Ligands

LPS from Escherichia coli O111:B4 (Sigma) was re-purified using two water/phenol extractions (11) or purchased from Invivogen (San Diego). LPS from Yersinia pestis pLpxL grown at 37 °C was purified as described (12). Poly(I:C) dsRNA was purchased purified from Invivogen. Synthetic diacylated (Pam₂Cys-based) macrophage-activating lipopeptide 2 kDa (MALP-2), Pam₃Cys-Ser-Lys₄ (Pam₃Cys), and fibroblast stimulating lipopeptide (FSL-1) were from EMC (Tübingen, Germany). R848 was from GLSynthesis (Worcester, MA) or Invivogen. Sendai virus (SV) Cantrell strain was from Charles River Laboratories (Wilmington, MA). Lipoteichoic acid (LTA) was a gift from Dr. Sonja von Aulock and Dr. Thomas Hartung (13).

Antibodies

Mouse anti-TLR2 mAb 6C2 was produced and purified as described (14). Mouse anti-CD86^PE-Cy5 and control rat IgG^PE-Cy5 antibody were from eBioscience (San Diego). TLR2 mAb 6C2 and rat IgG from Sigma or from Jackson ImmunoResearch (West Grove, PA) were labeled with an Alexa 488 kit from Molecular Probes (Eugene, OR).

Cells

Peritoneal macrophages were isolated and cultured as described previously (14). Human embryonic kidney 293 (HEK293) cells were cultured in 10% FBS/DMEM. HEK293 cell lines expressing TLR2^YFP were cultured with the selection antibiotic G418 (0.5 mg/ml).

Plasmids

The following expression vectors were used: pcDNA3 (Invitrogen); pFLAG-CMV2 (Sigma). pcDNA3-Cherry, provided by Dr. Ø. Halaas (NTNU, Norway), was cloned into pcDNA3 from pRSET-B-mCherry, provided by Prof. R. Y. Tsien (University of California). TLR2 tagged with yellow fluorescent protein (YFP) (TLR2^YFP) in pcDNA3 has been described (15). Murine TLR2^YFP was made using murine TLR2 originally cloned from RAW264.7 (16). Cherry-tagged TLR2 (TLR2^Cherry) was made by excising TLR2 from TLR2^YFP and ligating it into pcDNA3-Cherry. HA-tagged TLR2 in pcDNA3 (TLR2^HA) was from Dr. T. Fredsvik Gregers (University of Oslo, Norway). The following plasmids have been described: TRAM tagged with YFP (TRAM^YFP); TRIF tagged with CFP (TRIF^CFP); TRAMC117H (9); pEF-BOS, pEF-BOS-TRAM^FLAG, pEF-BOS-MAL^FLAG, and pEF-BOS-TRIF^FLAG (17); pEF-BOS-MyD88, MyD88-TIR, and TRIFΔNΔC (18); and early endosomal antigen-1 (Eea-1) tagged with CFP (Eea-1^CFP) (19). CCL4 (MIP-1β) luciferase (pGL3–1062WT) (20) was kindly provided by Prof. M. S. Reitz, Jr. (University of Maryland, Baltimore, MD). p65-pcDNA3, p65-pcDNA3^S536A, murine and human CCL5 (regulated on activation normal T cell expressed and secreted (RANTES)), and human ΔκB CCL5 luciferase reporter plasmids (CCL5-luc and ΔκB CCL5-luc) were kindly provided by Dr. Tom Maniatis (Harvard University, Cambridge, MA). pCMV-IRF3^Flag and -IRF5^Flag were from Prof. Paula M. Pitha (The Johns Hopkins University, Baltimore, MD). Renilla luciferase plasmid was from (Promega, Madison, WI). Transient transfections were performed using GeneJuice^TM transfection reagent (Novagen, Darmstadt, Germany) according to the manufacturer's instructions.

Mice

Wild-type (WT) C57Bl/6 and 129.B6 F2 mice were from the Jackson Laboratory (Bar Harbor, ME). myd88^−/− mice (MyD88 knock-out), tirap^−/− mice (denoted MAL^−/−), ticam-1^−/− (denoted TRIF^−/−), ticam-2^−/− mice (denoted TRAM^−/−), and tlr2^−/− and tlr4^−/− mice (denoted TLR2^−/− and TLR4^−/−, respectively) were provided by Dr. Shizuo Akira (Osaka University, Japan) and have been described (21,–24). Mice were back-crossed for six to eight (TLR4, TLR2, and MyD88), five to six (MAL), or four to seven (TRIF and TRAM) generations onto the C57BL/6 strain. MAL^−/−MyD88^−/−, TRIF^−/−MyD88^−/−, and TRAM^−/−TRIF^−/− double knock-out animals were generated from the single knockouts (KO) mentioned above. TRIF^Lps2/Lps2 mice generated on a C57/Bl6 background have been described (25). TRIF^Lps2/Lps2 animals were originally purchased from The Jackson Laboratory and were provided by Dr. Ian Rifkin, Dr. Robin Ingalls, and Anjali Nair. Alternatively, mouse legs from these animals were received from Dr. K. Hoebe and Dr. B. Beutler. ikke^−/− mice (denoted IKKϵ^−/−) were from Millennium Pharmaceuticals (Cambridge, MA). ifnar^−/− mice were obtained from Dr. J. Sprent (Scripps Institute, San Diego) and were backcrossed onto the C57BL/6 background. irf1^−/−, irf3^−/−, irf5^−/−, and irf7^−/− mice were provided by Dr. T. Taniguchi (University of Tokyo, Tokyo, Japan). tnfrsf1a^−/− mice (denoted tnfrI^−/− mice), tbk1^−/−/tnfrI^−/− mice and mouse embryonic fibroblasts (MEFs) from tbk1^−/− and tbk1^+/+ littermates were from Dr. T. Mak and W.-C. Yeh (University of Toronto, Canada) (26). Immortalized bone marrow-derived macrophages were generated with J2 recombinant retrovirus as described (27, 28).

Flow Cytometry Analysis and Cytokine Measurements

Cells were stimulated 16–20 h at 37 °C, 8% CO₂ before supernatant was harvested and assessed for Tnf or Ccl5 content using ELISA kits from R&D Systems (Oxon, UK) or Pharmingen. Stimulated cells were labeled as described (14) and analyzed by flow cytometry analysis on a BD-LSR II instrument.

Peritoneal macrophages from wild-type mice were also pretreated with medium, a DMSO control (diluted 1:375 in medium), or Dynasore (80 μm; 1:375 dilution in medium) for 30 min prior to stimulation with medium, LPS (10 ng/ml), Pam₃Cys (200 ng/ml), or FSL-1 (200 ng/ml) in 10% FBS/RPMI 1640 medium for 21 h. Supernatant was harvested and analyzed for Ccl5 and Tnf by ELISA.

In Vivo Experiments

WT or TRIF^Lps2/Lps2 mice were injected intraperitoneally with PBS or Pam₃Cys (25 μg). Whole blood was collected after 4 h by terminal cardiac puncture, after isoflurane anesthesia. Serum was separated from whole blood using pedi-serum separator tubes (BD Biosciences), which were placed on ice for 30 min, prior to centrifugation at 900 × g for 60 s. Ccl5 content in the serum was analyzed by ELISA.

Confocal Microscopy

HEK293 cells, seeded on 35-mm glass bottom γ-irradiated tissue cell dishes (MatTek Corp., Ashland, MA), were transiently transfected with TLR2^Cherry (1.25 μg/dish), TRAM^YFP (0.25 μg/dish), and early endosome marker EEA-1^CFP or TRIF^CFP (0.25 μg/dish) for 48 h. Cells were treated with medium or FSL-1 (200 ng/ml) for 1 h before cells were fixed and observed by confocal microscopy using an Axiovert 100-M inverted microscope, equipped with an LSM 510 laser scanning unit and a ×63 1.4-NA plan Apochromat oil-immersion objective (Zeiss, Jena, Germany). Co-localization maps showing co-localization events of TLR2 and/or TRAM and Eea-1 were created in Imaris 5.0.2, 64-bit version (Bitplane AG, Zurich, Switzerland). In these images, white denotes co-localization events between two channels, and pixels above the threshold that failed to co-localize were set to zero (black). User-defined thresholds were set conservatively in a rectangular selection mode chosen above the apparent noise level for each channel.

Luciferase Assays

HEK293-TLR2^YFP or HEK293 cells were plated in 96-well plates and transiently transfected with luciferase reporter genes (50 ng/well) and Renilla luciferase (10 ng/well) for 18–26 h prior to stimulation. In some experiments, HEK293 cells were transiently transfected with murine or human TLR2 (20 ng/well) and human CCL5 luciferase or human ΔkB CCL5 luciferase. In stimulation experiments, cells were stimulated in 10% FBS/DMEM for 18–20 h. In experiments where signaling molecules were overexpressed, cells were transfected with empty vector, MAL, MyD88, TRAM, and TRIF (2–20 ng/well) or the adaptor mutants MyD88-TIR, TRAMC117H, and TRIFΔNΔC (20 ng/well), or increasing concentrations of p65, p65^S536A (0.1–1-10⁻²⁰ ng/well), IRF3 (20–40-60–80 ng/well) or IRF5 (80 ng/well) for 24–48 h. Total amount of plasmid per well was kept constant. Following stimulation or overexpression, cells were lysed and assayed for luciferase reporter gene activity and Renilla activity. Luciferase results were normalized for Renilla activity and plotted as mean fold induction relative to medium/empty vector control.

Western

RAW264.7 mouse macrophages were stimulated with medium, FSL-1 (200 ng/ml) for 180, 90, 60, and 30 min, with LPS (100 ng/ml) for 90 or 60 min, or with poly(I:C) (50 μg/ml) for 60 min. Cells were lysed and run on a NuPAGE Novex 10% BisTris gel (Invitrogen) and transferred to a nitrocellulose membrane using the iBlot blotting system (Invitrogen). Membranes were blocked with 5% w/v BSA/TBS, 0.01% Tween 20 for 1 h before blots were incubated with antibodies against IRF3^Ser-396 (4D4G) or total IRF3 (Cell Signaling Technology, Danvers, MA) or alternatively antibody against total IRF3 (Santa Cruz, CA) for 48–72 h in 5% w/v BSA/TBS, 0.01% Tween 20 at 4 °C. Blots were subsequently stained with HRP-conjugated anti-rabbit (DAKO, DK) and subjected to SuperSignal West Femto ECL substrate.

Gene Expression Analysis

Nanostring nCounter gene expression analysis (NanoString Technologies) of selected inflammatory genes was performed on peritoneal macrophages from wild-type (WT) or TRIF^−/− mice treated with medium or FSL-1 lipopeptide for 4 h. Total RNA was isolated using RNeasy RNA isolation kit (Qiagen, Germany) and subjected to nCounter gene expression analysis of selected genes. Values are given as arbitrary units, normalized to internal GAPDH and hypoxanthine-guanine phosphoribosyltransferase standards. In other experiments, immortalized bone marrow macrophages (BMDM) from wild-type or from TRAM^−/−-, TRIF^−/−-, and TLR2^−/−- deficient C57Bl/6 mice were seeded in 24-well plates in triplicate and treated with medium or FSL-1 (200 ng/ml) for 3 h before cells were lysed. Total RNA was isolated with the NucleoSpin 96 RNA kit (Macherey-Nagel, Germany) and processed by vacuum. RNA quantity and purity were checked using NanoDrop (Thermo Scientific). cDNA was made using Maxima first-strand cDNA synthesis kit (Thermo Scientific). qPCR for murine ifnb1 (IFN-β) and tnf was done with GAPDH as endogenous control using TaqMan Probes (Mm0043552_s1, Mm00443258_m1, and Mm99999915_g1; Applied Biosystems). Real time thermal cycling was done with StepOnePlus (Applied Biosystems) using Perfecta qPCR FastMix (Quanta Biosciences) with uracil N-glycosylase and ROX reference dye. Analysis was done with the StepOne software version 2.1.

TIR Adaptors TRAM and TRIF Mediate Ccl5 Release Induced by TLR2 Ligands

We proceeded to test the role for TRAM in response to different TLR2 ligands. Ccl5 and Tnf induction was assessed in MyD88^−/−-, TRAM^−/−-, and Tlr2^−/−-deficient macrophages, in response to MALP-2, FSL-1, and LTA, as well as TLR2/1 ligand Pam₃Cys. We found that Ccl5 release was markedly reduced in TRAM^−/− macrophages in response to all TLR2 ligands tested (Fig. 2A), whereas Tnf induction in TRAM^−/− macrophages was normal (Fig. 2B). In contrast, the lack of MyD88 led to a sharp decline of both Tnf and Ccl5 release in response to all tested TLR2 ligands (Fig. 2, A and B). Tlr2-mediated Ccl5 induction was also impaired in MAL^−/− macrophages (data not shown). Ccl5 and Tnf release in response to Pam₃Cys, FSL-1, and LTA was completely abrogated in Tlr2^−/− macrophages, confirming the dependence on TLR2 in mediating these responses and indicating that the TLR2 ligands have high purity (Fig. 2, A and B). As TLR4 does not mediate the lipoprotein-induced Ccl5 (Fig. 1F), this promotes the view that TLR2 alone, and not TLR2/TLR4 heterodimers, triggers responses via TRIF and TRAM. LPS and SV induced normal levels of TNF and Ccl5 in TLR2^−/− macrophages (Fig. 2C).

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FIGURE 2.

TRAM and TRIF mediate Ccl5 release in response to TLR2 ligands. Peritoneal macrophages from wild-type (WT), MyD88^−/−, TRAM^−/−, and TLR2^−/− mice were exposed to medium (0), Pam₃Cys (P₃C) (100 ng/ml), or decreasing concentrations of MALP-2 (10–1 ng/ml), FSL-1 (20–2 ng/ml), or LTA (10–1 μg/ml) 18 h before supernatant was analyzed for Ccl5 content (A) and Tnf content (B) by ELISA. The results show mean with range of duplicates and are representative of three independent experiments. C, responses induced by TLR2 ligands are abolished in TLR2^−/− mice. Peritoneal macrophages from wild-type (WT) and TLR2^−/− mice were stimulated with medium or decreasing concentrations of Pam₃Cys (P₃C) (100–10 ng/ml), MALP-2 (100–1 ng/ml), LPS (100 ng/ml), or Sendai virus (SV) (100 HAU/ml) for 20 h before supernatant was harvested and analyzed for Ccl5 content by ELISA. The results show mean of quadruplicates ± S.E. Ccl5 (D) and Tnf (E) content in supernatant from macrophages from wild-type (WT), MyD88^−/−, and double knock-out MAL^−/−MyD88^−/−, TRIF^−/−MyD88^−/−, and TRAM^−/−TRIF^−/− mice stimulated with medium (0) or decreasing concentrations of LPS (100–10⁻¹ to 0.1 ng/ml), Pam₃Cys (P₃C) (100–10 ng/ml), R848 (100 ng/ml), or SV (100 HAU/ml) for 18 h before supernatant was analyzed by ELISA. Results show mean of triplicates ± S.D. ****, p < 0.0001 (two-way ANOVA with Bonferroni post-test).

Next, we assessed Ccl5 induction in response to TLR2 ligands in macrophages from MyD88^−/−, MAL^−/−MyD88^−/−, TRIF^−/−MyD88^−/−, and TRAM^−/−TRIF^−/− double-deficient mice. We observed that Ccl5 induction was impaired in TRAM^−/−TRIF^−/− cells in response to Pam₃Cys (Fig. 2D), confirming our observations in TRIF^−/− and TRAM^−/− macrophages (Figs. 1C and ​and22A). Ccl5 induction was also impaired in all the tested adaptor-deficient cells in response to the TLR2/TLR6 ligand MALP-2 (data not shown). Importantly, TRAM^−/−TRIF^−/− macrophages displayed normal Tnf expression in response to both Pam₃Cys and R848 (Fig. 2E). Combined, our results imply that the TRAM/TRIF-dependent pathway is involved in TLR2-induced Ccl5 release (Figs. 1C and ​and2,2, A, C, and D), but not in TNF release (Figs. 1D and ​and2,2, B and E). LPS-induced Ccl5 was completely abrogated in both TRIF^−/− MyD88^−/− and TRAM^−/−TRIF^−/− macrophages (Fig. 2D), emphasizing the recognized TRAM/TRIF dependence of this response. TNF release following LPS challenge was greatly reduced in both MAL^−/−MyD88^−/− and TRAM^−/−TRIF^−/− macrophages (Fig. 2E), implying that both of the signaling branches contribute considerably to LPS-induced TNF release. Responses toward SV were normal in TRIF^−/−MyD88^−/− macrophages (Fig. 2, C and D), showing that SV recognition is TLR-independent in these cells, as reported previously (6,–8).

TLR2 and TRAM Co-localize in Endosomes, and TLR2-mediated CCL5 Induction Is Dependent on Endocytosis

We proceeded to investigate the subcellular expression of TLR2 and TRAM to verify the involvement of TRAM in TLR2 signaling. TLR2 is expressed in early endosomes, as well as in recycling Rab11a-positive endosomes in human monocytes (30). TRAM has been reported to be expressed at the plasma membrane and in Rab5-positive endosomes (46). We therefore proceeded to investigate whether TLR2 co-localizes with TRAM in early endosomes. Overexpression of fluorescently tagged TLR2^Cherry, TRAM^YFP, and the early endosome marker Eea-1^CFP in HEK293 cells revealed that TLR2 and TRAM indeed do co-localize, both at the plasma membrane and in a portion of Eea-1-positive endocytic vesicles (Fig. 3A). Upon stimulation with FSL-1, TLR2 and TRAM accumulated and co-localized in the perinuclear area (Fig. 3B). Overexpression of TRAM^YFP and TRIF^CFP in HEK293 showed particularly good co-localization in endocytic vesicles (Fig. 3C). Minimal concentrations of plasmid were used to avoid cell activation because expression of high levels of these adaptor molecules alone can drive NFκB activation. However, we cannot exclude the possibility that the strong co-localization between TRAM and TRIF may partly be a consequence of transfection-driven cell activation. Nevertheless, we also observed TLR2 co-localizing with TRIF in TRAM-positive vesicles in HEK293 overexpressing TLR2^Cherry, TRIF^CFP, and TRAM^YFP (Fig. 3C), implying that TLR2 is recruited to a TRAM- and TRIF-containing complex.

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FIGURE 3.

TLR2 and TRAM co-localize in endosomes, and TLR2-mediated Ccl5 is impaired upon inhibition of endocytosis. HEK293 cells were transiently transfected with TLR2^Cherry (red), TRAM^YFP (green), and early endosome marker Eea-1^CFP (blue) (A and B) for 48 h prior to treatment with medium (A) or FSL-1 (B) (200 ng/ml) for 1 h before cells were fixed and observed by confocal microscopy. Overlays of TLR2^Cherry (red), TRAM^YFP (green), and early endosome marker Eea-1^CFP (blue) are shown in images to the left. C, TLR2 co-localizes with TRAM and TRIF. HEK293 cells were transiently transfected with TLR2^Cherry (red), TRAM^YFP (green), and TRIF^CFP (blue) for 48 h. Images to the right show single tracks of enlargements of the areas denoted by the square in the main picture. Co-localization between channels in this area are shown to the far right, both in color and by using co-localization maps. Maps show co-localization events between the two respective channels (white). Measure bar is 5 μm. D, Ccl5 and E, Tnf release in peritoneal macrophages from wild-type mice pretreated with medium, a DMSO control, or Dynasore (80 μm) for 30 min prior to stimulation with medium (0), LPS (10 ng/ml), Pam₃Cys (P₃C) (200 ng/ml), or FSL-1 (200 ng/ml) for 21 h. Supernatant was analyzed for cytokines by ELISA. Results show mean of triplicates ± S.D.

Because we observed TLR2 and TRAM co-localizing in endocytic compartments, we investigated the importance of endocytosis for Ccl5 induction by pretreating peritoneal macrophages with the dynamin GTPase inhibitor Dynasore prior to stimuli with LPS and TLR2 ligands. Dynasore efficiently inhibited CCL5 release induced by TLR2 ligands (Fig. 3D) but not TNF release (Fig. 3E), suggesting that endocytosis of ligand is required for Ccl5 induction in response to these ligands. These results suggest that signaling leading to Ccl5 induction likely occurs from early endosomes, where TLR2 and TRAM co-localize.

TLR2-mediated CCL5 Induction Is Impaired in TRIF^Lps2/Lps2 Mice in Vitro and in Vivo

TRIF^Lps2/Lps2 mice are derived from a C57Bl/6 background and contain a chemically induced base pair-deletion/frameshift in the gene encoding TRIF (25). These animals mimic mice with a gene targeted TRIF deletion. We observed that macrophages from TRIF^Lps2/Lps2 mice displayed reduced Ccl5 release in response to TLR2 ligands MALP-2 and FSL-1 (Fig. 4A). In contrast, Tnf release was not impaired in response to these TLR2 ligands (Fig. 4B), which is consistent with previous reports (25).Ccl5 and Tnf release were normal in response to R848 (Fig. 4, A and B).

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FIGURE 4.

TLR2-mediated Ccl5 induction is impaired in TRIF^Lps2/Lps2 mice in vitro and in vivo. Peritoneal macrophages from wild-type (WT) or TRIF^Lps2/Lps2 mice were treated with medium (0), and MALP-2 (1–0.1–0.01 μg/ml), FSL-1 (1–0.1–0.01 μg/ml), or R848 (10 ng/ml) for 20 h before supernatant was harvested and analyzed for Ccl5 (A) and TNF (B) release by ELISA. Results show mean of triplicates with standard deviations. ****, p < 0.0001; *, p < 0.05 (two-way ANOVA with Bonferroni post-test). C, in vivo induction of Ccl5 in the serum of wild-type mice (WT) or TRIF^Lps2/Lps2 mice injected intraperitoneally with PBS or Pam₃Cys (25 μg). Blood was collected by cardiac puncture 4 h after injection, and serum was analyzed for Ccl5 content by ELISA. Results show mean values of two PBS-injected mice and five Pam₃Cys-injected mice. ****, p < 0.0001 (unpaired Student's t test).

To determine whether TLR2 ligands can induce Ccl5 in a TRIF-dependent manner in vivo, WT or TRIF^Lps2/Lps2 mice received an intraperitoneal injection with Pam₃Cys. Serum was collected after 4 h and assayed for Ccl5. We found that Ccl5 levels were elevated in wild-type mice following Pam₃Cys injection (Fig. 4C). This Ccl5 induction in response to Pam₃Cys injection was impaired in TRIF^Lps2/Lps2 mice (Fig. 4C), confirming our in vitro findings and supporting a role for TRIF in TLR2-mediated Ccl5 induction.

TLR2-mediated CCL5 Activation Is Impaired upon Overexpression of Signaling Defective Mutant Versions of TRAM and TRIF

Our findings in peritoneal macrophages were verified by transiently transfecting HEK293 cells stably expressing TLR2 with a murine ccl5 luciferase reporter gene. Stimulation of TLR2-expressing HEK293 cells with TLR2 ligands activated the ccl5 promoter (Fig. 5A). Similar results were obtained using a human CCL5 luciferase reporter gene (data not shown). The different TIR-adaptor proteins were also assessed for their ability to activate the CCL5 promoter upon overexpression. This was done by transfecting HEK293 cells with TLR2 and the CCL5 luciferase and co-transfecting with MAL, MyD88, TRAM, or TRIF or the functionally defective adaptor mutants MyD88TIR, TRAMC117H, and TRIFΔNΔC. Transfection of MyD88, TRAM, or TRIF all resulted in activation of the CCL5 reporter in this system (Fig. 5B), whereas overexpression of TRAM C117H, TRIFΔNΔC, or MyD88TIR impaired CCL5 induction in response to TLR2 ligands Pam₃Cys and FSL-1 (Fig. 5C). Combined, these results confirm that TLR2 ligands induce CCL5 and that TRAM and TRIF mediate CCL5 induction in response to TLR2 ligands.

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FIGURE 5.

TLR2-mediated CCL5 activation is impaired upon overexpression of TRAM and TRIF. A, HEK293-TLR2^YFP cells were transiently transfected with a murine Ccl5 luciferase reporter gene for 24 h prior to stimulation with medium, Pam₃Cys (100–10 ng/ml), FSL-1 (100–10 ng/ml), LTA (10 μg/ml), or Sendai virus (SV) (50 HAU/ml) for 18 h and assayed for luciferase reporter gene activity. Results show fold induction relative to the medium control. B, HEK293 cells were transiently transfected with CCL5 luciferase reporter gene and co-transfected with empty vector (EV), MAL, MyD88, TRAM, or TRIF (20 ng/well) for 48 h and assayed for luciferase activity. ****, p < 0.0001; **, denotes p < 0.01; *, p < 0.05 (one-way ANOVA with Tukey post-test). C, HEK293 cells were transiently transfected with CCL5 luciferase, TLR2, and the adaptor mutants MyD88TIR, TRAMC117H, and TRIFΔNΔC for 48 h. Cells were then stimulated with medium, Pam₃Cys (100 ng/ml), or FSL-1 (200 ng/ml) for 16 h before luciferase reporter gene activity was assayed. D, HEK293 cells were transfected with CCL5 luciferase and empty vector (EV), IRF3-FLAG (80–60–40–20 ng), NF-κB subunit p65 (20–10 ng), or IRF5-FLAG (80 ng) for 26 h, before cells were lysed and assayed for luciferase activity. Activation of the CCL5 promoter occurs independent of NF-κB p65^S536. E, HEK293 cells were transfected with TLR2 and a CCL5 luciferase reporter gene (CCL5 Luc) or a CCL5 luciferase reporter gene with mutated NF-κB sites (ΔκB CCL5 Luc) overnight. Cells were stimulated with medium (0), FSL-1 (1–10–100 ng/ml), or Pam₃Cys (1–10–100–200 ng/ml) for 18 h before cells were lysed and assayed for luciferase activity. F, CCL5 luciferase activation upon overexpression of NF-κB subunit p65 (10–1–0.1 ng) or mutant p65^S536A (10–1–0.1 ng) for 27 h before cells were lysed and assayed for luciferase activity. All results show firefly luciferase values normalized to Renilla activity and display mean of triplicates ± S.D. Results are representative of three independent experiments.

The promoters of CCL5, CXCL10, as well as IFN-β contain transcription factor-binding elements for both NF-κB and IRFs (31, 32). We observed that overexpression of NF-κB p65 and IRF3, but not of IRF5, efficiently induced activation of the CCL5 promoter in HEK293 cells co-transfected with a CCL5 luciferase reporter (Fig. 5D). TLR2 ligands are considered poor activators of IRF3 (9, 33), so we initially investigated whether the observed TLR2-mediated CCL5 response could be mediated by NF-κB alone. Using HEK293 cells transiently expressing TLR2 and a CCL5 luciferase reporter containing mutated NF-κB sites (ΔκB CCL5 luciferase), we found that the fold induction activation of this reporter was similar to the activation of WT CCL5 luciferase reporter in response to TLR2 ligands (Fig. 5E). Overexpression of mutant NF-κB p65^S536A in HEK293 cells also activated the CCL5 luciferase reporter to the same extent as wild-type p65 (Fig. 5F). These results indicate that phosphorylation of p65^Ser-536 is not essential for TLR2-mediated CCL5 induction and prompted us to investigate a role for IRF3 in TLR2-mediated CCL5.

TLR2 Ligands Activate IRF3, and CCL5 Induction Is impaired in IRF3^−/− and IRF1^−/−Macrophages in Response to TLR2 Ligands

To investigate whether TLR2 ligands can activate IRF3, we initially applied a GAL4 luciferase reporter assay and a vector expressing yeast Gal4 DNA binding domain fused to IRF-3 lacking its own DNA binding domain (IRF3-Gal4). Activation of IRF3 in this assay leads to the expression of the GAL4 luciferase reporter (34, 35). We observed that TLR2 ligands activated IRF3 measured by this reporter system, and that the activation was impaired upon overexpression of the dominant negative version of TRAM, TRAMC117H (Fig. 6A). The FSL-1 lipopeptide also induced phosphorylation of IRF3^Ser-396 in RAW264.7 cells (Fig. 6B), confirming that TLR2 ligands can activate IRF3, although to a lesser extent than LPS and poly(I:C) (Fig. 6B). IRF1 also regulates TLR-dependent induction of type I interferon responses in myeloid dendritic cells and macrophages (36). We further investigated the role for IRF3 and IRF1, as well as IRF5 and IRF7, in TLR2-mediated CCL5 induction. Peritoneal macrophages from wild-type (WT), irf1^−/−-, irf3^−/−-, irf5^−/−-, or irf7^−/−-deficient mice were stimulated with TLR2 ligands, as well as LPS, poly(I:C), R848, or SV prior to assaying Ccl5 and Tnf release in these cells by ELISA. We found that Ccl5 release was impaired in peritoneal macrophages from both Irf1- and Irf3-deficient mice in response to the TLR2 ligands Pam₃Cys, Pam₂Cys, FSL-1, and MALP-2 (Fig. 6C). Tnf was, however, also partially impaired in irf1^−/− macrophages in response to TLR2 ligands (Fig. 6D), suggesting that irf1 plays a role in both Tnf and Ccl5 release in response to lipopeptides. LPS- and poly(I:C)-induced Ccl5 was normal in irf1^−/− macrophages, in line with previous reports (37). Importantly, IRF3 was found to be a key player in mediating the release of Ccl5 (Fig. 6C), but not Tnf (Fig. 6D), in response to TLR2 ligands. This correlates with our observations in TRAM^−/− and TRIF^−/− macrophages (Figs. 1 and ​and2).2). Ccl5 induction in response to TLR2 ligands was found to be normal in irf5^−/− peritoneal macrophages, whereas both Ccl5 and TNF were partially impaired in irf7^−/− cells in response to TLR2 ligands (Fig. 6, C and D). These results show that TLR2 ligands activate Irf3, and TLR2-mediated Ccl5 is mediated by Irf3, as well as by Irf1, and they suggest that TLR2-induced Irf3 activation is mediated by the TRAM/TRIF pathway.

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FIGURE 6.

TLR2 ligands activate IRF3, and TLR2-mediated Ccl5 release is impaired in IRF3^−/− cells. A, HEK293 cells were transiently transfected overnight with empty vector (EV) or TRAMC117H (20–2-0.2 ng), and co-transfected with TLR2 and IRF3-Gal4 and a luciferase reporter containing the Gal4 activation sequence (Gal4 luciferase). Cells were subsequently stimulated with Pam₃Cys (200 ng/ml), FSL-1 (200–20 ng/ml), or Sendai virus (SV) (100 HAU/ml) for 16 h before cells were assayed for luciferase activity. Results are normalized for Renilla activity and show mean fold induction ± S.D. of triplicates. B, Western blots of lysate of RAW264.7 stimulated with medium (0), with FSL-1 (200 ng/ml) for 180–90-60 and 30 min, with LPS (100 ng/ml) for 90 and 60 min, or poly(I:C) (50 μg/ml) for 60 min and stained with anti-IRF3^Ser-396 (pIRF3) or anti-Total-IRF3. Graph shows quantification of band intensities of blot shown below stained with anti-IRF3^Ser-396 (pIRF3) (top blot) and total IRF3 (TOT IRF3) (bottom blot) relative to α-tubulin staining. TLR2-mediated Ccl5 induction is impaired in IRF1^−/− and IRF3^−/− mice. Peritoneal macrophages from wild-type (WT), irf1^−/−, irf3^−/−, irf5^−/−, and irf7^−/− mice were exposed to medium (0), Pam₃Cys (1000–100–10 ng/ml), Pam₂Cys (1000–100–10 ng/ml), MALP-2 (1000–100–10 ng/ml), FSL-1 (1000–100-10 ng/ml), LPS (10 ng/ml), poly(I:C) (pIC) (50 μg/ml), R848 (100 ng/ml), or Sendai virus (SV) (100 HAU/ml) for 18 h before supernatant was harvested and analyzed for Ccl5 content (C) and Tnf content (D) by ELISA. Results show mean with range of duplicate samples and are representative of two experiments.

TLR2-mediated Ccl5 Induction Is Impaired in TBK1-deficient Cells

The noncanonical IκB kinases IKKϵ and TBK1 are important mediators of TRIF-dependent IRF3 activation leading to type I IFN and CCL5 induction in response to viral components and LPS (38). To determine the role of IKKϵ, as well as the type I interferon receptor I (IFNAR) in TLR2-mediated Ccl5, we assayed Ccl5 induction in response to TLR2 ligands in peritoneal macrophages from WT, ikke^−/− (IKKϵ) and ifnar^−/− mice, as well as irf3^−/− mice. Ccl5 release in response to TLR2 ligands was normal in IKKϵ-deficient cells after 18 h of stimulation (Fig. 7A). The receptor for type I IFNs, Ifnar, did not appear to be important for TLR2-mediated Ccl5 release either (Fig. 7A), in contrast to Irf3 (Fig. 7A), indicating that TLR2-mediated Ccl5 induction is a primary response that occurs independent of type I IFN signaling. TLR2-induced ccl5 mRNA induction was also unaffected by inhibition of protein synthesis with cycloheximide (data not shown), further suggesting that this Ccl5 induction is a primary response.

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FIGURE 7.

TLR2-mediated Ccl5 induction is impaired in tbk1-deficient cells. A, peritoneal macrophages from wild-type (WT), ikkϵ^−/−, irf3^−/−, and ifnar1^−/− mice were exposed to medium (0), Pam₃Cys (P₃C) (100 ng/ml), FSL-1 (100–10 ng/ml), MALP-2 (100 ng/ml), LTA (10 μg/ml), or Sendai virus (SV) (10 HAU/ml) for 18 h before supernatant was harvested and analyzed for Ccl5 content by ELISA. Results show mean with range of duplicate samples. Ccl5 (B) and Tnf (C) induction in immortalized BMDM from tnfrI^−/−-deficient or tbk1^−/−/tnfrI^−/− double-deficient mice stimulated with medium (0), Pam₃Cys (100–10⁻¹ ng/ml), or FSL-1 (100–10⁻¹ ng/ml) for 6 h. Supernatant was harvested and analyzed for Ccl5 and Tnf by ELISA. Results show mean ± S.D. of quadruplicates. D, Ccl5 induction in tbk1^+/+ or tbk1^−/− MEFs stimulated for 18 h with medium (0), Pam₃Cys (200–100–50 ng/ml), FSL-1 (200–100-50 ng/ml), or LPS (100 ng/ml) before supernatant was assayed for Ccl5 by ELISA. Results show mean ± S.D. of triplicate samples.

IKKϵ and TBK1 have both been shown to participate in mediating TRIF-IRF3 signaling (17), so we proceeded to investigate the role of TBK1 in TLR2-mediated CCL5. Homozygous deletion of tbk1 is lethal in C57BL/6, but because this embryonic lethality is TNF-dependent, mice survive upon deletion of tnfrsf1a (tnfrI) (17). We consequently assayed Ccl5 induction in response to TLR2 ligands in immortalized BMDM from tbk1^−/−/tnfrI^−/− double-deficient and tnfrI^−/−-deficient mice. tbk1^−/−/tnfrI^−/− cells displayed impaired Ccl5 induction in response to TLR2 ligands at early time points, following 6 h of stimulation (Fig. 7B), whereas the same cells displayed similar TNF release (Fig. 7C). After longer stimulation (>12 h) TLR2-mediated Ccl5 induction appeared normal in tbk1^−/−/tnfrI^−/− BMDM (data not shown), possibly due to redundancy between IKKϵ and TBK1 in their ability to activate IRF3 (39, 40). The role for TBK1 in mediating TLR2-induced Ccl5 was confirmed in tbk1^−/− MEFs (Fig. 7D). LPS-induced Ccl5 was also impaired in tbk1^−/− MEFs (Fig. 7D), in accordance with previous reports (38). These data are consistent with a role for TBK1 in TLR2-mediated CCL5 induction.