively regulates IFN-β and IL-12 in macrophages and conventional DCs, but not in pDCs. BMDMs, BMDCs, and pDCs generated from WT and Tpl-2^−/− mice were stimulated with CpG (500 nM) for 24 h. Cytokine production in culture supernatants was determined by ELISA (mean ± SD; n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student's t test. Results are representative of at least three independent experiments.

TPL-2 negatively regulates TLR-induced IFN-β mRNA expression via a de novo protein synthesis–dependent mechanism and independent of IL-10. (a) Before stimulation with 500 nM CpG for 3 h, BMDMs generated from WT and Tpl-2^−/− mice were incubated with cycloheximide or left untreated. Total RNA was extracted and the indicated cytokine mRNA expression was measured by quantitative PCR (normalized to HPRT mRNA). The graph shows mean ± SD from two individual mice assayed in triplicate cell cultures. Results are representative of two independent experiments. (b) Before stimulation with 500 nM CpG, BMDMs derived from IL10^−/− mice were incubated for 30 min with 2.5 µM U0126 or vehicle control (DMSO). After 24 h stimulation, cytokine production in culture supernatants was assayed by ELISA (mean ± SD; n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student's t test. (c) BMDMs generated from WT and Tpl-2^−/− mice were stimulated with 500 nM CpG for the times indicated and total RNA was extracted. c-fos mRNA expression was measured by quantitative PCR (normalized to HPRT mRNA). *, P < 0.05; **, P < 0.01; and ***, P < 0.001; two-way analysis of variance (ANOVA) with Bonferroni correction. (d) BMDMs generated from WT and Tpl-2^−/− mice were stimulated with 500 nM CpG for the times indicated, and nuclear extracts were prepared. c-fos binding activity was measured by ELISA. Graph shows mean ± SD; n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-way ANOVA with Bonferroni correction. Data in b–d are representative of at least three independent experiments.

TPL-2 activation in BMDMs and BMDCs after LPS stimulation negatively regulates IFN-β and IL-12, but is required for optimal IL-10 production. BMDMs (a) and BMDCs (b) generated from wild-type (+/+) and Tpl-2^−/− (−/−) mice were stimulated with 100 ng/ml LPS. Cytokine levels in culture supernatants were determined by ELISA after 24 h stimulation. Total RNA was extracted from cells stimulated for 3 h, and cytokine mRNA expression was measured by quantitative PCR (normalized to ubiquitin mRNA). Graphs show mean of individual cell cultures ± SD (protein, n = 4; mRNA, n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student's t test. Data are representative of at least three independent experiments with similar results. Whole-cell extracts of BMDMs (a, right) and BMDCs (b, right) were generated after indicated stimulation times and analyzed by immunoblotting with the antibodies shown. Results are representative of at least four independent experiments.

TPL-2 is essential for TLR-induced ERK signaling in pDCs. Macrophages (a), myeloid DCs (b), and pDCs (c) were generated from WT and Tpl-2^−/− mice and stimulated with 500 nM CpG for times indicated. Whole-cell extracts were analyzed by immunoblotting with the indicated antibodies. Luminescence signals in pDCs blots were visualized using a high sensitivity substrate. Results are representative of three independent experiments with similar results.

c-fos reduces TLR up-regulation of IFN-β and IL-12 in Tpl-2^−/−, Il-10^−/−, and WT BMDCs, independent of IL-10. BMDCs were generated from Tpl-2^−/− (a), IL-10^−/− (b), and WT (c) mice and transfected with control GFP or c-fos-IRES-GFP encoding vectors. GFP⁺ BMDCs were sorted by MoFlo and stimulated with 500 nM CpG for 24 h. Cytokine production in culture supernatants was assayed by ELISA (mean ± SD; n = 3). *, P < 0.05; **, P < 0.01; and ***, P < 0.001; Student's t test. Results are representative of five (a and b) or two (c) independent experiments with similar results.