TLR2 signaling subpathways regulate TLR9 signaling for the effective induction of IL-12 upon stimulation by heat-killed Brucella abortus

Abstract

Brucella abortus is a Gram-negative intracellular bacterium that induces MyD88-dependent IL-12 production in dentritic cells (DCs) and a subsequent protective Th1 immune response. Previous studies have shown that the Toll-like receptor 2 (TLR2) is required for tumor-necrosis factor (TNF) production, whereas TLR9 is responsible for IL-12 induction in DCs after exposure to heat-killed Brucella abortus (HKBA). TLR2 is located on the cell surface and is required for optimal microorganism-induced phagocytosis by innate immune cells; thus, phagocytosis is an indispensable preliminary step for bacterial genomic DNA recognition by TLR9 in late-endosomal compartments. Here, we hypothesized that TLR2-triggered signals after HKBA stimulation might cross-regulate TLR9 signaling through the indirect modulation of the phagocytic function of DCs or the direct modulation of cytokine gene expression. Our results indicate that HKBA phagocytosis was TLR2-dependent and an essential step for IL-12p40 induction. In addition, HKBA exposure triggered the TLR2-mediated activation of both p38 and extracellular signal-regulated kinase 1/2 (ERK1/2). Interestingly, although p38 was required for HKBA phagocytosis and phagosome maturation, ERK1/2 did not affect these processes but negatively regulated IL-12 production. Although p38 inhibitors tempered both TNF and IL-12 responses to HKBA, pre-treatment with an ERK1/2 inhibitor significantly increased IL-12p40 and abrogated TNF production in HKBA-stimulated DCs. Further experiments showed that the signaling events that mediated ERK1/2 activation after TLR2 triggering also required HKBA-induced Ras activation. Furthermore, Ras-guanine nucleotide-releasing protein 1 (RasGRP1) mediated the TLR2-induced ERK1/2 activation and inhibition of IL-12p40 production. Taken together, our results demonstrated that HKBA-mediated TLR2-triggering activates both the p38 and ERK1/2 signaling subpathways, which divergently regulate TLR9 activation at several levels to induce an appropriate protective IL-12 response.

TLR2 and TLR4 signaling pathways are required for recombinant Brucella abortus BCSP31-induced cytokine production, functional upregulation of mouse macrophages, and the Th1 immune response in vivo and in vitro

“…These data also agree with a previous report indicating that p38 inhibitors temper IL-12p40 responses, whereas an ERK1/2 inhibitor significantly increases IL-12p40 in heat-killed B. abortus-stimulated DCs. 70 Taken together, these findings suggest that rBCSP31 may activate several innate immune signaling pathways, culminating in cytokine production. In addition, we observed that the phosphorylation of MAPK in response to rBCSP31 is mediated primarily by TLR2 and TLR4.…”

TLR9 is required for MAPK/NF-κB activation but does not cooperate with TLR2 or TLR6 to induce host resistance to Brucella abortus

“…Furthermore, no apparent cooperative interplay was observed between TLR2-TLR9 or TLR6-TLR9 receptors concerning bacterial load in infected mice. However, crosstalk between TLR2 and TLR9 in DCs stimulated with HKBa has been reported in vitro [37]. In such a scenario, TLR2 can drive the TLR9 response in DCs upon HKBa stimulation, leading to IL-12 production [37].…”

Ephedrine hydrochloride inhibits PGN-induced inflammatory responses by promoting IL-10 production and decreasing proinflammatory cytokine secretion via the PI3K/Akt/GSK3β pathway

“…The different regulatory mechanisms affected by EH to control IL-10 expression may be due to differences in crosstalk between EH to the signaling pathways downstream of different TLRs. 28 Regardless of whether mice were challenged with LPS or PGN, DXM had a small impact on anti-inflammatory IL-10 expression. In contrast, EH enhances IL-10 secretion significantly in both LPS-and PGN-challenged mouse models.…”