NF-κB activation is critical for bacterial lipoprotein tolerance-enhanced bactericidal activity in macrophages during microbial infection

Tolerance to bacterial components represents an essential regulatory mechanism during bacterial infection. Bacterial lipoprotein (BLP)-induced tolerance confers protection against microbial sepsis by attenuating inflammatory responses and augmenting antimicrobial activity in innate phagocytes. It has been well-documented that BLP tolerance-attenuated proinflammatory cytokine production is associated with suppressed TLR2 signalling pathway; however, the underlying mechanism(s) involved in BLP tolerance-enhanced antimicrobial activity is unclear. Here we report that BLP-tolerised macrophages exhibited accelerated phagosome maturation and enhanced bactericidal activity upon bacterial infection, with upregulated expression of membrane-trafficking regulators and lysosomal enzymes. Notably, bacterial challenge resulted in a strong activation of NF-κB pathway in BLP-tolerised macrophages. Importantly, activation of NF-κB pathway is critical for BLP tolerance-enhanced antimicrobial activity, as deactivation of NF-κB in BLP-tolerised macrophages impaired phagosome maturation and intracellular killing of the ingested bacteria. Finally, activation of NF-κB pathway in BLP-tolerised macrophages was dependent on NOD1 and NOD2 signalling, as knocking-down NOD1 and NOD2 substantially inhibited bacteria-induced activation of NF-κB and overexpression of Rab10 and Acp5, two membrane-trafficking regulators and lysosomal enzymes contributed to BLP tolerance-enhanced bactericidal activity. These results indicate that activation of NF-κB pathway is essential for BLP tolerance-augmented antimicrobial activity in innate phagocytes and depends primarily on both NOD1 and NOD2

Activation of both TLR and NOD signaling confers host innate immunity-mediated protection against microbial infection

The detection of microbial pathogens relies on the recognition of highly conserved microbial structures by the membrane sensor Toll-like receptors (TLRs) and cytosolic sensor NOD-like receptors (NLRs). Upon detection, these sensors trigger innate immune responses to eradicate the invaded microbial pathogens. However, it is unclear whether TLR and NOD signaling are both critical for innate immunity to initiate inflammatory and antimicrobial responses against microbial infection. Here we report that activation of both TLR and NOD signaling resulted in an augmented inflammatory response and the crosstalk between TLR and NOD led to an amplified downstream NF-kB activation with increased nuclear transactivation of p65 at TNF-a and IL-6 promoters. Furthermore, co-stimulation of macrophages with TLR and NOD agonists maximized antimicrobial activity with accelerated phagosome maturation. Importantly, administration of both TLR and NOD agonists protected mice against polymicrobial sepsis-associated lethality with increased serum levels of inflammatory cytokines and accelerated bacterial clearance from the circulation and visceral organs. These results demonstrate that activation of both TLR and NOD signaling synergizes to induce efficient inflammatory and antimicrobial responses, thus conferring protection against microbial infection

B7-H3 Augments Inflammatory Responses and Exacerbates Brain Damage via Amplifying NF-κB p65 and MAPK p38 Activation during Experimental Pneumococcal Meningitis.

The costimulatory protein B7-H3 has been shown to play a contributory role in the development and progression of experimental pneumococcal meningitis by augmentation of the innate immunity-associated inflammatory response via a TLR2-dependent manner. This study aimed to clarify the component(s) of TLR2-mediated signal transduction pathways responsible for B7-H3-augmented inflammatory response and subsequent brain damage during experimental pneumococcal meningitis. Administration of B7-H3 did not augment expression of TLR2 and other TLR2 upstream components, but led to an enhanced formation of MyD88-IRAK immunocomplex in the brain of S. pneumoniae-infected mice. Furthermore, B7-H3 substantially augmented S. pneumoniae-induced activation of TLR2 downstream NF-κB p65 and MAPK p38 pathways in the brain of S. pneumoniae-infected mice. Notably, blockage of NF-κB p65 and/or MAPK p38 with their specific inhibitors strongly attenuated B7-H3-amplified inflammatory response with significantly reduced proinflammatory cytokine and chemokine production, and markedly ameliorated B7-H3-exacerbated disruption of blood-brain barrier and severity of disease status in S. pneumoniae-infected mice. These results indicate that targeting NF-κB p65 and/or MAPK p38 may represent a promising therapeutic option for amelioration of overwhelming inflammatory response-associated brain injury frequently observed during pneumococcal meningitis

Gene-specific PCR primers.

<p>Gene-specific PCR primers.</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

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

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