Frizzled1 is a marker of inflammatory ... - The FASEB Journal

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Frizzled1 is a marker of inflammatory macrophages, and its ligand Wnt3a is involved in reprogramming Mycobacterium tuberculosis-infected macrophages Jan Neumann,* Kolja Schaale,* Katja Farhat,†,1 Tobias Endermann,*,2 Artur J. Ulmer,† Stefan Ehlers,‡,§ and Norbert Reiling*,3 *Division of Microbial Interface Biology, †Division of Cellular Immunology, and ‡Microbial Inflammation Research, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel, Germany; and §Molecular Inflammation Medicine, Christian-Albrechts University, Kiel, Germany Wnt/Frizzled signaling, essential for embryonic development, has also recently been implicated in the modulation of inflammatory processes. In the current study, we observed a reciprocal regulation of the Toll-like receptor (TLR)/nuclear factor-␬B (NF␬B) and the Wnt/␤-catenin pathway after aerosol infection of mice with Mycobacterium tuberculosis: whereas proinflammatory mediators were substantially increased, ␤-catenin signaling was significantly reduced. A systematic screen of Fzd homologs in infected mice identified Fzd1 mRNA to be significantly up-regulated during the course of infection. In vitro infection of murine macrophages led to a strong induction of Fzd1 that was dependent on TLRs, the myeloid differentiation response gene 88 (MyD88), and a functional NF-␬B pathway. Flow cytometry demonstrated an elevated Fzd1 expression on macrophages in response to M. tuberculosis that was synergistically enhanced in the presence of IFN-␥. Addition of the Fzd1 ligand Wnt3a induced Wnt/␤-catenin signaling in murine macrophages that was inhibited in the presence of a soluble Fzd1/Fc fusion protein. Furthermore, Wnt3a reduced TNF release, suggesting that Wnt3a promotes antiinflammatory functions in murine macrophages. The current data support the notion that evolutionarily conserved Wnt/Fzd signaling is involved in balancing the inflammatory response to microbial stimulation of innate immune cells of vertebrate origin.—Neumann, J., Schaale, K., Farhat, K., Endermann, T., Ulmer, A. J., Ehlers, S., Reiling, N. Frizzled1 is a marker of inflammatory macrophages, and its ligand Wnt3a is involved in reprogramming Mycobacterium tuberculosis-infected macrophages. FASEB J. 24, 4599 – 4612 (2010). www.fasebj.org

ABSTRACT

Key Words: innate immunity 䡠 Wnt signaling 䡠 inflammation

The frizzled (FZD) family of proteins represents a highly conserved group of 10 membrane proteins that have been shown to function as receptors for some or all of the Wnt family members in invertebrates and 0892-6638/10/0024-4599 © FASEB

vertebrates (1, 2). Wnts are palmitoylated glycoproteins (3, 4) that regulate essential steps of early development, including embryonic patterning, cell proliferation, and cell fate determination (4, 5). Wnts bind to the cysteinrich domain of Fzd proteins, which are 7-transmembrane domain cell surface receptors that phylogenetically belong to the large family of G-protein-coupled receptors (reviewed in ref. 6). Depending on the receptor context, Wnt ligands can initiate at least 3 different intracellular signaling cascades that regulate various cellular events: the Wnt/␤-catenin pathway, the Wnt/Planar cell polarity pathway, and the Wnt/Ca2⫹ pathway (recently reviewed in ref. 7). In addition to its essential role in developmental processes, the Wnt/␤catenin pathway is critical for maintaining stem cell and organ homeostasis (reviewed in ref. 8), represented by, e.g., controlled cell proliferation and cellular differentiation. Organ function and homeostasis are significantly altered during inflammation or infection. In the latter, cells of the innate immune system, such as macrophages and dendritic cells, recognize conserved microbial structures, also known as pathogen-associated molecular patterns (PAMPs). PAMPS are expressed by many microbes, but not found in higher eukaryotes. These structures are recognized by members of the Toll-like receptor (TLR) family (9). TLR-dependent signaling pathways can directly induce macrophage antimicrobial programs, but they also initiate inflammatory cell recruitment and help to prime cells of the adaptive immune system in order to amplify bactericidal effector mechanisms. Immunity to infections de1

Current address: Department of Cardiovascular Physiology, Georg-August University, Go¨ttingen, Germany. 2 Institute for Experimental Endocrinology, Charite´ Universita¨tsmedizin, Berlin, Germany. 3 Correspondence: Division of Microbial Interface Biology, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany. E-mail: [email protected] doi: 10.1096/fj.10-160994 4599

pends on the successful integration of innate and adaptive defense strategies (10). Experimental infections with microorganisms have been successfully used to uncover the intricacies governing the interplay between innate and adaptive immunity (11). For example, cell wall components of Mycobacterium tuberculosis, the causative organism of tuberculosis, critically depend on TLR-2 and -4 to induce secretion of the proinflammatory cytokines TNF and IL-12 (12). Subsequently, mycobacteria-primed T cells secrete IFN-␥ as a critical macrophage-activating agent. Eradication of mycobacteria is only achieved when both arms of the immune system are fine tuned for full antimicrobial potency. Only very recently has it become evident that Wnt and Fzd homologs are expressed not only by developing immune cells (13) but also by differentiated cells of the innate and the adaptive immune system: we (14) have demonstrated that Wnt5a and Fzd5 are induced in a TLR-dependent manner and regulate critical antimicrobial effector functions, e.g., the formation of proinflammatory cytokines such as IFN-␥ and IL-12. Similar data were subsequently obtained by Pereira et al. (15), who identified Wnt5a to be up-regulated in human myeloid cells during septicemia. Wnt signaling has also been shown to induce extravasation of murine effector T cells (16). The overall concept that factors of the Wnt/Fzd pathway act as novel regulators of inflammatory processes has only recently been reviewed (17). The current study focuses on the regulation of the Wnt/␤-catenin signaling in a model of chronic inflammation induced by experimental aerosol infection with M. tuberculosis: whereas inducible nitric oxide synthase (NOS2) and IFN-␥ formation are increased on infection with M. tuberculosis in vivo, ␤-catenin levels and the mRNA expression of the ␤-catenin-dependent target gene Axin2 are significantly reduced. These inflammatory conditions, however, lead to a sustained up-regulation of mRNA for Fzd1, a receptor known to mediate Wnt/␤-catenin signaling, in M. tuberculosis-infected mice and macrophages. The observation that activation of the ␤-catenin pathway by Wnt3a, which is constitutively present in lung epithelium, can be inhibited by soluble Fzd1 suggests a novel role of Fzd1 in a Wntmediated feedback mechanism that may be involved in preserving cell homeostasis during microbe-induced inflammation.

MATERIALS AND METHODS

Bacteria and conserved bacterial structures Mycobacterium avium (strain SE01) and M. tuberculosis (strain H37Rv, ATCC 27294; Manassas, VA, USA) were grown as described previously (19, 20). For in vitro experiments, bacterial aliquots were thawed, centrifuged for 10 min at 835 g, and resuspended in PBS. Bacterial LPS (Salmonella enterica serotype Friedenau H909) was kindly provided by H. Brade (Research Center Borstel). The synthetic lipopeptide Pam3CSK4 was kindly provided by K.-H. Wiesmu¨ller (EMC Microcollections, Tu¨bingen, Germany). In vitro infection BMDMs (0.5⫻106) were cultured in DMEM (PAA, Pasching, Austria) [supplemented with 10% heat-inactivated FCS (Biochem, Berlin, Germany), 4 mM l-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate (all PAA)] in 24-well flat-bottom microtiter plates (Nunc, Roskilde, Denmark) at 37°C in a humidified atmosphere containing 5% CO2. Macrophages were infected with M. tuberculosis and M. avium at the indicated multiplicities of infection (MOI). LPS, Pam3CSK4, and recombinant TNF (PeProTech, Rocky Hill, NY, USA) were used as controls. IFN-␥ (250 U/ml; PeProTech) was added 24 h before infection. Wnt3a stimulation assays and inhibition of Wnt3a by soluble Fzd1/Fc chimera For Wnt3a stimulation assays, conditioned medium (CM) was derived from Wnt3a transgenic L929 and nontransfected L929 cells (American Type Culture Collection, Manassas, VA, USA). Only cells of passage 3 to 13 were used for the generation of CM. Cells were cultivated in 10 ml culture medium in the absence of antibiotics for 4 d. The medium was removed and filtered (0.45 ␮m; Sarstedt, Nu¨mbrecht, Germany). For stimulation experiments, 5 ⫻ 105 BMDMs were seeded on a 24-well flat-bottom microtiter plate (Nunc) and incubated in 500 ␮l of CM or Wnt3-CM for 4 h. The GSK3␤ inhibitor SB216763 was obtained from Tocris (Bristol, UK). Aerosol infection and colony enumeration assay Pulmonary infection of experimental animals with M. tuberculosis (100 CFU) was performed as described previously (20). To determine the bacterial load, mice were killed at different time points after infection. Lungs of infected mice were removed aseptically and weighed, and 1 lobe per mouse was homogenized in PBS containing protease inhibitors (Complete; Roche Applied Science, Mannheim, Germany). Tenfold serial dilutions of lung homogenates were plated in duplicates onto Middlebrook 7H10 agar plates (Difco, Heidelberg, Germany) containing 10% heat-inactivated bovine serum (Biowest, Paris, France) and incubated at 37°C for 21 d. Colonies on plates were enumerated, and bacterial burden was expressed as log10 CFU per lung.

Mice and cultivation of macrophages Quantitative real-time PCR (qRT-PCR) C57BL/6, MyD88⫺/⫺, TLR2⫺/⫺, and TNF⫺/⫺ mice were raised and maintained under specific pathogen-free conditions. All knockout mice were backcrossed for at least 10 generations to a C57BL/6 background. Bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages were generated as described previously (18, 19). All animal experiments were approved by the Ministry of Environment (Kiel, Germany). 4600

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Total RNA was isolated from BMDMs (0.5⫻106 cells) or 100 – 150 mg lung tissue of pulmonary-infected mice using TriFast FL (Peqlab, Erlangen, Germany). For reverse transcription, the Transcriptor High Fidelity cDNA synthesis kit (Roche Applied Science) was used. The following gene-specific primer pairs were used in SYBR green qRT-PCRs: Fzd1 fwd 5⬘-TTCCTGCTGGCCGGTTTCGTGTCA-3⬘, rev 5⬘-CTGGGCT-

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at 4°C. Cells were washed and resuspended in PBS containing 1% (v/v) PFA (Merck) until analysis in a FACSCalibur flow cytometer (BD Bioscience). For intracellular FACS staining, macrophages were fixed for 10 min at 37°C by resuspending the cells in FACS buffer containing 3% (v/v) PFA. After fixation, the cells were washed and permeabilized for 30 min at 4°C in FACS buffer containing 90% (v/v) methanol as previously decribed (21). For simultaneous detection of intracellular Fzd1 and TNF, cells were incubated with 1 ␮l/ml GolgiPlug (BD Bioscience) and LPS (100 ng/ml) and IFN-␥ (250 U/ml) for 12 h. Fixation and permeabilization were done using Cytofix/Cytoperm solution (BD Bioscience). Cells were labeled with phycoerythrin(PE)-conjugated antimouse TNF (eBioscience, San Diego, CA, USA) and FITCconjugated anti-human/mouse Frizzled-1 (R&D Systems). PE-labeled rat IgG1␬ (eBioscience) and FITC rat IgG2a (Caltag Laboratories, Burlingame, CA, USA) were used as isotype controls.

CATGGGCGGGTGTGG-3⬘; TNF fwd 5⬘-CTGTAGCCCACGTCGTAGC-3⬘, rev 5⬘-TTGAGATCCATGCCGTTG-3⬘; and hypoxanthine-guanine phosphoribosyl transferase (HPRT) fwd 5⬘-GCAGTACAGCCCCAAAATGG-3⬘, rev 5⬘-AACAAAGTCTGGCCTGTATCCAA-3⬘. Melting curve analysis was performed at the end of each PCR reaction to ensure specificity. The following gene-specific primer pairs and UniversalProbeLibrary probes were used in a TaqMan assay (Roche Applied Science): Arg1 fwd 5⬘-CCTGAAGGAACTGAAAGGAAAG-3⬘, rev 5⬘-TTGGCAGATATGCAGGGAGT-3⬘, probe 2; Axin2 fwd 5⬘GCAGGAGCCTCACCCTTC-3⬘, rev 5⬘-TGCCAGTTTCTTTGGCTCTT-3⬘, probe 50; Fzd1 fwd 5⬘-GAGGAGGGAGCCGAAAAA3⬘, rev 5⬘-TTCGGCACAAAGTTCCAA-3⬘, probe 102; Fzd2 fwd 5⬘-GCTCTTCGTATACCTGTTCATCG-3⬘, rev 5⬘-GATGCGGAAGAGTGACACG-3⬘, probe 4; Fzd3 fwd 5⬘-TCCATGGGCGTAGGAAAA-3⬘, rev 5⬘-TCCCTCAGGAGTGACTGAGC-3⬘, probe 1; Fzd4 fwd 5⬘-ACTTTCACGCCGCTCATC-3⬘, rev 5⬘-TGGCACATAAACCGAACAAA-3⬘, probe 19; Fzd5 fwd 5⬘-TGTCACTGGGATTCTTGGTG-3⬘, rev 5⬘-GGGCCGGTAGTCTCATAGTG-3⬘, probe 76; Fzd6 fwd 5⬘-TTAAGCGAAACCGCAAGC-3⬘, rev 5⬘-TTGGAAATGACCTTCAGCCTA-3⬘, probe 81; Fzd7 fwd 5⬘-GGGTATCTCTGTGTAGCCCTGA-3⬘, rev 5⬘-AGAGGCAGGTGGATGTCTGT-3⬘, probe 21; Fzd8 fwd 5⬘-CTAGAGGTGCACCAGTTTTGG-3⬘, rev 5⬘-TGCTACACAGAAAGAACTTGAGGT-3⬘, probe 22; Fzd9 fwd 5⬘-CACAGAGGGTCCCAGGATAA-3⬘, rev 5⬘-CATCATTAAATAACTCTGCACTGGAC-3⬘, probe 29; Fzd10 fwd 5⬘ATGGAAACCAAGCCAGTGTG-3⬘, rev 5⬘-CTCCCCCTTCCTCTCCAC-3⬘, probe 95; HPRT fwd 5⬘-TCCTCCTCAGACCGCTTTT-3⬘, rev 5⬘-CCTGGTTCATCATCGCTAATC-3⬘, probe 95; TNF fwd 5⬘CTGTAGCCCACGTCGTAG-3⬘, rev 5⬘-TTGAGATCCATGCCGTTG-3⬘, probe 102; and Wnt3a fwd 5⬘-CTTAGTGCTCTGCAGCCTGA-3⬘, rev 5⬘-GAGTGCTCAGAGAGGAGTACTGG-3⬘, probe 76. qRT-PCR amplification was performed using the LightCycler 480 II system (Roche Diagnostics, Mannheim, Germany). Cp values of target and reference gene (HPRT) were determined by the second derivative maximum method. The relative gene expression was calculated considering the individual efficiency of each PCR determined by a standard curve. The term “fold induction” is defined as the cDNA ratio between target gene and reference gene (HPRT) normalized to untreated control.

Whole proteins from the phenol fractions of TriFast FL (Peqlab) and cell lysates (0.5⫻106 cells) were purified by ethanol, guanidine/HCl extraction as recommended by the manufacturer. Cellular protein was solubilized in sample buffer, and SDS-Page and Western blot were performed as described previously (19). The following mAb were used: rabbit anti-mouse iNOS/NOS2 (Upstate, Millipore, Schwalbach, Germany), mouse anti-mouse ␤-catenin (BD Bioscience), mouse anti-mouse GAPDH (Hytest, Turku, Finland), and biotinylated rat anti-human/mouse Fzd1 (R&D Systems). Blots were washed 3 times for 15 min in TTBS and incubated with secondary antibody (Streptavidin AlexaFluor 680; Invitrogen, Karlsruhe, Germany) at an appropriate dilution in TTBS containing 1% (v/v) Roti-ImmunoBlock (Roth, Karlsruh, Germany) for 1 h at 25°C. Signal detection and quantification was done using the Odyssey infrared imaging system (software ver. 2.1; Li-Cor Biotechnology, Lincoln, NE, USA).

Immunohistological analysis

Statistical analyses

One lung lobe per mouse was fixed in 4% formalin-PBS and set in paraffin blocks. Then, 2–3 ␮m tissue sections were prepared, and the staining was performed by the sequential use of a monoclonal rat anti-Wnt3a antibody (R&D Systems, Minneapolis, MN, USA) and a biotin-conjugated goat anti-rat IgG (Jackson Immunoresearch, Newmarket, UK). The reaction was visualized using streptavidin conjugated to horseradish peroxidase (Jackson Immunoresearch) followed by incubation with 3,3⬘-diaminobenzidine (Dako, Glostrup, Denmark). Tissue sections were counterstained with Mayer’s hematoxylin (Merck, Darmstadt, Germany) and mounted.

For statistical analyses, the mean values of technical replicates of ⱖ3 independent in vitro experiments were log transformed according to Willems et al. (22) and analyzed using the Student’s 2-tailed t test (confidence interval 95%) for paired observations. At least 4 biological replicates were used for statistical analyses of in vivo experiments assuming nonparametric distribution using the Mann-Whitney U test. All values shown represent the mean ⫾ se.

Flow cytometric analysis Cells were washed in FACS buffer [PBS, 10% (v/v) heatinactivated FCS] and incubated with a mixture containing anti-Fc␥RIII/II mAb (clone 2.4G2; BD Biosciences, San Jose, CA, USA) and mouse and rat serum to block nonspecific binding for 20 min at 4°C. Cells were then labeled with biotinylated rat anti-human/mouse Fzd1 (R&D Systems) and Cy5-conjugated streptavidin (Jackson ImmunoResearch), followed by FITC rat anti-mouse F4/80 (AbD Serotec, Du¨sseldorf, Germany). Biotin-labeled rat IgG2a␬, and FITC rat IgG2b␬ (BioLegend, San Diego, CA, USA) were used as isotype controls. Each staining step was performed for 25 min MACROPHAGE EXPRESSION OF Fzd1

SDS-PAGE and Western blot analysis

RESULTS Differential regulation of Fzd1 and Wnt/␤-catenin signaling in M. tuberculosis-infected mice Mice were infected with 100 CFU M. tuberculosis H37Rv via the aerosol route. A significant increase in CFU was detected in lung homogenates at d 21 and 42 postinfection (Fig. 1) as described previously (20). Containment of bacterial replication was paralleled by a substantial augmentation of NOS2 levels as detected by Western blot (data not shown). Quantitative RT-PCR revealed a significant increase of IFN-␥ and TNF mRNA 4601

Figure 1. Analysis of Fzd expression, ␤-catenin (␤-cat) stabilization, and Axin2 expression in lung homogenates of M. tuberculosis-infected mice. C57BL/6 mice were aerogenically infected with M. tuberculosis (strain H37Rv; 100 CFU). Mice were killed at indicated time points, and lung homogenates were prepared. A, D) Total RNA was isolated and reverse transcribed. mRNA expression of Fzd homologs 1–10 was determined by qRT-PCR analysis (TaqMan assay), showing the cDNA ratio of the target gene related to HPRT. Results represent means ⫾ se of 4 mice. Statistical analysis was performed by MannWhitney U test (infected vs. uninfected). *P ⬍ 0.05. n.s., not significant. B) Bacterial growth (CFU/lung) was determined by colony enumeration assay. Results represent means ⫾ se of 5 mice. C, D) Analysis of ␤-catenin protein levels and Axin2 mRNA expression. C) Equal protein amounts of 5 lung homogenates per time point were pooled. After SDS-PAGE and Western blot, blots were incubated with antibodies against ␤-catenin or GAPDH used as loading control. After incubation with secondary reagents, the blot was analyzed using an Odyssey near-infrared detection system. D) cDNA ratio of Axin2 and HPRT as determined by qRT-PCR analysis.

expression (data not shown), documenting the upregulation of inflammation-related target genes. A systematic screen of all known 10 Fzd homologs by qRT-PCR revealed that the mRNA levels of the majority of Fzd receptors, namely Fzds 3, 4, and 7–10, were significantly down-regulated during infection (Fig. 1A). This was paralleled by a decrease of ␤-catenin levels in the infected lung at d 21 and 42, as demonstrated by Western blot (Fig. 1C). In addition, the mRNA expression of the ␤-catenin dependent target gene Axin2 was reduced by 70% at d 21 and 42 after M. tuberculosis infection (Fig. 1D), indicating that Wnt/␤-catenin signaling is down-regulated during the M. tuberculosis induced inflammatory response. In contrast, the same analysis identified Fzd1 and Fzd5 to be significantly up-regulated during the course of M. tuberculosis infection in vivo. Presence of the Fzd1 ligand Wnt3a in the lungs of M. tuberculosis-infected mice Fzd1 predominantly mediates cellular responses to Wnt3a (23, 24). To address the question of whether Wnt3a is present or up-regulated during the course of infection, we monitored its expression in lung homogenates of M. tuberculosis-infected mice. Wnt3a was expressed at constant levels in the infected mouse lung on d 21 and 42 postinfection (Fig. 2A). To corroborate this finding on the protein level, immunohistological anal4602

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yses were performed: staining of paraffin-embedded lung tissue of uninfected mice and of mice d 42 postinfection, using an anti-Wnt3a antibody, revealed the presence of Wnt3a in the bronchiolar epithelium of uninfected (Fig. 2C, D) and infected mice (Fig. 2E, F). Infection of macrophages with mycobacteria dose dependently up-regulates Fzd1 mRNA To analyze whether Fzd1 expression was induced in macrophages, the known host cell of mycobacteria, murine BMDMs were infected with increasing MOI of M. tuberculosis and M. avium for 4 h and the expression of Fzd1 mRNA was analyzed by qRT-PCR. Fzd1 mRNA was dose dependently up-regulated in response to M. tuberculosis and M. avium by 15- to 60-fold (Fig. 3A). In the same samples, TNF mRNA expression was upregulated (35- to 750-fold), which demonstrates that enhanced Fzd1 transcription correlates with macrophage activation (Fig. 3B). Fzd1 mRNA induction was apparent 1 h after stimulation, peaked after 4 h, and was detectable up to 8 h, whereas 24 h after infection Fzd1 mRNA had decreased to minimum transcript levels (Fig. 3C). A similar transcriptional regulation was observed when the cells were treated with LPS, the major constituent of the outer cell wall of gram-negative bacteria, indicating that up-regulation of Fzd1 is induced by other microbial components.

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Figure 2. Wnt3a expression in lungs of M. tuberculosis-infected mice. C57BL/6 mice were aerogenically infected with M. tuberculosis (strain H37Rv; 100 CFU). A) Mice were killed at indicated time points, and lung homogenates were prepared. cDNA ratio of Wnt3a and HPRT was determined by qRT-PCR analysis (TaqMan assay). Results represent means ⫾ se of 4 mice. B–E) Immunohistological analysis of Wnt3a in lung tissue of M. tuberculosis-infected mice; 2-␮m sections were cut from paraffin-embedded lung tissue derived from uninfected (C, D) or infected (E, F) mice (100 CFU, d 42 postinfection). Wnt3a was detected by the use of a monoclonal rat anti-Wnt3a antibody. B) Lung section of an infected mouse stained in the absence of the anti-Wnt3a antibody. Representative staining from 1 of 4 infected mice or uninfected mice. Scale bars ⫽ 100 ␮M (C, E); 50 ␮M (B, D, F).

Fzd1 transcription is induced through TLRs The family of TLRs, in particular TLR2 and TLR4, mediates macrophage activation in response to conserved bacterial structures of gram-positive and gramnegative bacteria, as well as mycobacteria (9). Gene expression studies using 100 nM FSL-1 and 100 nM PamOct2C-(VPGVG)4VPGKG, 2 synthetic TLR2/TLR6 and TLR2/TLR1 agonists (25), also identified upregulation of Fzd1 mRNA 2 and 6 h after stimulation of

murine macrophages (Table 1). A transcriptional regulation of the Fzd homologs (Fzd2–10) in response to these TLR agonists was not observed (data not shown). We next compared the Fzd1 mRNA expression of BMDMs derived from TLR2-deficient mice and C57BL/6 wild-type mice that were stimulated with mycobacteria or LPS for 4 h. Compared to wild-type cells, Fzd1 mRNA in TLR2-deficient macrophages was reduced by ⬃75% in response to M. avium, whereas stimulation with LPS was not affected (Fig. 4A). Again, Fzd1 mRNA induction

Figure 3. Mycobacteria- and LPS-induced Fzd1 and TNF mRNA expression in murine macrophages. A, B) C57BL/6 BMDMs were incubated with indicated MOI of M. tuberculosis (M. tb.) and M. avium (M. av.) and increasing concentrations of LPS. After 4 h, cells were lysed and total RNA was isolated and reverse transcribed. qRT-PCR analysis of Fzd1 (A) and TNF (B) mRNA expression levels shown as fold induction. C) Kinetics of Fzd1 expression in BMDMs exposed to M. tuberculosis, M. avium (MOI⫽3), and 10 ng/ml of LPS for indicated time intervals. Results are means ⫾ se of triplicate measurements. MACROPHAGE EXPRESSION OF Fzd1

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TABLE 1. Fzd1 induction by synthetic TLR agonists Receptor

TLR2/TLR6 TLR2 TLR2/TLR1

Agonist

2h

6h

FSL-1 Pam2CSK4 PamOct2C-(VPGVG)4VPGKG

5.11 ⫾ 1.04 4.34 ⫾ 1.00 4.91 ⫾ 2.65

6.25 ⫾ 4.34 6.75 ⫾ 5.01 6.92 ⫾ 5.20

C57BL/6-derived BMDMs were stimulated with TLR2/TLR6-specific FSL-1, TLR2-specific Pam2CSK4, and TLR2/TLR1-specific PamOct2C-(VPGVG)4VPGKG (100 nM each). After 2 and 6 h, mRNA was isolated and processed for hybridization to Affymetrix MG 430 2.0 microarray (24). Data shown for Fzd1 represent mean ⫾ sd of duplicate determinations of 2 experiments.

correlated with the expression of TNF (data not shown). In a similar set of experiments, the functional role of the adaptor protein MyD88 (myeloid differentiation primary response gene 88) in microbially induced Fzd1 induction was analyzed: BMDMs from MyD88-deficient mice and wild-type mice were stimulated with M. tuberculosis, M. avium, and LPS for 4 h. Fzd1 mRNA expression in MyD88-deficient cells was reduced by ⬎70% (M. tuberculosis), 85% (M. avium), and 65% (LPS), when compared to wild-type cells (Fig. 4B).

initiation of Fzd1 mRNA expression, BMDMs were stimulated with M. tuberculosis, M. avium, and LPS in the presence or absence of a specific inhibitor (BAY11–7082), which blocks I␬B␣ phosphorylation (26). In mycobacteria-, LPS-, and TNF-treated macrophages, the presence of the inhibitor led to a significant decrease in Fzd1 expression (Fig. 4D), indicating that Fzd1 mRNA expression is critically dependent on NF-␬B function. Cell viability in the presence of the inhibitor was measured by trypan blue exclusion and was 95% of the viability of control (0.1% DMSO treated) cultures.

Fzd1 expression depends on TNF and NF-␬B The experiments using MyD88-deficient macrophages demonstrated that Fzd1 can be induced independently of TLR signaling, and TNF alone is sufficient to induce Fzd1 mRNA expression (Fig. 4B). We therefore analyzed whether and to what extent Fzd1 mRNA expression would be TNF dependent. Figure 4C shows that in TNF⫺/⫺ macrophages, expression of Fzd1 in response to M. avium, M. tuberculosis, and LPS was completely abrogated, demonstrating that TNF is an essential mediator of microbially induced Fzd1 expression. To examine whether NF-␬B plays a part in the

Macrophage surface expression of Fzd1: IFN-␥ enhances TLR-mediated Fzd1 up-regulation in murine macrophages To demonstrate Fzd1 expression on the protein level, macrophages were stained with anti-Fzd1 and antiF4/80 (as a macrophage marker) before flow cytometric analysis. Fzd1 was present on the cell surface of ⬃10 –15% of unstimulated cells. After infection of macrophages with M. tuberculosis or stimulation with LPS for 24 h, ⬃18 –25% of the F4/80⫹ cells expressed Fzd1 on the cell surface (Fig. 5A, C, D). Next, we

Figure 4. Fzd1 mRNA expression depends on TLR2, MyD88, TNF, and NF-␬B. BMDMs were incubated with M. tuberculosis (M. tb), M. avium (M. av; MOI⫽3), 100 ng/ml Pam3CSK4, 10 ng/ml LPS, and 50 ng/ml recombinant (r)TNF as indicated. After 4 h, cells were lysed and total RNA was isolated and reverse transcribed. Fzd1 mRNA expression levels determined by qRT-PCR are shown as fold induction. A–C) Comparative analysis of macrophages from C57BL/6 wild-type (WT) and TLR2⫺/⫺ mice (A), C57BL/6 WT and MyD88⫺/⫺ mice (B), and C57BL/6 WT and TNF⫺/⫺ mice (C). D) C57BL/6 BMDMs were treated in the presence or absence of BAY 11-7082 (3 ␮M) after 45 min preincubation with the inhibitor. Results represent means ⫾ se of 3 independent experiments performed in technical duplicates. Data were log transformed and analyzed by Student’s 2-tailed, paired t test. *P ⬍ 0.05; *P ⬍ 0.01. 4604

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Figure 5. Cell surface expression of Fzd1 on murine and human macrophages. A, B) BMDMs incubated in the absence (A) or presence (B) of IFN-␥ (250 U/ml, 24 h before stimulation) were infected with M. tuberculosis (MOI 0.5) or treated with 10 ng/ml LPS for 24 h. Cells were stained with anti-Fzd1 and anti-F4/80 monoclonal antibodies and analyzed by flow cytometry. Isotype controls were used to determine the percentage of Fzd1/F4/80 positive cells by quadrant plot analysis. C, D) Data are means ⫾ se of the percentage of Fzd1/F4/80-positive cells, summarizing the results of 5 (M. tuberculosis; C) and 3 (LPS; D) independent experiments. For statistical analysis, data were log transformed and analyzed by Student’s 2-tailed, paired t test. **P ⬍ 0.005 vs. control. E, F) Human monocyte-derived macrophages were stimulated as described above. Cells were stained with anti-Fzd1 and anti-CD14 monoclonal antibodies. Based on isotype controls, percentage of cells that stained positive for Fzd1 and CD14 was monitored by dot plot analysis. Results represent 1 of 2 independent experiments.

addressed the role of IFN-␥, a key cytokine facilitating full macrophage activation (27). Treatment with IFN-␥ alone did not affect Fzd1 surface expression. The addition of M. tuberculosis or LPS to IFN-␥treated cells resulted in a 2-fold increase of Fzd1positive cells compared with cells stimulated with M. tuberculosis or LPS alone (Fig. 5B–D). These data demonstrate a significant synergism between IFN-␥ and TLR signaling with respect to Fzd1 expression on macrophages (Fig. 5C). To also address the expression of Fzd1 on human macrophages, human monocyte-derived macrophages were stimulated as described above, stained for Fzd1 and CD14, as a human monocyte/macrophage marker, and subsequently analyzed by flow cytometry. The analysis of unstimulated human macrophages identified cells expressing Fzd at a high level (Fzd1high) and at a low level (Fzd1low; Fig. 5E). Following infection with M. tuberculosis, a 3.8-fold increase in the percentage of Fzd1⫹/CD14⫹ cells was observed compared to untreated control cells. Stimulation with IFN-␥ further enhanced the size of this population by 62% (Fig. 5F). Treatment of human macrophages with LPS caused an up-regulation of Fzd1⫹/CD14⫹ positive gated cells by 2.7-fold. Yet, IFN-␥ had no further impact on the percentage of LPS-stimulated Fzd1⫹/CD14⫹ cells. Macrophages treated solely with IFN-␥ showed no difference in the Fzd1⫹/CD14⫹ population when compared to unstimulated control cells. The overall increase of Fzd1 surface expression was primarily due to an inMACROPHAGE EXPRESSION OF Fzd1

crease of the Fzd1low/CD14⫹ cell population (Fig. 5E, F). The percentage and the MFI of Fzd1high/CD14⫹ remained nearly unchanged in response to stimulation. The experiments also identified a subpopulation of CD14⫺/FZD1⫹⫹-expressing macrophages. Fzd1 protein is redistributed to the macrophage surface on microbial stimulation These experiments prompted us to monitor the relationship between surface expression and total amount of Fzd1 protein in macrophages. In Western blot experiments, Fzd1 was detectable as a double band of ⬃70 kDa (Fig. 6A). A Fzd1/Fc fusion protein was used as positive control to ensure the specificity of the antibody. In cell lysates, no significant increase of total Fzd1 protein was observed in M. tuberculosis/IFN-␥ stimulated macrophages in a time frame from 4 to 24 h, whereas NOS2 protein levels were substantially enhanced (Fig. 6A). These findings were corroborated by intracellular flow cytometry measurements (Fig. 6B, C): the mean fluorescence intensity (MFI) of Fzd1 expression differed significantly when untreated and M. tuberculosis/IFN-␥-treated cells were compared (Fig. 6B). In contrast, intracellular staining showed an identical MFI independently of whether the cells were stimulated or not (Fig. 6C). Next, cells were stimulated in the presence of GolgiPlug, a brefeldin A containing inhibitor, which blocks intracellular protein transport processes, 4605

Figure 6. Analysis of total Fzd1 protein levels in murine macrophages. BMDMs were treated as described in Fig. 5. A) Cells were lyzed at the indicated time points, and proteins were subsequently separated by SDSPAGE and transferred to a nitrocellulose membrane by Western blot. A recombinant murine Fzd1/Fc fusion protein (50 ng) served as control. After incubation with anti-Fzd1 and antiGAPDH antibodies, near-infrared dye labeled secondary reagents were applied for detection by a near-infrared imaging system. B, C) Cell surface and intracellular Fzd1 expression of murine macrophages analyzed by flow cytometry. Histograms of Fzd1 expression of gated BMDMs; 1 representative experiment of 2 independent experiments performed is shown. Shaded histograms, isotype controls; black solid line, control unstimulated cells; gray solid line, M. tuberculosis/IFN␥-treated cells. D, E) For simultaneous detection of intracellular Fzd1 and TNF, cells were incubated with 1 ␮l/ml GolgiPlug (GPlug; BD Bioscience) and LPS (100 ng/ml) and IFN-␥ (250 U/ml) for 12 h. Shaded histograms, isotype controls; black solid line, no GolgiPlug added; gray solid line, GolgiPlug-treated cells.

resulting in the accumulation of cytokines and other proteins in the Golgi complex. Flow cytometric analysis identified substantially enhanced intracellular levels of TNF in GolgiPlug-treated cells, when compared with control cells (Fig. 6D). Staining for Fzd1 in the same set of experiments also revealed enhanced Fzd1 protein levels in GolgiPlug-treated, LPS/IFN-␥-activated cells (Fig. 6E). These data suggest that Fzd1 is primarily present intracellularly in macrophages and is redistributed to the cell surface on macrophage activation. The total Fzd1 protein level in the macrophage, however, appears to remain constant, which might be due to a rapid turnover of Fzd1 protein, as indicated by the accumulation of intracellular protein when protein transport is impaired. A Fzd1/Fc fusion protein blocks Wnt3a-mediated activation of ␤-catenin signaling in macrophages Wnt3a has been shown to induce ␤-catenin signaling, resulting in Axin2 expression (28). To analyze whether Wnt3a induces ␤-catenin signaling in BMDMs, Wnt3a CM derived from stably Wnt3a transfected L929 fibroblasts (L929-Wnt3a) was used. Western blot analysis of cell lysates using an anti-Wnt3a antibody led to the detection of a band of ⬃40 kDa. Recombinant Wnt3a protein (15 ng) was used as positive control to ensure the specificity of the antibody (Fig. 7A). 4606

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BMDMs where incubated with Wnt3a CM or control CM derived from L929 cells. After 4 h, Wnt3a-CMtreated macrophages showed an accumulation of ␤-catenin when compared with CM-treated cells as shown by Western blot (Fig. 7B). A similar effect was observed when cells were incubated with the GSK3␤ inhibitor SB216763. qRT-PCR analysis of Wnt3a-CMtreated macrophages revealed a significant increase in Axin2 mRNA expression when compared with the CM control (Fig. 7C). Increasing concentrations of a soluble Fzd1/Fc fusion protein were added to the CM before the addition to macrophages to allow an interaction of Wnt3a and Fzd1/Fc. Stimulation of the cells with Wnt3a in the presence of the Fzd1/Fc fusion protein led to a dose-dependent reduction of Axin2 mRNA expression levels (Fig. 7D), without affecting TNF expression (Fig. 7E). These results indicate that Wnt3a-induced ␤-catenin stabilization and Tcf/Lef-dependent target gene expression in murine macrophages can be blocked by a soluble Fzd1/Fc fusion protein. Wnt3a inhibits proinflammatory cytokine secretion of murine macrophages To examine whether the Fzd1 ligand Wnt3a may modulate the effector functions of macrophages, we monitored the TNF production of M. tuberculosis-infected

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Figure 7. Wnt3a-induced ␤-catenin stabilization and Axin2 mRNA expression in murine macrophages. A) Detection of Wnt3a by Western blot in stably transfected cell lines expressing Wnt3a and Wnt5a (L929-Wnt3a and L929-Wnt5a) and L929 control cells. rWnt3a recombinant Wnt3a (15 ng). After SDS-PAGE and Western blot, blots were incubated with anti-Wnt3a and anti-GAPDH antibodies, and secondary antibodies were subsequently applied for the analysis in an Odyssey near-infrared detection system; 1 of 2 representative experiments is shown. B) BMDMs were exposed to control CM, Wnt3a-CM, and the GSK3␤ inhibitor SB216763 (30 mM). After 4 h, cell lysates were analyzed by immunoblot using a monoclonal anti-␤-catenin antibody. Results shown are representative for 1 out of 3 independent experiments. C) BMDMs were incubated with control CM and Wnt3a-CM for 4 h. After mRNA isolation and reverse transcription, the cDNA ratio of Axin2/HPRT was determined by qRT-PCR analysis. Indicated are the results of 5 independent experiments. For statistical analysis, the data were log transformed and analyzed by Student’s 2-tailed, paired t test. **P ⬍ 0.01. D, E) Influence of a soluble Fzd1/Fc fusion protein on Wnt3a-induced Axin2 and TNF expression in murine macrophages. First, Wnt3a CM was preincubated with the indicated concentrations of a soluble Fzd1/Fc fusion protein for 45 min at 4°C. Subsequently, BMDMs were incubated with Wnt3a CM or Fzd1/Fc-treated Wnt3a-CM. After 4 h, cells were lyzed and total RNA was analyzed and reverse transcribed. Depicted is fold induction of Fzd1 (D) and TNF (E) expression as determined by qRT-PCR (TaqMan assay) normalized to the CM control. Results are means ⫾ se of 3 independent experiments performed in technical duplicates. Data were log transformed and analyzed by Student’s 2-tailed, paired t test. *P ⬍ 0.05 vs. untreated cells.

macrophages in the presence or absence of Wnta3a. After 1.5 h of preincubation of the macrophages with Wnt3a CM or control CM, cells were infected with M. tuberculosis. At 12 h postinfection, equal amounts of TNF were detected in supernatants of M. tuberculosisinfected cells treated with control CM or Wnt3a CM. However, at 24 h postinfection, TNF levels in supernatants from Wnt3a-treated macrophages were significantly reduced by ⬃45% (Fig. 8A). Similarly, increasing concentrations of recombinant Wnt3a led to a dosedependent and significant decrease of TNF formation when cells were infected with M. tuberculosis (Fig. 8C, D). In a different setting, we first stimulated the macrophages with IFN-␥ and infected them with M. tuberculosis to induce high surface expression of Fzd1 and subsequently exposed the cells to Wnt3a CM or control CM. After an additional 4 h of incubation, qRT-PCR analysis revealed that in cells treated with M. tuberculosis/IFN-␥ the presence of Wnt3a CM led to a significant decrease of TNF mRNA expression by ⬃50% (Fig. 8B). In a separate approach, we stimulated macrophages with M. avium in the presence or absence of the GSK3␤ inhibitor SB216763, which has been widely used to mimic Wnt signaling (29). In Fig. 8E, it is shown that TNF expression is dose dependently down-regulated with increasing concentrations of SB216763, whereas Axin2 expression is significantly up-regulated, when GSK3␤ activity is blocked. These data sets clearly demonstrate that Wnt/␤-catenin signaling can substantially MACROPHAGE EXPRESSION OF Fzd1

affect the proinflammatory response of macrophages after infection with mycobacteria. Based on predominantly pro- or anti-inflammatory effector functions, macrophages were assigned a M1 or M2 phenotype (30). To analyze whether Wnt3a would affect macrophage polarization, the expression of the murine M2-associated marker gene arginase 1 (Arg1) was monitored after exposure of the cells to Wnt3a. Figure 8F shows that Wnt3a CM led to a significant increase of Arg1 transcription in primary murine macrophages.

DISCUSSION The current study identifies murine Fzd1 as a novel marker associated with inflammatory macrophage activation. Fzd1 is dose dependently up-regulated in response to mycobacteria and conserved bacterial structures. Fzd1 transcription induced via TLRs and MyD88 strictly depends on TNF. Surface expression of Fzd1 protein on murine macrophages was increased in response to M. tuberculosis infection and synergistically enhanced in the presence of IFN-␥. The Fzd1 ligand Wnt3a is present in the lungs of M. tuberculosis-infected mice, and soluble Fzd1 inhibits Wnt3a-induced ␤-catenin-dependent target gene transcription in macrophages. Finally, Wnt3a influences macrophage effector function by decreasing TNF formation 4607

Figure 8. Influence of Wnt3a on the macrophage response. A) BMDMs were incubated with Wnt3a CM for 1.5 h, followed by M. tuberculosis infection (MOI 0.5). At 12 and 24 h postinfection, TNF concentration in supernatant medium was determined by ELISA. B) BMDMs were preincubated in the presence or absence of 250 U/ml IFN-␥ for 24 h and subsequently infected with M. tuberculosis (MOI 0.5) for another 24 h. Culture medium was completely removed, and control CM or Wnt3a CM was added. Subsequently, mycobacteria and IFN-␥ were again added to the cells as indicated. After 4 h, cells were lyzed for total RNA extraction, reverse transcribed, and analyzed by qRT-PCR (TaqMan assay). Shown is the fold induction of TNF expression normalized to the untreated control. A, B) Results represent the mean ⫾ se of 3 independent experiments performed in technical duplicates. Data were log transformed and analyzed by Student’s 2-tailed, paired t test. C) BMDMs were incubated with increasing concentrations of rWnt3a and subsequently infected with M. tuberculosis (MOI 3). After 24 h, TNF concentrations in supernatants were analyzed by ELISA. Means ⫾ sd of 1 of 2 independent experiments are depicted. D) M. tuberculosis-induced TNF release (ELISA) of BMDMs (MOI 3) in the presence of 400 ng/ml rWnt3a 24 h postinfection; 3 independent experiments were compared by normalizing control conditions to 100%. E) TNF and Axin2 mRNA expression (fold induction) in BMDMs preincubated with SB216763 for 1 h and infected with M. avium SE01 for 4 h (MOI 3). F) BMDMs were incubated with control CM and Wnt3a-CM for 4 h. After mRNA isolation and reverse transcription the cDNA ratio of arginase 1/HPRT was determined by qRT-PCR analysis. Indicated are the results of 5 independent experiments. Data were log transformed and analyzed by Student’s 2-tailed, paired t test. *P ⬍ 0.05; ***P ⬍ 0.001.

after microbial stimulation. Taken together, these findings support a role for Fzd1 in regulating the activation status of infected macrophages. A screen of the transcriptional regulation of murine Fzd homologs identified Fzd1 (Fzd1) as significantly up-regulated during the course of M. tuberculosis infection, suggesting that it may play a role in the context of inflammation. Only little is known about the expression and function of Fzd1: Fzd1 and the structurally related Fzd2 were found to be widely expressed with the highest steady-state levels of mRNA in kidney, lung, liver, heart, uterus, and ovary (31). Fzd1 was shown to mediate Wnt3a-induced cellular responses, resulting in the activation of ␤-catenin signaling in murine but also in human cells (23, 24). Initially, up-regulation of Fzd1 was observed after treatment of osteoblast-like UMR106-H5 cells with bone resorbing agents, including parathyroid hor4608

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mone, epidermal growth factor, and 1,25-dihydroxyvitamin D3 (31). Also, bone morphogenetic protein 2 (Bmp2), a member of the TGF-␤ superfamily of proteins, induced an increased Fzd1 expression in murine mesenchymal cells (32). In the female reproductive tract, Fzd1 was induced by luteinizing hormone in ovulating follicles of rodent ovaries (33) and in the Mu¨llerian duct mesenchyme (34). In malignant tissue, immunohistochemical analysis of Fzd1 and Fzd2 identified both receptors to be up-regulated in breast cancer (35). Also, in poorly differentiated colon tumors, a high degree of Fzd1 receptor expression, especially at the margin of cellular invasion, has been demonstrated (36). To date, there have been no detailed studies on the expression and regulation of Fzd1 on immune cells, in particular in terms of a functional involvement in inflammatory processes. The current study identifies

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an up-regulation of Fzd1 after M. tuberculosis infection or LPS stimulation of macrophages in vitro, which suggests a novel and unexpected role in immune regulation. It demonstrates that TLR2 and MyD88 are critically involved in mycobacteriainduced Fzd1 expression. Our findings are corroborated by a very recent report by Lattin et al. (37), who studied G-protein-coupled receptors in murine macrophages using microarrays and found Fzd1 to be up-regulated in response to LPS. The use of TNFdeficient mice clearly demonstrates that TNF is the key factor in regulating Fzd1 transcription in macrophages. This may indicate that Fzd1 is generally up-regulated under proinflammatory conditions, suggesting a broader role in feedback regulation. Microbially induced Fzd1 transcription expression peaks already at 4 h and is back down to baseline levels after 24 h. Very similar observations were made when preovulatory follicles were treated with PMSG cells as described previously (33). In contrast to soluble mediators that are primarily regulated at the transcriptional or translational level, 7 transmembrane-spanning receptors are regulated by additional mechanisms, in particular receptor internalization and recycling (6). Indeed, our own Western blot analyses documented no changes in total Fzd1 protein levels in stimulated macrophages. However, in TLR-activated and IFN-␥-stimulated cells, we did see a major change in the cellular localization of the Fzd1 protein from intracellular locations to the cell surface as demonstrated by flow cytometry. We did not determine the intracellular localization and storage pool for Fzd1 in detail, but it has been proposed that Frizzleds are retained in the Golgi apparatus (38). When intracellular protein transport was blocked by brefeldin A, an enhanced Fzd1 mRNA expression in TLR/IFN-␥-stimulated cells was accompanied by higher Fzd1 protein levels in macrophages. Our observation may be indicative for a rapid Fzd1 turnover, although further detailed studies are needed to characterize Fzd1 mRNA and protein stability as well as Fzd1 receptor recycling in macrophages in full detail. Fzd1 has been shown to mediate Wnt3a and Wnt7b signals (23, 24, 39). Wnt3a was constitutively expressed in the lungs before and during infection. As stimulated BMDMs did not express Wnt3a (data not shown), we presume that nonimmune cells are responsible for Wnt3a formation. Wnt3a appears to be primarily expressed by bronchoepithelial cells of the murine lung. This finding corroborates a recent report by Ko¨nigshoff et al. (40), who demonstrated the presence of Wnt3a in human alveolar and bronchial epithelium. With respect to Wnt7b, our recent preliminary data reveal an upregulation of this Fzd1 ligand during experimental tuberculosis (data not shown). Wnt3a, in contrast to Wnt5a, was assigned to the group of transforming Wnts (41). Addition of Wnt3a to murine macrophages led to ␤-catenin stabilization and the mRNA expression of Axin2. Axin2 has previously been shown to be up-regulated in reMACROPHAGE EXPRESSION OF Fzd1

sponse to increased ␤-catenin concentrations and serves to limit the duration and intensity of the Wnt signal (42, 43). Axin2 binding to ␤-catenin promotes the phosphorylation of ␤-catenin by CKI␣ and GSK3, which is required for the ubiquitination and subsequent degradation of ␤-catenin. Thus, Axin2 is an integral part of a negative feedback loop that acts to restrain or desensitize Wnt signaling (reviewed in ref. 44). The fact that the macrophage responses to Wnt3a were inhibited in the presence of a soluble Fzd1/Fc fusion protein, which contains the extracellular N-terminal cystein-rich domain of Fzd1, which is essential for Wnt-ligand binding, suggests that Fzd1 is involved in mediating Wnt3a signaling in macrophages. Wnt3a-treated cells produced less TNF in response to mycobacterial infection, which demonstrates that Wnt3a reduces proinflammatory effector mechanisms of macrophages. In contrast to Wnt5a, which has been shown to up-regulate proinflammatory effector functions of macrophages (14, 15), one may consider Wnt3a as an anti-inflammatory Wnt homologue. This notion is supported by observations of Tickenbrock et al. (45), who described that Wnt3a reduced the transendothelial migration of human monocytes. In addition, we observed a Wnt3a-induced transcription of arginase 1, a known marker enzyme of alternatively activated murine macrophages (reviewed in ref. 30). Taken together, these data suggest that Wnt3a is actively reprogramming macrophage functions. We propose that inflammation-induced up-regulation of Fzd1 may render the macrophage sensitive to Wnt3a signals. In an in vivo situation, macrophages that enter areas with high local Wnt3a concentrations may thereby change their sensitivity/reactivity to exogenous proinflammatory stimuli. This may be of particular relevance in barrier organs, e.g., the lung or the intestine, where a balanced inflammatory response may prevent excessive tissue damage. This line of argument is supported by the recent finding that Fzd1 mediates the Wnt3a-induced neuroprotective effect against the toxicity of Alzheimer’s amyloid-␤-peptide (46). A protective role of Wnt/␤-catenin signaling has also been observed in cancer therapy and NO-induced apoptosis (4, 47). The Wnt/␤-catenin pathway is a key signaling pathway that controls the genetic programs of embryonic development and tissue homeostasis (reviewed in ref. 4, 8). For the lung, it was demonstrated that ablation of the ␤-catenin gene in epithelial cells resulted in an enlargement of distal airways and an inhibition of proximal airways (48). Overexpression of ␤-catenin led to the opposite phenotype (49) and is accompanied by a significant increase in bronchioalveolar stem cell numbers (50). This indicates that ␤-catenin signaling dramatically influences the architecture of the lung and thereby also lung function. Very little, however, is known about how the Wnt/␤catenin pathway is regulated under inflammatory conditions. In the gut, Salmonella spp. activate ␤-catenin signaling in epithelial cells and interfere with NF-␬B signaling (51). In the current study, we set out to investigate how key components of the ␤-catenin sig4609

nificant reduction of epithelial ␤-catenin levels, which in turn leads to an enhanced permeability of murine and also human lung epithelial layers (52, 53). This is of relevance since a controlled migration of immune cells to the site of inflammation or infection is essential. The down-regulation of ␤-catenin levels seen in M. tuberculosis infection may reflect a pathological situation of chronic and excessive inflammation. In this situation, Wnt3a signaling via Fzd1 may fine tune the activation status of macrophages, thereby protecting the immune cell from hyperactivation during microbial stimulation (summarized in Fig. 9). This feedback mechanism of Wnt3a, operative in inflammation, may serve similar homeostatic functions as other Wnt signals do in tissue and organ development.

naling are affected during experimental M. tuberculosis infection in mice, a well-established model of chronic inflammation. ␤-Catenin levels were significantly decreased in lung lysates of M. tuberculosis-infected mice, and mRNA expression of Axin2, a well-known indicator gene for ␤-catenin-dependent target gene expression (28), was reduced. We observed that WNT/␤-catenin signaling affects TLR/NF-␬B-mediated signaling and cellular functions and vice versa. The activity of both pathways needs to be carefully balanced: in a homeostatic or uninfected situation, active Wnt/␤-catenin signaling in vivo may limit the proinflammatory response, thus protecting the lung or the cell from unwanted inflammation. The strength of the TLR/NF-␬〉 signal may provide a checkpoint after which a homeostatic Wnt signaling threshold is overcome, which then leads to a down-regulation of homeostatic Wnt/␤-catenin signaling, opening the window for inflammation and the immune response. It was previously demonstrated that TNF mediates a sig-

Figure 9. Role of Fzd1 and Wnt3a-induced ␤-catenin signaling in macrophage effector functions and inflammatory processes. A, B) Mycobacteria and conserved bacterial structures activate TNF release (A) that in an auto- or paracrine manner triggers the induction of Fzd1 mRNA (B). Synergistic effects between IFN-␥ (secreted by activated lymphocytes) and inflammatory macrophages further enhance the presence of Fzd1 within the plasma membrane. C) Wnt3a (primarily expressed by epithelial cells) binds to macrophage Fzd receptors, inducing a ␤-catenin signaling cascade that can be blocked by soluble Fzd1 (Fzd1/Fc). D) Wnt3a affects macrophage effector functions by selectively decreasing TNF mRNA expression and protein release, causing a feedback loop that negatively regulates the expression of Fzd1.

This work was supported in part by Deutsche Forschungsgemeinschaft grants SFB 415-C7 and EXC306 to N.R. and S.E. The authors declare that they have no conflicting financial interests. The authors gratefully acknowledge K.

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Kopp, K. Seeger, S. Kro¨ger, and L. Dost for expert technical assistance. 21.

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Received for publication April 15, 2010. Accepted for publication July 15, 2010.

NEUMANN ET AL.