miR-146a participates in BLP-induced cross-tolerance to gram-negative bacteria.

<p>(<b>A</b> and <b>B</b>) Human THP-1 cells were pre-incubated with either culture medium (naive) or 100 ng/ml BLP (BLP-tolerised) for 18 h, and further stimulated with 1×10⁶ CFU/ml heat-killed <i>S. typhimurium</i> (<i>S. typhi</i>) or 1×10⁷ CFU/ml heat-killed <i>S. aureus</i> for 6 h. (<b>C</b> and <b>D</b>) THP-1 cells were pre-incubated with various doses of BLP for 18 h, and further stimulated with 1×10⁶ CFU/ml <i>S. typhimurium</i> for 6 h. (<b>E</b> and <b>F</b>) THP-1 cells were transfected with 40 nM of either miR-146a mimic or miRNA negative control and then stimulated with 1×10⁶ CFU/ml <i>S. typhimurium</i> (<b>E</b>) or 1×10⁷ CFU/ml <i>S. aureus</i> (<b>F</b>) for 6 h. TNF-α concentrations in the culture supernatants were assessed by ELISA, and miR-146a levels in THP-1 cells were detected by real-time PCR and expressed as fold expression. Data are presented as mean ± SE of three independent experiments, and each experiment was conducted in triplicate. *<i>p</i><0.05, **<i>p</i><0.01 compared with naive cells or miRNA negative control-transfected cells.</p

miR-146a is involved in BLP-induced self-tolerance.

<p>(<b>A</b> and <b>B</b>) Human THP-1 cells were pre-incubated with either culture medium (naive) or 100 ng/ml BLP (BLP-tolerised) for 18 h, and further stimulated with 1,000 ng/ml BLP for 6 h (<b>A</b>) or 24 h (<b>B</b>). (<b>C</b> and <b>D</b>) THP-1 cells were transfected with 40 nM of either miR-146a mimic or miRNA negative control (<b>C</b>) and then stimulated with 1,000 ng/ml BLP for 6 h (<b>D</b>). TNF-α concentrations in the culture supernatants were assessed by ELISA and miR-146a levels in THP-1 cells were detected by real-time PCR. Data are presented as mean ± SE of three independent experiments and each experiment was conducted in triplicate. *<i>p</i><0.05, **<i>p</i><0.01 compared with naive cells or miRNA negative control-transfected cells.</p

Gene-specific PCR primers.

<p>Gene-specific PCR primers.</p

Blockage of NF-κB p65 and/or MAPK p38 attenuates B7-H3-amplified proinflammatory cytokine and chemokine mRNA expression in brain tissues of <i>S</i>. <i>pneumoniae</i>-infected mice.

<p>Mice were challenged with PBS as the control, live <i>S</i>. <i>pneumoniae</i> (SP), or live <i>S</i>. <i>pneumoniae</i> plus B7-H3 (SP+B7-H3) 1 hr after mice pretreated with the MAPK p38 inhibitor SB203580, the NF-κB p65 inhibitor PDTC, or their combination (SB203580+PDTC) as described in the Methods. Brain samples were collected at 12 hrs after challenges for detecting mRNA expression of TNF-α (<b>A</b>), IL-1β (<b>B</b>), IL-6 (<b>C</b>), and MCP-1 (<b>D</b>) by quantitative real-time PCR. Data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments. **<i>p</i><0.01 compared with mice treated with PBS, ^≠<i>p</i><0.05 compared with mice treated with SP alone or SP+B7-H3, ^ε<i>p</i><0.05 compared with SP alone.</p

Gene-specific PCR primers.

<p>Gene-specific PCR primers.</p

Administration of B7-H3 up-regulates phosphorylation of NF-κB p65 and MAPK p38 in brain tissues of <i>S</i>. <i>pneumoniae</i>-infected mice.

<p>Mice were challenged with PBS as the control, recombinant mouse B7-H3, live <i>S</i>. <i>pneumoniae</i> (SP), or live <i>S</i>. <i>pneumoniae</i> plus B7-H3 (SP+B7-H3) via intracerebral ventricular injection as described in the Methods. Brain samples were collected at the indicated time points after challenges. Phosphorylated p65 at Ser536 (<b>A</b>), total p65 (<b>A</b>), phosphorylated p38 at Thr180/Tyr182 (<b>B</b>), and total p38 (<b>B</b>) was detected by Western blot analysis. Data shown represent one experiment from a total of four separate experiments. Density ratios of p-p65/p65 (n = 4) (<b>C</b>) and p-p38/p38 (n = 4) (<b>D</b>) were quantified by densitometry analysis. **<i>p</i><0.01 compared with mice treated with PBS, ^≠<i>p</i><0.05 compared with mice treated with SP alone.</p

Administration of B7-H3 causes increased MyD88-IRAK immunocomplex formation in brain tissues of <i>S</i>. <i>pneumoniae</i>-infected mice.

<p>Mice were challenged with PBS as the control, recombinant mouse B7-H3, live <i>S</i>. <i>pneumoniae</i> (SP), or live <i>S</i>. <i>pneumoniae</i> plus B7-H3 (SP+B7-H3) via intracerebral ventricular injection as described in the Methods. Brain samples were collected at the indicated time points after challenges. IRAK-1 protein expression was detected by Western blot analysis (<b>A</b>) and MyD88-IRAK immunocomplex formation was detected by immunoprecipitation (<b>B</b>). Data shown represent one experiment from a total of three or four separate experiments. Density ratios of IRAK1/GAPDH (n = 4) (<b>C</b>) and MyD88-IRAK/GAPDH (n = 3) (<b>D</b>) were quantified by densitometry analysis. *<i>p</i><0.05, **<i>p</i><0.01 compared with mice treated with PBS, ^≠<i>p</i><0.05 compared with mice treated with SP alone.</p

B7-H3 does not augment TLR2 expression in brain tissues of <i>S</i>. <i>pneumoniae</i>-infected mice.

<p>Mice were challenged with PBS as the control, recombinant mouse B7-H3, live <i>S</i>. <i>pneumoniae</i> (SP), or live <i>S</i>. <i>pneumoniae</i> plus B7-H3 (SP+B7-H3) via intracerebral ventricular injection as described in the Methods. Brain samples were collected at the indicated time points after challenges. TLR2 mRNA expression was assessed by quantitative real-time PCR (<b>A</b>) and data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments. **<i>p</i><0.01 compared with mice treated with PBS. TLR2 protein expression (<b>B</b>) was detected by Western blot analysis and data shown represent one experiment from a total of four separate experiments. Density ratios of TLR2/GAPDH (n = 4) (<b>C</b>) were quantified by densitometry analysis.</p

Blockage of NF-κB p65 and/or MAPK p38 attenuates B7-H3-amplified proinflammatory cytokine and chemokine production in brain tissues of <i>S</i>. <i>pneumoniae</i>-infected mice.

<p>Mice were challenged with PBS as the control, live <i>S</i>. <i>pneumoniae</i> (SP), or live <i>S</i>. <i>pneumoniae</i> plus B7-H3 (SP+B7-H3) 1 hr after mice pretreated with the MAPK p38 inhibitor SB203580, the NF-κB p65 inhibitor PDTC, or their combination (SB203580+PDTC) as described in the Methods. Brain samples were collected at 18 hrs after challenges for detecting protein levels (<b>B</b>) of TNF-α (<b>A</b>), IL-1β (<b>B</b>), IL-6 (<b>C</b>), and MCP-1 (<b>D</b>) by ELISA. Data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments. **<i>p</i><0.01 compared with mice treated with PBS, ^≠<i>p</i><0.05, ^≠≠<i>p</i><0.01 compared with mice treated with SP alone or SP+B7-H3, ^ε<i>p</i><0.05 compared with SP alone.</p

Neither <i>S</i>. <i>pneumoniae</i> nor B7-H3 enhances mRNA expression of MyD88, IRAK-1, TIRAP, and TRAF6 in brain tissues of <i>S</i>. <i>pneumoniae</i>-infected mice.

<p>Mice were challenged with PBS as the control, recombinant mouse B7-H3, live <i>S</i>. <i>pneumoniae</i> (SP), or live <i>S</i>. <i>pneumoniae</i> plus B7-H3 (SP+B7-H3) via intracerebral ventricular injection as described in the Methods. Brain samples were collected at the indicated time points after challenges and MyD88 (<b>A</b>), IRAK-1 (<b>B</b>), TIRAP (<b>C</b>), and TRAF6 (<b>D</b>) mRNA expression was assessed by quantitative real-time PCR. Data are expressed as mean ± SD of five to six mice per time point and represent two separate experiments.</p