TLR2 signaling subpathways regulate TLR9 signaling for the ... - Nature

Cellular & Molecular Immunology (2012) 9, 324–333 ß 2012 CSI and USTC. All rights reserved 1672-7681/12 $32.00 www.nature.com/cmi

RESEARCH ARTICLE

TLR2 signaling subpathways regulate TLR9 signaling for the effective induction of IL-12 upon stimulation by heat-killed Brucella abortus Chun-Yan Zhang*, Nan Bai *, Zhu-Hong Zhang, Ning Liang, Lan Dong, Rong Xiang and Cheng-Hu Liu 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. Cellular & Molecular Immunology (2012) 9, 324–333; doi:10.1038/cmi.2012.11; published online 28 May 2012 Keywords: ERK; interleukin-12; phagocytosis; p38; TLR2; TLR9; TNF

INTRODUCTION Microbial recognition is the first step in initiating successful innate immune responses against invading pathogens, which, in turn, stimulate the adaptive immune response and elicit effective host resistance. The recognition of microorganisms occurs primarily through the interaction between pathogen-associated molecular patterns and their cognate pattern recognition receptors. Toll-like receptors (TLRs), numbered TLR1–11, are one of the most important families of pattern recognition receptors, participating in most microbial recognition that functions through MyD88- and/or TRIF-dependent signaling pathways1,2 and leading to the activation of innate immune cells and subsequent proinflammatory responses.2,3 Previous studies on the interactions between microorganismderived TLR ligands and TLRs were primarily limited to interactions between a single ligand and its corresponding TLR. However, microorganisms such as bacteria and fungi express more than one TLR

ligand; thus, their interactions with TLRs are more complex and probably involve more than one TLR. Here, we investigated the effect on heat-killed Brucella abortus (HKBA) through the costimulation of two TLRs, particularly because it is known to activate both TLR2 and TLR9 in mouse dentritic cells (DCs).4,5 We paid particular attention to the possibility that the cross-talk between two signal transduction pathways might require regulated integration for the effective induction of proinflammatory cytokines. B. abortus is an intracellular pathogen that resides mainly in macrophages and causes disease in livestock and humans.6,7 Clinical brucellosis in humans is characterized by either acute or insidious onset that is manifested through undulating fever, profound weakness, arthralgia and weight loss. However, chronic disease with granuloma formation, lymphadenopathy and splenomegaly could also occur, thereby indicating the long-lasting recruitment of proinflammatory mechanisms in response to Brucella-derived products. Similar to the live organism,

Department of Immunology, Medical School, Nankai University, Tianjin, China *These authors contributed equally to this work. Correspondence: CH Liu, Department of Immunology, Medical School, Nankai University, Tianjin 30071, China. E-mail: [email protected] R Xiang, Department of Immunology, Medical School, Nankai University, Tianjin 30071, China. E-mail: [email protected] Received 11 January 2012; revised 19 March 2012; accepted 20 March 2012

TLR2-triggered p38 and ERK1/2 by HKBA-regulated TLR9 signaling CY Zhang et al 325

HKBA can activate both the innate and the adaptive immune system, thereby leading to proinflammatory responses.8 During B. abortus infection, DCs are considered the main sentinel cells that sense invading pathogens and secrete IL-12 to induce a beneficial Th1 immune response. In fact, it was shown that DCs phagocytose HKBA injected into mice and then migrate to T-cell areas in the spleen secreting IL-12.9 Moreover, HKBA activates DCs to secrete tumor-necrosis factor (TNF) and IL-12 in a MyD88-dependent way.5 Importantly, it has been demonstrated that TLR2 is required for HKBA-stimulated TNF production, whereas HKBA induced the production of IL-12 in a TLR9-dependent manner in DCs.4 TLR9 functions in the endosomes of innate immune cells and recognizes distinct patterns of nucleic acids in late-endosomal compartments;10–12 its activation requires HKBA phagocytosis.4 TLR2 and TLR4 localize to the plasma membrane, and during phagocytosis, they are recruited to the contact site with the pathogen and are highly enriched on phagosomes. The recruitment of TLRs to phagosomes provides a mechanism for the association of phagocytosis with inflammatory responses.13 In fact, in the absence of TLR2/TLR4 or MyD88, the phagocytosis of bacteria is reduced due to impaired phagosome formation and maturation.14 Adequate TLR recognition leads to the production of proinflammatory mediators such as TNF and IL-12. These responses depend on intracellular signaling pathways that connect receptor-mediated events to transcriptional responses within the nucleus. Mitogen-activating protein kinases (MAPKs) are one of the key components of TLR intracellular signaling cascades and are also implicated in bacterial pathogenesis, as demonstrated by the induction or inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPKs upon infection with Salmonella enterica serovar Typhimurium,15 Yersinia spp.,16,17 Listeria monocytogenes,18,19 Mycobacterium spp.20 and Brucella spp.21,22 Based on previous studies, it was of interest to characterize the role of signaling cascades initiated after the HKBA-mediated activation of TLR2 and TLR9 in terms of bacterial uptake, phagolysosome maturation and triggering of IL-12 and TNF responses. A complete understanding of the molecular interactions of these steps and the possible cross-talk between TLRs is crucial for the successful development of new therapeutic strategies and vaccines for brucellosis that induce the activation of Th1 immune responses. Here, we demonstrate that TLR2 plays a dual role in regulating TLR9-dependent IL-12p40 production in DCs. On the one hand, TLR2 mediates the activation of p38 MAPK, which is a critical step for HKBA phagocytosis, consequently inducing IL-12 production via TLR9 activation. However, TLR2 signaling also induces the activation of the Ras-guanine nucleotide-releasing protein 1 (RasGRP1)–ERK1/ 2 signaling pathway, which exerts an inhibitory effect on TLR9mediated IL-12 production. We demonstrate that TLR2-deficient cells fail to induce Ras activity. Moreover, we show that HKBA-stimulated RasGRP1-deficient DCs do not affect p38, but decrease ERK1/2 activation, resulting in the elevated IL-12 production by these cells. Thus, we propose that the cross-talk between TLR2 and the TLR9 signaling pathway is a critical mechanism for inducing proper protective cytokine production in response to B. abortus. MATERIALS AND METHODS Mice WT controls (C57BL/6), TLR22/2, C3H/HeJ and C3H/HeOuJ were obtained from The Jackson Laboratory. All animals were bred and maintained under pathogen-free conditions at a Duke University

animal facility (Durham, NC, USA) using a protocol approved by the Duke University Animal Care and Use Committee. Reagents SB202190, a specific p38 inhibitor (IC50 of 350 nM) that does not affect c-Jun N-terminal kinase (JNK) or ERK, was purchased from Calbiochem. PD98059 (ERK inhibitor, IC50 of 2 mM), a specific inhibitor of MEK and an upstream MAPK kinase that phosphorylates ERK, was also purchased from Calbiochem. HKBA and soluble Toxoplasma gondii tachyzoite Ag (STAg) HKBA 1119.3 was prepared as indicated. T. gondii tachyzoites (RH88 strain) were cultured in the human fibroblasts cell line Hs27, and STAg was prepared from sonicated tachyzoites as previously described. Isolation of splenic DCs Splenic DCs were isolated as previously described.23 Briefly, the spleens from mice were digested with Liberase CI. Low-density leukocytes were harvested from the digested spleens using a dense-bovine serum albumin gradient. The enriched DCs were further purified using anti-CD11c MACS beads, followed by passage over a MACS column (Miltenyi Biotec, Germany), and the cells purified from the column were routinely 70%–85% CD11c1, as determined using flow cytometry. Cytokine detection IL-12p40 and TNF levels were measured using commercial ELISA kits (R&D Systems). Immunoblot assay Whole-cell extracts were generated upon lysing WT, TLR2- and RasGRP1-deficient DCs or thymocytes in buffer containing 50 mM HEPES, pH 7.9, 0.25 M NaCl, 5 mM EDTA, 0.1% NP-40, 1 mM PMSF and 13 HALT protease inhibitor cocktail (Pierce Biotechnology Inc., Rockford, IL, USA). For the detection of the phosphorylated-proteins, Na3VO4 and NaF were added to the lysis buffers. The detection of protein expression using western blot was performed with primary antibodies against p38, ERK1/2, RasGRP1 and HSP70, which were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Phospho-p38 and phospho-ERK1/2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Each antibody was used according to the recommendations of the manufacturer. The western blot experiments were performed as described earlier. Bacterial phagocytosis, phagosome acidification and maturation Total splenic CD11c1 cells were purified as described above and incubated in vitro with ALEXA 488- or ALEXA350-labeled HKBA (using Molecular Probe kits) (108/ml) in complete RPMI (RPMI-1640, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 10 mM HEPES, antibiotic/antimycotic, 1 mM sodium pyruvate and 53 mM 2-ME) at 37 uC and 5% CO2 for 1 h. In some experiments, the DCs were incubated in the presence of DMSO, cytochalasin D (CyD) or chloroquine (CQ) (all from Sigma Chemical Co., St Louis, Mo, USA) 1 h prior to HKBA, STAg (100 ng/ml) or peptidoglycan (Sigma) stimulation. The cells were washed three times with cold phosphate-buffered saline to remove the extracellular HKBA or drugs. Flow cytometric analysis was used to evaluate phagocytosis. For the acidification studies, a LysoTracker (Molecular Probe) was used, containing a red fluorescent weak base that accumulates in acidic compartments, concomitant with ALEXA350-labeled HKBA. For Cellular & Molecular Immunology


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Ras GTPase activity assay The activation of Ras was measured using a Ras GTPase Activation ELISA Kit (Upstate). Briefly, WT and TLR2-deficient DCs were purified as described above and exposed to medium alone or with HKBA for increasing intervals of time (0–30 min). Following the incubation period, the cells were solubilized with lysis buffer, and Ras activation was quantified according to the manufacturer’s instructions. The Ras GTPase Chemi ELISA Kit contained a Raf-RBD protein that was fused to GTS on a 96-well plate coated with glutathione. The GST-Raf-RBD was incubated in the wells for 1 h to immobilize the captured probe. The cell lysate (50 mg/well) was added and incubated for 1 h. A primary antibody to anti-Ras was added to the wells for 1 h. The wells were washed, and secondary antibody conjugated to HRP was added to each well, incubated and washed. Finally, the product was detected using chemiluminescent reagents. The plate was read on a luminometer, which provided a sensitive and quantitative chemiluminescent readout of activated Ras.

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the maturation studies, after stimulation with ALEXA350-labeled HKBA, the DCs were cytospun on slides and a two-step method was used involving primary incubation with rat anti-mouse lysosomal associated membrane protein-2 (LAMP-2; Calbiochem), followed by a second double incubation with Alexa488-conjugated anti-rat IgG (Molecular Probes). Images were acquired with an Apotome system (Zeiss). The determination of colocalization was performed as described by Magarian et al.24

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* Statistical analysis The statistical significance of the differences in the mean values between the experimental and control or vehicle treated samples was evaluated using Student’s t-test. Differences were considered significant at P,0.05. RESULTS HKBA-induced IL-12 production depends on phagocytosis and phagosome acidification It is well accepted that for pathogen genomic DNA triggering of TLR9, microbe internalization and phagosome acidification are required steps for initiating the intracellular events that elicit cytokine responses.25 Therefore, to determine whether HKBA-induced IL-12 production requires phagocytosis and/or phagosome acidification, wildtype splenic DCs were pre-treated for 1 h with micromolar concentrations of CyD or CQ prior to an 18-h incubation with HKBA or STAg. CyD is a drug that affects actin polymerization and particle internalization and also inhibits the binding stage of phagocytosis,26 and CQ is a lysosomotropic weak base that inhibits the acidification of endosomes and lysosomes.10,27–29 As shown in Figure 1, both CyD and CQ inhibited the release of IL-12p40 induced by HKBA in a dose-dependent manner, as measured using ELISA. In contrast, the stimulation of IL-12p40 by STAg, which is CCR5- and MyD88-dependent,30 was unaffected by pre-treatment with CyD and CQ, as compared with the untreated cells (data not shown). The CyD- and CQ-induced inhibition of IL-12p40 was not secondary to cell death, as the viability of the untreated and CyD- or CQ-pre-treated DCs was 96.8%61.3% and 95.4%61.6% versus 93.9%62.7% and 92.8%63%, respectively. These data confirm the indispensable role of HKBA phagocytosis and digestion in IL-12p40 induction by DCs and further supports previous data4 suggesting that IL-12p40 secretion by HKBA-stimulated DCs is important for the activation of TLR9 localization to the endosomes. Cellular & Molecular Immunology

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Figure 1 HKBA-induced IL-12 production depends on phagocytosis and phagosome acidification. Total splenic CD11c1 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 mean6s.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.

TLR2-, but not TLR4-deficient DCs have defective HKBA phagocytosis Prior studies reported that different TLRs are involved in the modulation of IL-12p40 and TNF production. Indeed, TLR2 is required for TNF but not IL-12p40 production.5 In contrast, TLR4 is not required for either TNF or IL-12p40 induction by HKBA.5 Moreover, HKBA stimulation of macrophages elicits IL12p40 secretion, which is MyD88 dependent but only partially TLR9-dependent. These observations suggest that the optimal activation of IL-12p40 production likely depends on the cooperation of two or more receptors. The fact that CyD and CQ inhibited the effect of HKBA on IL-12p40 secretion by DCs supports the notion that HKBA DNA and TLR9 interact within mature endosomes. We hypothesized that TLR2 and TLR4, which are expressed in the plasma membrane, are important for the phagocytic process and consequently facilitate TLR9 stimulation. In fact, it is known that TLR2 and TLR4 are recruited to sites of pathogen contact and are highly enriched on phagosomes.13 We investigated the role of TLR2 and TLR4 in HKBA phagocytosis by incubating WT and


TLR2- or TLR4-deficient DCs in the presence of ALEXA488-labeled HKBA. The results shown in Figure 2b and c indicate that the TLR2-deficiency caused a significant reduction of HKBA internalization (45%–60%) in both CD11c1CD81 and CD11c1CD82 DC subsets after 1-h incubation. In contrast, ALEXA488-labeled HKBA phagocytosis was not affected by the lack of functional TLR4 (Figure 2e and f). Figure 2g and h shows the calculated MFI of HKBA-ALEXA488. To address whether the lack of TLR2 could intrinsically compromise the DC phagocytic response, we tested whether the uptake of inert latex beads would be affected by TLR2 deficiency. Figure 2i shows that the phagocytosis of the biologically inert latex beads was similar in the WT and TLR2-deficient DCs. In addition, the paraformaldehyde-fixed DCs of both strains showed low background phagocytosis levels, indicating that spontaneous bacterial adherence to the DC membrane was not a relevant factor in the mechanisms of uptake (Figure 2j). These results indicate that DC HKBA phagocytosis is mediated by stimulation with TLR2, but not TLR4. HKBA triggers a TLR2-dependent p38 and ERK phosphorylation It is well known that TLR2 stimulation with Pam3Cys induces the activation of MAPKs (p38 and ERK).31 MAPKs are signaling molecules that are essential for both phagocytosis and IL-12p40 production.13,31 Therefore, we examined the role of HKBA-triggered TLR2 production for the activation of p38 and ERK1/2. Figure 3a and b shows that in TLR2-deficient DCs, the HKBA-induced phosphorylation of p38 and ERK1/2 was impaired and the kinetics of the expression were decreased and delayed as compared with the phosphorylation detected in the HKBA-stimulated WT DCs. The TLR2deficient DCs exposed to the TLR2 ligand, peptidoglycan (30 min) also showed decreased ERK1/2 and p38 phosphorylation as compared with the WT cells (Figure 3a and b). These data indicate that HKBA triggers a TLR2-mediated signaling pathway involving p38 and ERK1/2 phosphorylation. No HKBA

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p38 but not ERK controls TLR2-dependent HKBA phagocytosis, phagosome maturation and acidification To identify the molecular mechanism underlying the effects of p38 and ERK1/2 on TLR2-mediated HKBA internalization, we analyzed the effects of the p38 inhibitor (SB202190) and ERK1/2 inhibitor (PD98059) on HKBA phagocytosis by WT DCs. The HKBA phagocytosis in both the CD11c1CD81 and CD11c1CD82 DCs was severely reduced after pre-exposure to the p38 inhibitor (Figure 4a– c and g). In contrast, prior incubation with the ERK inhibitor had no effect on the HKBA phagocytosis detected in both DCs subsets (Figure 4d–f and h). These data clearly indicate that TLR2mediated HKBA phagocytosis is dependent on p38 but not on ERK1/2 activity in DCs. Following internalization, the degradation of the phagocytic body occurs upon phagosome maturation and acidification.32 The recruitment of cysteine proteases, lysosomal glycosidases and other lysosomal enzymes to phagosomes and the modification of the phagosomal pH are important steps in this maturation process.13 To determine whether the HKBA-mediated TLR2-triggered MAPK (p38 and ERK1/2) pathway is involved in the maturation process of phagosomes in DCs, WT and TLR2deficient DCs were pre-treated with SB202190 or PD98059 inhibitors, before incubation with ALEXA350-labeled HKBA together with LysoTracker, a red fluorescent weak base that accumulates in acidic compartments. As shown in Figure 5, approximately 46% of the ALEXA350-HKBA colocalized with LysoTracker in WT DCs after a 60-min incubation (Figure 5a–c and m). This colocalization was decreased to 10% in TLR2-deficient DCs (Figure 5d–f and m) incubated for the same amount of time. Pre-treatment with the p38 inhibitor SB202190, but not with the ERK1/2 inhibitor PD98059, decreased the colocalization percentage of ALEXA-350 HKBA with LysoTracker in the WT DCs, mirroring the results observed with the TLR2-deficient DCs (Figure 5g–m). Moreover, we assessed whether phagosome maturation in HKBA-stimulated DCs is dependent on TLR2-triggered

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Figure 2 TLR2, but not TLR4-deficient, DCs have defective HKBA phagocytosis. C57BL/6 (WT), TLR22/2, 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 TLR22/2 (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 TLR22/2 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 mean6s.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. Cellular & Molecular Immunology


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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.

p38 and/or ERK1/2. To this end, we quantified the frequency of cells stained with an antibody specific to the late phagosome marker, LAMP-2. In TLR2-deficient DCs, approximately 8% of ALEXA350labeled HKBA and LAMP-2 colocalized (Figure 6d–f and m). However, 38% colocalization was observed in the WT DCs (Figure 6a–c and m). Pre-treatment with SB202190 (Figure 6g–i and m), but not PD98059 (Figure 6j–m), decreased the colocalization of HKBA/LAMP-2 in the WT DCs, mimicking the results

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Figure 4 p38, but not ERK1/2, controls TLR2-dependent HKBA phagocytosis. Total splenic CD11c1 cells were purified from WT mice and incubated in the presence of medium alone, DMSO, SB202190 (0.1 or 1 mM) (a–c, g) or PD98059 (0.1 or 1 mM) (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 mM solid line and 1 mM 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 mean6s.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. Cellular & Molecular Immunology


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Figure 5 HKBA phagosome acidification depends on TLR2 and p38, but not ERK1/2. Total splenic CD11c1 cells were purified from WT (a–c, g–l) and TLR2deficient mice (d–f) and incubated in the presence of DMSO (a–f), 1 mM SB202190 (g–i) or 1 mM 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, 363 original magnification). (m) The percentage of colocalization of ALEXA350-HKBA and LysoTracker was calculated. The data shown are mean6s.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. TLR2deficient or WT-treated cells, respectively. ERK, extracellular signal-regulated kinase; HKBA, heat-killed Brucella abortus; TLR, Toll-like receptor; WT, wild type.

Figure 6 p38, but not ERK1/2, mediates TLR2-dependent HKBA phagosome maturation. Total splenic CD11c1 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 mM SB202190 (g–i) or 1 mM 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, 363 original magnification). (m) The percentage of colocalization of ALEXA350-HKBA and LAMP2 was calculated. The data shown are mean6s.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 signalregulated kinase; HKBA, heat-killed Brucella abortus; LAMP2, lysosomal-associated membrane protein 2; TLR, Toll-like receptor; WT, wild type.

ERK1/2 and p38 have opposing roles in TLR2-triggered IL-12, but not TNF, induction by HKBA Thus far, the results presented here indicate that upon HKBA stimulation, DC cytokine induction results from several signaling pathways,

including TLR2-triggered intracellular production of MAPKs and bacterial uptake. Although TLR9 has been directly associated with the induction of IL-12 production in response to HKBA incubation, Cellular & Molecular Immunology


it is still possible that TLR2-dependent p38-mediated phagocytosis and endosome maturation are critical steps for exposure of DNA stimulatory motifs and their interaction with TLR9. Interestingly, DC TNF production is dependent on TLR2, but not TLR-9, after HKBA stimulation.9 Based on these findings and the results presented here, we decided to investigate the role of the two TLR2-triggered signaling pathways unveiled here (p38 and ERK1/2) in determining cytokine induction after exposure to HKBA. To address this question, we pre-treated splenic WT DCs with SB202190 (p38 inhibitor) and PD98059 (ERK inhibitor) prior to HKBA stimulation. Figure 7a shows that upon HKBA stimulation, the secretion of TNF and IL-12 were concordantly highly sensitive to pre-treatment with the p38 inhibitor. As seen in Figure 7b, pre-treatment with the ERK1/2 inhibitor had a discordant effect in abrogating TNF but significantly increased IL12p40 secretion from HKBA-stimulated DCs. The results shown here further establish the role of TLR2-triggered p38 activity as a critical step for both TLR2-dependent TNF and TLR9-dependent IL-12 responses. However, these results also reveal unexpected cross-talk between the two TLRs involved in HKBA IL-12 induction, whereby

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RAS-GRP1 regulates TLR2-induced ERK1/2 activation and inhibition of IL-12 induction by HKBA in DCs It has been suggested that the activation of ERK MAPK in many cell types is under the control of RasGRPs.33,34 In fact, RasGRP1-deficient mice are defective in TCR-induced ERK phosphorylation in T cells, suggesting that RasGRP1 is an important Ras activator downstream of TCR.35 In view of the defective ERK1/2 phosphorylation in the TLR2deficient DCs and the modulatory effects of ERK activity on IL-12 responses, we hypothesized that RasGRP1 acts downstream of TLR2 and that TLR2 deficiency results in the failure to activate RasGRP1 in HKBA-stimulated DCs, consequently abrogating ERK1/2 phosphorylation and enhancing IL-12p40 production. However, we could not find any prior evidence indicating that RasGRP1 is indeed expressed by DCs. As shown in Figure 8a, RasGRP1 can be detected in splenic mouse DC protein extracts, albeit at decreased levels as compared with mouse thymocytes. The HSP70 expression was similar between the DCs and thymocytes and was used as a loading control (Figure 8a). Next, we performed a number of experiments to determine whether the HKBA-mediated TLR2 production occurs via the activation of a RasGRP1-dependent pathway in DCs. Interestingly, we found that although TLR2 stimulation in WT DCs resulted in a large increase in the phosphorylation of ERK1/2, such activation was greatly reduced in RasGRP1-deficient cells (Figure 8b). Moreover, the levels of p38 phosphorylation induced by HKBA were similar between the WT and RasGRP1-deficient DCs (Figure 8b). Splenic DCs from WT or TLR2deficient mice were incubated in the presence of HKBA and analyzed for Ras activation using a Ras GTPase Activation ELISA Kit (Figure 8c). Indeed, TLR2 did induce RasGRP1activation. These findings indicate that HKBA-mediated TLR2 activation triggers a signaling pathway that results in ERK activation. Furthermore, we found that RasGRP1-deficient DCs produced elevated levels of IL-12p40 in response to HKBA stimulation when compared with the levels detected in HKBA-stimulated WT cells (Figure 8d). These results are consistent with previous studies (see above) showing that a specific inhibitor of ERK phosphorylation likewise produced an increased IL12 induction following HKBA stimulation. Notably, the baseline levels of IL-12p40 production were indistinguishable between WT and RasGRP1-deficient DCs. Finally, to address whether RasGRP1 indirectly modulated IL-12p40 responses through enhancing phagocytic responses, we examined the phagocytosis of HKBA by RasGRP1deficient DCs. These DCs did not show detectable changes in HKBA phagocytosis within both DCs subsets, CD11C1CD81 (Figure 8e and f) and CD111CD82 (Figure 8g and h), as compared with their WT controls, indicating that the RasGRP1/ERK2 pathway plays a direct role in HKBA internalization.

HKBA

Figure 7 TLR2-triggered ERK1/2 and p38 have opposing roles in IL-12, but not TNF, induction by HKBA. Total splenic CD11c1 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 mean6s.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. Cellular & Molecular Immunology

the TLR2-triggered ERK1/2 activity down-modulates TLR9-dependent IL-12 responses.

DISCUSSION Microbial recognition in DCs constitutes a major step in the activation of immune mechanisms that lead to the development of resistance against many infectious agents. These recognition events are primarily mediated through a class of receptors (TLRs) that exhibit moderate microbial ligand specificity. A full understanding of the molecular interactions of this step is crucial for the successful development of vaccine strategies. Most studies on the effects of TLR ligands are limited to single TLR interactions. This report investigates the effect of the engagement of more than one TLR by one microorganism, which is probably the more likely scenario, considering that microorganisms such as


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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 TLR2deficient mice incubated in the presence of HKBA and the Ras activation were analyzed using a Ras GTPase Activation ELISA Kit. (d) Total splenic CD11c1 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 RasGRP12/2 (f) CD11c1CD81 cells, and for WT (g) and RasGRP12/2 (h) CD11c1CD82 cells. The data represent mean6s.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.

bacteria and fungi are complex structures bearing several potential TLR ligands on the cell surface as nucleic acids. We used HKBA as a probe in this study, because it is known to activate TLR2 and TLR9 in DCs. HKBA behaves as a Th1-like activator of the immune system in both humans and mice.8,36,37 The ability of HKBA to generate T helper cellindependent antibody responses and CD41 T cell-independent CTL responses can be harnessed for vaccine development, especially for individuals with compromised CD41 T-cell function. Peptides or proteins can be conjugated to HKBA and used to elicit strong antibody and CTL responses even in the absence of CD41 T cells. Thus far, this approach has been used successfully in mice and monkeys to elicit

neutralizing systemic and mucosal antibodies and CTLs against HIV1.8 In addition, recent studies show that HKBA activates macrophages and DCs by stimulating TNF and IL-12p40 production via two distinct TLRs, TLR2 and TLR9.4,5 TLR9 is expressed in the endosomes of innate immune cells and recognizes distinct patterns of nucleic acids in late endosomal compartments.10–12 TLRs are reportedly involved in phagocytosis,13 and the recruitment of TLRs to phagosomes provides a mechanism by which phagocytosis and associated inflammatory responses can be linked.13 In the present study, we demonstrate that HKBA-induced production of IL-12p40 depends on phagocytosis and phagosome acidification Cellular & Molecular Immunology


for the delivery of HKBA to intracellular TLR9 and supports previous data4 showing that IL-12p40 secretion by HKBA-stimulated DCs is dependent on the activation of TLR9 localized in the endosomal compartment. HKBA phagocytosis is mediated by TLR2, because DCs that are deficient in TLR2-, but not TLR4-, have defective HKBA phagocytosis. During phagocytosis, TLR2, which is expressed in the plasma membrane, is recruited to sites of contact by microorganisms and becomes enriched on phagosomes. TLR2 is recruited to phagosomes during the recognition of yeast particles and the phagocytosis of zymosan particles.38 TLR2 was reported to induce MAPKs activation (p38 and ERK).31,39,40 MAPKs are essential molecules involved in innate immune responses to microbes.39 We demonstrated here that HKBA triggers the phosphorylation of p38 and ERK (Figure 9). However, only the action of p38 controls TLR2-dependent HKBA phagocytosis and phagosome acidification and maturation. The p38 pathway likely facilitates the activation of TLR9, which explains the observed increase in IL-12p40 induction following HKBA stimulation. In contrast, the ERK1/2 pathway acts on a different aspect of the DC response, namely, ERK 1/2 inhibits IL-12p40 induction. Thus, ERK1/ 2- or p38-mediated TLR2 production plays opposing roles in IL12p40 induction through HKBA. Interestingly, HKBA activates DCs to secrete TNF and IL-12p40 through a p38-dependent pathway. The induction of TNF also requires ERK1/2 activity. It is unknown how the ERK1/2-dependent pathway down-modulates IL-12p40 levels

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. Cellular & Molecular Immunology

produced in response to HKBA in DCs; however, this down-modulation occurs without affecting the uptake of bacteria. These findings indicate that the stimulation of the two TLRs, TLR2 and TLR9, in response to a single microorganism results in activation and cross-talk between the respective signal transduction pathways. Following exposure to an infectious agent, it is likely that the activation of more than one TLR occurs because many microorganisms express more than one TLR ligand. An understanding of the cross-talk between different pathways stimulated in parallel downstream of TLRs is important to assess the host immune response. In addition, this particular knowledge could aid in the design of vaccines that might consist of more than one TLR ligand. Different upstream signals lead to the activation of MAPK. However, prominent roles for small G proteins have been identified. Thus, the ERK pathway can be activated in many cell types by RasGRPs.41–43 In contrast, the Rho family of GTPases, including Rac and Cdc42, activate p38 and JNK.41,43 It was originally suggested that RasGRP1 was selectively expressed in the lymphocytes and brain, kidney and skin cells.34 RasGRP1-deficient mice are defective in TCRinduced ERK phosphorylation, indicating that RasGRP1 is an important Ras activator that acts downstream of TCR in T cells.35 Interesting, it was recently reported that RasGRP1 plays an important role in phosphatidyl inositol-3 phosphate kinase activation and mast cell function, as RasGRP1-deficient mice fail to mount mast cell-dependent anaphylaxis.44 Here, we demonstrate that RasGRP1 is expressed in splenic DCs and that both Ras and RasGRP1 are activated downstream of TLR2. In fact, HBKA-mediated TLR2-triggered phosphorylation of ERK1/2 was greatly reduced in RasGRP1-deficient cells. Notably, RasGRP1-deficient lymphocytes are also defective in TCR-induced ERK phosphorylation,35 suggesting that RasGRP1 is an important Ras activator that acts downstream of TCR and TLR in lymphocytes and DCs, respectively. Consistent with these findings, Raf-1 kinases are essential in DC-SIGN-induced signaling pathways leading to the modulation of TLR signaling through an interaction with the active form of Ras in human DCs.45 However, Raf-1 activation by DC-SIGN through the TLR2-ligand ManLAM (a component from Mycobacterium tuberculosis) does not lead to ERK kinase activity. Although RasGRP1-deficient DCs were defective in ERK1/2 activation, they did not have defective phagocytosis. However, failure to activate ERK1/2 in RasGRP1-deficient DCs greatly impacted IL12p40 secretion, resulting in enhanced expression in response to HKBA. In light of our results and those from other studies, a mechanism emerges for the effect of stimulation of DCs in response to HKBA: (i) TLR2 is involved in the recognition of HKBA and in phagocytosis, phagosome maturation/acidification and the induction of TNF via p38; (ii) TLR2 engagement also activates ERK2 via RasGRP1, contributing to TNF induction while downregulating IL-12p40 secretion; (iii) DC maturation is followed by migration to T cell areas; (iv) DCs in Tcell areas, bearing internalized HKBA, are stimulated by DNA from HKBA via TLR9 to secrete IL-12p40, which, in turn, activates a strong Th1 immune response, with high levels of IFN-c production by T cells; and (v) TLR9-triggered IL-12p40 induction in DCs stimulated with HKBA is downregulated by ERK1/2 activation. Our results suggest that HKBA-stimulated TLR2 signaling requires RasGRP1 to activate ERK1/2, which, in turn, inhibits HKBA-induced IL-12p40 production in DCs. Because the phosphorylation of ERK1/2 was only partially reduced in RasGRP1-deficient DCs stimulated with HKBA, we considered the possibility that another protein and/or pathway might act as an ERK activator. It is reasonable to speculate


that the serine/threonine kinase Cot/Tpl2, which is indispensable for signal-regulated ERK activation and the production of TNF in LPSstimulated macrophages, could be a potential activator. Cot/Tpl2 is also activated through other TLR ligands, including bacterial DNA. Macrophages and bone marrow-derived DCs from Cot/Tpl2-deficient mice produced significantly more IL-12 in response to CpG-DNA than those from WT mice.46 However, the induction of Cot/Tpl2 and its role in the modulation of ERK1/2 activation in DCs stimulated with HKBA remains unknown. Finally, the insights gained from this study may help to design future adjuvants that can be used effectively in humans. For example, a Th1 stimulus such as HKBA can be used in conjunction with an inhibitor of RasGRP1 or ERK1/2. This combination would increase IL-12p40 induction, thereby enhancing the Th1 response while decreasing TNF secretion and possibly averting the toxic side effects caused by TNF. Furthermore, RasGRP1 inhibition could be considered a potential target for the immune therapy of diseases, such as auto-immunity, allergy, infectious disease and cancer, whereby the Th1/Th2 imbalance plays an essential role.

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This work was supported through funding from a Tianjin Municipal Science and Technology commission grant (No. 08ZCGYSH04700).

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Cellular & Molecular Immunology