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. 2012 Jul;9(4):324-33.
doi: 10.1038/cmi.2012.11. Epub 2012 May 28.

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

Chun-Yan Zhang  1 , Nan Bai, Zhu-Hong Zhang, Ning Liang, Lan Dong, Rong Xiang, Cheng-Hu Liu
Affiliations

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

Chun-Yan Zhang et al. Cell Mol Immunol. 2012 Jul.

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.

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Figures

Figure 1
Figure 1
HKBA-induced IL-12 production depends on phagocytosis and phagosome acidification. Total splenic CD11c+ cells were purified from WT mice and incubated in the presence of medium alone, DMSO, CyD (a) or CQ (b) 1 h prior to stimulation with HKBA (108/ml) or STAg. After 15 h, the supernatants were harvested, and the IL-12p40 levels were assayed using ELISA. The data shown are mean±s.d. of triplicate samples from three independent experiments. The asterisks indicate statistically significant (P<0.05) differences between the means of the values obtained with CyD-, CQ-treated vs. untreated cells. CQ, chloroquine; CyD, cytochalasin D; HKBA, heat-killed Brucella abortus; STAg, soluble Toxoplasma gondii tachyzoite Ag.
Figure 2
Figure 2
TLR2, but not TLR4-deficient, DCs have defective HKBA phagocytosis. C57BL/6 (WT), TLR2−/−, C3H/HeJ (TLR4-deficient) or C3H/HeOuJ (TLR4-sufficient) mouse splenic DCs were purified and incubated in vitro with HKBA-labeled ALEXA 488 for 1 h. The cells were subjected to flow cytometry to assess HKBA-ALEXA 488 phagocytosis. Histograms for (a–c) WT (solid line) and TLR2−/− (dashed line) and (d–f) C3H/HeJ (solid line) and C3H/HeOuJ (dashed line). (a, d) Control no bacteria and (b, c, e, f) HKBA-ALEXA 488. (g, h) MFIs of HKBA-ALEXA 488. WT and TLR2−/− unfixed (i) or fixed (j) DCs were incubated in vitro with ALEXA-488-labeled beads (1 h, approximately 10 beads/cell). The data shown are mean±s.d. of triplicate samples from three independent experiments. The asterisks indicate statistically significant (P<0.05) differences between the means of the values obtained with TLR2-deficient vs. WT control mice. DC, dentritic cell; HKBA, heat-killed Brucella abortus; MFI, mean fluorescence intensity; STAg, soluble Toxoplasma gondii tachyzoite Ag; TLR, Toll-like receptor; WT, wild type.
Figure 3
Figure 3
HKBA triggers p38 and ERK1/2 in a TLR2-dependent manner. Splenic DCs from WT and TLR2-deficient mice were incubated in the presence of HKBA (108/ml). After different time points, whole-cell extracts were prepared, and the levels of phospho-38 (a) and phospho-ERK1/2 (b) were determined. The data shown in the graphs represent arbitrary units of increase in p38 (a) or ERK1/2 (b) phosphorylation. The data shown are representatives from three independent experiments with similar results. The asterisks indicate statistically significant (P<0.05) differences between the means of the values obtained in WT and TLR2-deficient cells after stimulation. DC, dentritic cell; ERK, extracellular signal-regulated kinase; HKBA, heat-killed Brucella abortus; TLR, Toll-like receptor; WT, wild type.
Figure 4
Figure 4
p38, but not ERK1/2, controls TLR2-dependent HKBA phagocytosis. Total splenic CD11c+ cells were purified from WT mice and incubated in the presence of medium alone, DMSO, SB202190 (0.1 or 1 µM) (a–c, g) or PD98059 (0.1 or 1 µM) (d–f, h) 1 h prior to ALEXA488-labeled HKBA (108/ml) stimulation for 60 min. After 60 min, the cells were subjected to flow cytometry to assess HKBA-ALEXA 488 phagocytosis. Histograms for DMSO bold solid line (a–f), SB202190 (a–c) and PD98059 (d–f) 0.1 µM solid line and 1 µM dashed line. (a, d) Control no bacteria and (b–f) HKBA-ALEXA 488. (g, h) The phagocytosis index corresponds to the mean number of HKBA-ALEXA 488 phagocytosed per cell. The data shown are mean±s.d. of triplicate samples from three independent experiments. The asterisks indicate statistically significant (P<0.05) differences between the means of the values obtained with untreated vs. treated cells. ERK, extracellular signal-regulated kinase; HKBA, heat-killed Brucella abortus; TLR, Toll-like receptor; WT, wild type.
Figure 5
Figure 5
HKBA phagosome acidification depends on TLR2 and p38, but not ERK1/2. Total splenic CD11c+ cells were purified from WT (a–c, g–l) and TLR2-deficient mice (d–f) and incubated in the presence of DMSO (a–f), 1 µM SB202190 (g–i) or 1 µM PD98059 (j–l) 1 h prior to ALEXA350-labeled HKBA (107/ml) and LysoTracker stimulation. After 1 h, the cells were cytospun onto slides, and the colocalization of ALEXA350-labeled HKBA (blue) and LysoTracker (red) was analyzed. (a–l, ×63 original magnification). (m) The percentage of colocalization of ALEXA350-HKBA and LysoTracker was calculated. The data shown are mean±s.d. of triplicate samples from three independent experiments. The asterisks indicate statistically significant (P<0.05) differences between the means of the values obtained with WT or WT untreated vs. TLR2-deficient or WT-treated cells, respectively. ERK, extracellular signal-regulated kinase; HKBA, heat-killed Brucella abortus; TLR, Toll-like receptor; WT, wild type.
Figure 6
Figure 6
p38, but not ERK1/2, mediates TLR2-dependent HKBA phagosome maturation. Total splenic CD11c+ cells were purified from WT (a–c, g–l) and TLR2-deficient mice (d–f) and incubated in the presence of DMSO (a–f), 1 µM SB202190 (g–i) or 1 µM PD98059 (j–l) 1 h prior to ALEXA350-labeled HKBA (107/ml) stimulation. After 1 h, the cells were cytospun onto slides and immunofluorescently stained for LAMP2, and the colocalization of ALEXA350-labeled HKBA (blue) and LAMP2 (green) was analyzed. (a–l, ×63 original magnification). (m) The percentage of colocalization of ALEXA350-HKBA and LAMP2 was calculated. The data shown are mean±s.d. of triplicate samples from three independent experiments. The asterisks indicate statistically significant (P<0.05) differences between the means of the values obtained with WT or WT untreated vs. TLR2-deficient or WT-treated cells, respectively. ERK, extracellular signal-regulated kinase; HKBA, heat-killed Brucella abortus; LAMP2, lysosomal-associated membrane protein 2; TLR, Toll-like receptor; WT, wild type.
Figure 7
Figure 7
TLR2-triggered ERK1/2 and p38 have opposing roles in IL-12, but not TNF, induction by HKBA. Total splenic CD11c+ cells were purified from WT mice and incubated in the presence of medium alone with different concentrations of SB202190 (a) or PD98059 (b) 1 h prior to HKBA (108/ml) stimulation. After overnight incubation, the supernatants were harvested and assayed for TNF and IL-12p40 using ELISA. The data represent mean±s.d. of triplicate samples from three independent experiments with similar results. The asterisks indicate statistically significant (P<0.05) differences between the means of the values obtained with untreated stimulated vs. treated stimulated cells. ERK, extracellular signal-regulated kinase; HKBA, heat-killed Brucella abortus; TLR, Toll-like receptor; TNF, tumor-necrosis factor; WT, wild type.
Figure 8
Figure 8
RasGRP1 regulates TLR2-induced ERK activation and inhibition of IL-12 induction by HKBA in DCs. Thymocytes (a) and splenic DCs (a–h) were harvested from WT and RasGRP1-deficient mice. (a) The presence of RasGPR1 and HSP70 was determined with immunoblot analysis of whole-cell extracts prepared from WT and RasGRP1-deficient thymocytes and DCs. (b) Splenic DCs from WT and RasGRP1-deficient mice were incubated at different time points in the presence of HKBA (108/ml). Whole-cell extracts were prepared, and the levels of p38, ERK1/2, phosho-p38 and phospho-ERK1/2 were determined. (c) Splenic DCs from WT or TLR2-deficient mice incubated in the presence of HKBA and the Ras activation were analyzed using a Ras GTPase Activation ELISA Kit. (d) Total splenic CD11c+ cells were purified from WT and RasGRP1-deficient mice and incubated in the presence of medium alone with HKBA (106 or 107/ml). After overnight incubation, the supernatants were harvested and assayed for IL-12p40 using ELISA. (e–h) WT and RasGRP1-deficient DCs were incubated with HKBA-labeled ALEXA 488 for 1 h. The cells were subjected to flow cytometry to assess HKBA-ALEXA 488 phagocytosis. Histograms for WT (e) and RasGRP1−/− (f) CD11c+CD8+ cells, and for WT (g) and RasGRP1−/− (h) CD11c+CD8− cells. The data represent mean±s.d. of triplicate samples from three independent experiments with similar results. The asterisks indicate statistically significant (P<0.05) differences between the means of the values obtained with WT vs. RasGRP1-deficient cells. DC, dentritic cell; ERK, extracellular signal-regulated kinase; HKBA, heat-killed Brucella abortus; RasGRP1, Ras-guanine nucleotide-releasing protein 1; TLR, Toll-like receptor; WT, wild type.
Figure 9
Figure 9
Model of activation of DCs by HKBA showing the stimulation of TLR2 and TLR9 and cross-talk between their respective signal transduction pathways. Upon exposure to HKBA, TLR2 present at the surface of the cell membrane of DCs initiates several signal transduction pathways. P38 MAPK provides a stimulatory signal for TNF induction, endocytosis and phagolysosome fusion, with subsequent exposure of bacterial DNA and TLR9 activation. Simultaneously, a Ras/RasGRP1 pathway mediates ERK activation. This signaling pathway is essential for TNF induction; however, it also provides negative feedback for TLR9-induced IL-12. DC, dentritic cell; ERK, extracellular signal-regulated kinase; HKBA, heat-killed Brucella abortus; MAPK, mitogen-activating protein kinase; RasGRP1, Ras-guanine nucleotide-releasing protein 1; TLR, Toll-like receptor; TNF, tumor-necrosis factor.
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