Wnt6 Is Expressed in Granulomatous Lesions of Mycobacterium tuberculosis−Infected Mice and Is Involved in Macrophage Differentiation and Proliferation This information is current as of September 27, 2021. Kolja Schaale, Julius Brandenburg, Andreas Kispert, Michael Leitges, Stefan Ehlers and Norbert Reiling J Immunol 2013; 191:5182-5195; Prepublished online 11 October 2013; doi: 10.4049/jimmunol.1201819 Downloaded from http://www.jimmunol.org/content/191/10/5182

Supplementary http://www.jimmunol.org/content/suppl/2013/10/11/jimmunol.120181 http://www.jimmunol.org/ Material 9.DC1 References This article cites 60 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/191/10/5182.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Wnt6 Is Expressed in Granulomatous Lesions of Mycobacterium tuberculosis–Infected Mice and Is Involved in Macrophage Differentiation and Proliferation

Kolja Schaale,*,1,2 Julius Brandenburg,*,1 Andreas Kispert,† Michael Leitges,‡ Stefan Ehlers,x,{ and Norbert Reiling*

The Wnt signaling network, an ancient signaling system governing ontogeny and homeostatic processes, has recently been identified to exert immunoregulatory functions in a variety of inflammatory and infectious disease settings including tuberculosis. In this study, we show that Wnt6 is expressed in granulomatous lesions in the lung of Mycobacterium tuberculosis–infected mice. We identified foamy macrophage-like cells as the primary source of Wnt6 in the infected lung and uncovered a TLR–MyD88–NF-kB–

dependent mode of induction in bone marrow–derived macrophages. Analysis of Wnt6-induced revealed Downloaded from a pertussis toxin–sensitive, ERK-mediated, but b-catenin–independent induction of c-Myc, a master regulator of cell proliferation. Increased Ki-67 mRNA expression levels and enhanced thymidine incorporation in Wnt6-treated macrophage cultures demon- strate a proliferation-promoting effect on murine macrophages. Further functional studies in M. tuberculosis–infected macro- phages using Wnt6 conditioned medium and Wnt6-deficient macrophages uncovered a Wnt6-dependent induction of macrophage Arginase-1 and downregulation of TNF-a. This identifies Wnt6 as a novel factor driving macrophage polarization toward an M2- like phenotype. Taken together, these findings point to an unexpected role for Wnt6 in macrophage differentiation in the M. http://www.jimmunol.org/ tuberculosis–infected lung. The Journal of Immunology, 2013, 191: 5182–5195.

acrophages are the primary host cells for Mycobacte- replication (reviewed in Ref. 2). The immune response to myco- rium tuberculosis. The pathogenesis of tuberculosis is bacterial infections has been studied in great detail; however, still M characterized by the formation of granulomatous le- little is known about how pathogenic mycobacteria manipulate sions in the lung, which temporally and spatially coincides with their host cell to achieve persistence. immune-mediated containment of the infection. The granuloma One successful approach to identify novel factors that influence can proceed either to localized sterilization of the infection and antimicrobial effector mechanisms has been the use of systematic by guest on September 27, 2021 mineralization of the lesion or to localized caseation and necrosis expression profiling of macrophages infected with myco- that culminates in the release of infectious bacteria into the air- bacteria or treated with conserved bacterial structures (3). We and ways (recently reviewed in Ref. 1). Aerosol infection models in others have recently identified a regulatory role for components of mice have been used successfully to identify key mechanisms the Wnt signaling network to be operative at the interface between governing granuloma formation and integrity, as well as bacterial innate and adaptive immunity. In essence, Wnts can exert both proinflammatory and anti-inflammatory functions on macrophages

*Division of Microbial Interface Biology, Research Center Borstel, Leibniz Center and other cells of the immune system (reviewed in Refs. 4, 5). for Medicine and Biosciences, 23845 Borstel, Germany; †Institut fu¨r Moleku- Wnt signaling is well-known as an ancient and highly conserved larbiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany; principle among metazoan species, which executes essential func- ‡Biotechnology Centre of Oslo, University of Oslo, N-0317 Oslo, Norway; xPriority Research Area Infections, Research Center Borstel, Leibniz Center for Medicine and tions in embryogenesis and tissue homeostasis (reviewed in Refs. Biosciences, 23845 Borstel, Germany; and {Molecular Inflammation Medicine, 6, 7). In the adult, dysregulation of Wnt signaling is often asso- Christian-Albrechts University, 24118 Kiel, Germany ciated with cancer and degenerative diseases. In mice and men, 19 1 K.S. and J.B. contributed equally to this work. ligands, secreted palmitoylated glycoproteins of the Wnt family, 2Current address: Group Sweet, Institute for Molecular Bioscience, University of 10 seven-transmembrane receptors of the (Fzd) family, as Queensland, St. Lucia, QLD, Australia. well as several coreceptors or alternative receptors are known. Received for publication June 29, 2012. Accepted for publication September 5, 2013. Wnt can fuel at least three distinct signal transduction This work was supported in part by Deutsche Forschungsgemeinschaft Grants pathways (Wnt/b-catenin, Wnt/planar cell polarity and Wnt/Ca2+ SFB415-C7andEXC306(toN.R.andS.E.). signaling; reviewed in Ref. 6). Address correspondence and reprint requests to Dr. Norbert Reiling, Division of Microbial Interface Biology, Research Center Borstel, Leibniz Center for Med- Wnt5a was the first member of the Wnt family that was described icine and Biosciences, Parkallee 22, 23845 Borstel, Germany. E-mail address: to be induced in multiple inflammatory disease settings such as rheu- [email protected] matoid arthritis (8), tuberculosis (9), sepsis (10), psoriasis (11), ath- The online version of this article contains supplemental material. erosclerosis (12), and very recently also in obesity (13) and asthma Abbreviations used in this article: Arg-1, Arginase-1; BMDM, bone marrow–derived (14). Our group described Wnt5a and its Fzd5 to be in- macrophage; BODIPY 493/503, 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s- indacene; CCM, control conditioned medium; CM, conditioned medium; GSK, glycogen duced in human macrophages challenged with M. tuberculosis or synthase kinase; MOI, multiplicity of infection; MRC-1, mannose receptor 1; NOS, NO conserved bacterial structures (9). Furthermore, we could assign a synthase; p.i., postinfection; PKC, kinase C; pMF, peritoneal macrophage; PTX, proinflammatory function to Wnt5a, a finding later also reported by pertussis toxin; RT-qPCR, real-time quantitative PCR; ZN, Ziehl-Neelsen. other groups (10, 15): Wnt5a exerts proinflammatory functions on Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 macrophages (4, 9, 10), endothelial cells (15) and adipocytes (13). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201819 The Journal of Immunology 5183

Unlike Wnt5a, Wnt3a has been referred to as a “classical” or Aerosol infection “canonical” member of the Wnt family characterized by the in- High- (1000 CFU) and low-dose (100 CFU) infection of C57BL/6 mice with duction of the Wnt/b-catenin signaling pathway. In an inflam- M. tuberculosis H37Rv via the aerosol route was performed under BSL3 matory context, activation of the Wnt/b-catenin pathway has conditions as described previously (22). Mice were sacrificed at days 1, 21, been associated with a reduced migration of monocytes (16) and and 42 postinfection (p.i.). Analyses were performed as previously de- lately with the induction of tolerance in dendritic cells (17). A key scribed (20). b player in Wnt/ -catenin signaling is the glycogen synthase kinase Real-time quantitative PCR 3b (GSK3b), which is also a well-known regulator of NF-kB– b Total RNA was isolated from lung tissue (∼20 mg homogenized in lysis dependent gene transcription. GSK3 inhibition, as it happens 3 6 3 6 F 3 b buffer) or macrophages (BMDM: 0.4 10 to 1.5 10 cells; pM :1 during activation of Wnt/ -catenin signaling, was shown to sup- 106 cells) using the High Pure RNA Isolation Kit, the High Pure RNA press proinflammatory cytokine expression in different experi- Tissue Kit (Roche Applied Science, Mannheim, Germany), or the Direct- mental settings (18, 19). This holds true also in mycobacterial zol RNA MiniPrep (Zymo Research, Irvine, CA). For reverse transcription, infections, as we recently demonstrated the suppression of TNF-a the Maxima First Strand cDNA Synthesis Kit for real-time quantitative PCR (RT-qPCR; Fermentas, St-Leon-Rot, Germany) was used. Gene-specific expression in mycobacteria-infected macrophages by Wnt3a or primer pairs and TaqMan probes (Universal Probe Library, Roche Applied inhibition of GSK3b, respectively (4, 20). Science) were designed with the UPL assay design center (ProbeFinder We have now systematically screened the expression of the 19 Version 2.45 and earlier versions; sequences and probes are given in Sup- Wnt homologs in mice experimentally infected with M. tubercu- plemental Table I). RT-qPCR was performed using the LightCycler 480 losis via the aerosol route. In this study, we identified Wnt6 to be Probe Master Kit and the LightCycler 480 II system (Roche Applied Sci- ence) (20). Cp values of target and reference gene (hypoxanthine-guanine induced in the lung of M. tuberculosis–infected mice and localized phosphoribosyltransferase) were determined by the second derivative max- Downloaded from Wnt6 expression exclusively to macrophage clusters within granu- imum method. Relative was calculated considering the lomatous lesions. In vitro experiments using bone marrow–derived individual efficiency of each PCR setup determined by a standard curve. macrophages (BMDMs) uncovered a TLR-, MyD88-, and NF-kB– Wnt mRNA expression in lungs and macrophages was analyzed using the 2ΔΔCT method. dependent, but TNF-a–independent, induction of Wnt6 gene ex- pression. Studies on Wnt6 function identified a new role for this SDS-PAGE and Western blot analysis

Wnt homolog in murine macrophage differentiation and prolifer- http://www.jimmunol.org/ BMDMs (0.5 3 106 cells) were lysed in 125 ml23 SDS sample buffer ation, suggesting a distinct immunomodulatory potential. (125 mM Tris pH 6.8, 4% SDS, 20% glycerol, 100 mM DTT). SDS-PAGE and Western blot were performed as previously described (25). Primary Abs Materials and Methods were phospho–NF-kB p65 (Ser536), phospho-SAPK/JNK (Thr183/Tyr185; G9), phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204; 20G11), phospho-p38 Mice and macrophages MAPK (Thr180/Tyr182), phospho–protein kinase C (PKC) (pan; beta II Ser660; C57BL/6, TLR22/2, Myd882/2, TNF-a2/2, NMRI Wnt6+/+, Wnt6+/2, and all , Danvers, MA), purified anti–b-catenin (BD Bioscience, Wnt62/2 (21) mice were raised and maintained under specific pathogen- Heidelberg, Germany), GAPDH Ab (HyTest, Turku, Finland), or Human free conditions. Murine BMDMs were generated as previously described Wnt-6 Affinity Purified Polyclonal Ab Sheep IgG (R&D Systems, Wiesbaden, (22). Cells were allowed to attach overnight before proceeding further. Germany). Secondary Abs were AlexaFluor 680 Donkey Anti-Mouse IgG Resident peritoneal cells, mainly consisting of in vivo differentiated mac- (Invitrogen, Darmstadt, Germany), IRDye800 Conjugated Affinity Purified by guest on September 27, 2021 rophages (pMF), were isolated as described previously (23). Human Anti-Rabbit IgG (Rockland, Gilbertsville, PA), or peroxidase-conjugated monocyte-derived macrophages were generated from peripheral blood F(ab)2 Fragment Donkey Anti-Sheep (Jackson Immunoresearch, Suffolk, monocytes (purity consistently .92%) obtained by counterflow centrifu- U.K.). Western blots were analyzed using the Odyssey infrared imaging gation from PBMCs of healthy blood donors (24) by a 7-d culture in Teflon system (Ver. 2.1; Li-Cor Biotechnology, Lincoln, NE) or the Amersham bags (VueLife 72C; Cellgenix, Freiburg, Germany) in the presence of 10 ng/ ECL Western blotting Detection Reagent, Amersham Hyperfilm ECL (GE ml recombinant human M-CSF as described previously (25). All animal Healthcare, Buckinghamshire, U.K.) and the X-Omat M35 film processor experiments were approved by the Ministry of Agriculture, Environment (Kodak, Stuttgart, Germany). and Rural Areas (Kiel, Germany) and a local ethics committee. All experi- ments performed with human cells were approved by the Ethics Committee Immunohistochemistry and immunofluorescence analysis of the University of Lu¨beck (Lu¨beck, Germany; permit 12/116). Two-micrometer formalin-fixed, paraffin-embedded lung tissue sections Stimuli and inhibitors were deparaffinized, blocked with PBS containing 5% (v/v) FCS, and stained for Wnt6. Biotin-SP–conjugated F(ab)2 Fragment Rabbit Anti- M. tuberculosis (strain H37Rv, ATCC 27294; American Type Culture Sheep and Peroxidase-conjugated Streptavidin (Jackson Immunoresearch) Collection, Manassas, VA), GFP-expressing M. tuberculosis H37Rv, and was used for detection. To visualize M. tuberculosis andWnt6inthe M. avium (strain SE01) were grown as previously described (25, 26). GFP- same lung section, we stained deparaffinized tissue sections for acid-fast expressing M. tuberculosis was generated using the plasmid 32362: bacteria using a Ziehl-Neelsen (ZN) carbol-fuchsin solution (Merck, pMN437 (Addgene, Cambridge, MA), kindlyprovidedbyProf.M.Niederweis Darmstadt, Germany) and stained for Wnt6 as described earlier. Slides (University of Birmingham, Birmingham, AL) (27). For in vitro experi- were counterstained with Gills hematoxylin and analyzed with a BX41 ments, bacterial aliquots were centrifuged for 10 min at 835 3 g and microscope (Olympus, Hamburg, Germany) and the NIS-Elements software resuspended in the appropriate cell-culture medium using a syringe and (Nikon, Badhoevedorp, The Netherlands). Frozen tissue sections (5 mm) a 26-gauge syringe needle. LPS (Salmonella enterica serotype Friedenau were air-dried, fixed (acetone/chloroform or 10% [v/v] ice-cold formalin), H909) was kindly provided by Prof. H. Brade (Research Center Borstel, and blocked with 5% FCS or 10% normal donkey serum (Pan-Biotek, Borstel, Germany). The synthetic lipopeptide Pam3CSK4 was kindly pro- Aidenbach, Germany) with a combined permeabilization step with 0.1% vided by Prof. K.-H. Wiesmu¨ller (EMC Microcollections, Tu¨bingen, Triton X-100. After staining for Wnt6, slides were stained with 4,4-difluoro- Germany). rTNF-a was purchased from PeproTech (Rocky Hill, NJ). 1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503; Conditioned medium (CM) was derived from Wnt6-transfected (Wnt6 Life Technologies) to visualize lipid droplets (29) or with an anti-CD68 Ab CM) or LacZ-transfected NIH3T3 cells (control CM [CCM]), which were (FA-11; Abcam, Cambridge, U.K.). Macrophages grown on glass cov- a kind gift of Prof. S. Vainio (University of Oulu, Oulu, Finland) (28) and erslip were fixed (4% PFA), permeabilized using 90% methanol, and Prof. R. Kemler (University of Freiburg, Freiburg, Germany), respectively. stained for Wnt6 (see earlier) or anti–Ki-67 (Clone MIB-5; DAKO, In brief, cells were seeded in equal numbers and cultured for 4 d. Cell culture Glostrup, Denmark). The following secondary Abs were used: DyLight supernatants were filtered through a 0.2-mm filter and stored at 280˚C until 488–conjugated goat anti-rat and 549-conjugated donkey anti-sheep (Dia- further usage. The GSK3 inhibitor SB216763 was obtained from Tocris nova, Hamburg, Germany) or Cy5-conjugated AffiniPure F(ab)2 Fragment (Bristol, U.K.). Inhibitors of MAPK and NF-kB signaling (SB 203580, Goat Anti-Mouse (Jackson Immunoresearch). Nuclei were stained with U0126, and Bay 11-7082) were obtained from Calbiochem (Schwalbach, DAPI (Roche Applied Science). Slides were analyzed with an Axio Ob- Germany). Pertussis toxin (PTX) was purchased from Alexis Biochemicals server microscope with ApoTome and the AxioVision Software (Carl Zeiss (San Diego, CA). AG, Oberkochen, Germany). 5184 WNT6 IN M. TUBERCULOSIS INFECTION

Proliferation assays for two groups or one-way ANOVA for multiple-group comparison (*p , 0.5, **p , 0.01, ***p , 0.01). All data are shown as mean 6 SEM. Real-time proliferation assays (1.25 3 104 BMDMs) were performed with the xCELLigence System (Roche Applied Science). Impedance measure- ment was carried out using plates with incorporated sensor array (E-Plate) Results and the Real-Time Cell Analyzer SP instrument. Data obtained were an- Expression of Wnt in the lung of M. tuberculosis– alyzed using the Real-Time Cell Analyzer Software 1.2 (Roche Applied infected mice Science). After 3 d, an additional 100 ml fresh medium (including stimuli) was added. For [3H]thymidine incorporation assays, 1.25 3 104 BMDMs C57BL/6 mice were infected with 1000 CFU M. tuberculosis H37Rv were pulsed on day 4 of the experiment for 12 h with tritiated thymidine in an aerosol infection chamber. As previously described (22), a ([3H]TdR; 2 Ci/mmol, 0.2 mCi/culture). Cells were detached and harvested on glass-filter mats. Differences in amounts of incorporated radioactive rapid rise of the bacterial burden was observed between days 1 and material were expressed as cpm/per culture. 21 p.i. followed by a plateau of M. tuberculosis replication until day 42 (data not shown). A systematic screen for the expression of a Determination of TNF- and nitrite release all 19 Wnt genes was carried out using RT-qPCR. All 19 Wnt Cell-culture supernatants were harvested at the times indicated and frozen genes were detected at all time points analyzed. Statistically sig- at 220˚C until analysis. The concentrations of TNF-a were determined nificant changes in expression levels are shown in Fig. 1 (uninfected a using quantitative ELISA (Mouse TNF- DuoSet; R&D Systems) as rec- mice [day 0] versus day 21 or 42 p.i., respectively). mRNA ex- ommended by the manufacturer. The formation of nitrite was performed using the Griess reaction as previously described (30). pression of Wnt2, Wnt3a, Wnt4, Wnt5a, Wnt7a, and Wnt10b was significantly reduced at days 21 and 42 p.i. (Fig. 1A). mRNA Statistical analysis levels of Wnt8a and Wnt2b were reduced during the course of Downloaded from Statistical analysis was performed using GraphPad Prism 5 software infection, but statistically significant differences were only ob- (GraphPad Software, La Jolla, CA). For real-time PCR data from in vivo tained at day 21 or 42, respectively (Fig. 1A). For Wnt5b, Wnt8b, experiments, four to five mice per time point were analyzed with a two- Wnt9a, Wnt9b, Wnt11, and Wnt16, a tendency to lower expression tailed Mann–Whitney U test not assuming parametric distribution (*p , 0.5, **p , 0.01). Real-time PCR data of three to five independent in vitro during the course of infection was observed, whereas Wnt3 and experiments were log-transformed according to Willems et al. (31), and Wnt7b showed no clear tendency to higher or lower expression statistical comparison was carried out using either Student two-tailed t test (data not shown). Wnt1 and Wnt10a were expressed to a signifi- http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. mRNA expression of Wnt ligands and Wnt/b-catenin signaling target genes in the lung of M. tuberculosis–infected mice. RNA was isolated from lung tissue of C57BL/6 mice infected with 1000 CFU M. tuberculosis H37Rv and analyzed by RT-qPCR for Wnt ligands, Axin2, c-Myc, and NOS2. Samples from five mice per time point (day 0 [uninfected mice] and days 21 and 42 p.i.) were analyzed. Relative expression levels of Wnt ligands that were found to be significantly downregulated or upregulated at day 21 and/or 42 p.i. compared with day 0 are shown in (A) and (B), respectively. Fold induction of the Wnt/b-catenin target genes Axin2 and c-Myc, as well as of inducible NOS2, a common marker for M. tuberculosis/Th1–induced macrophage ac- tivation (C). All data are presented as mean 6 SEM. Statistical analysis was performed using the Mann–Whitney U test (day 0 versus day 21 or 42, respectively; *p , 0.05, **p , 0.01). n.s., Not significant. The Journal of Immunology 5185 cantly higher extent at day 42 p.i. (Fig. 1B). However, expression were located within these granulomatous structures. Wnt6+ cells levels of Wnt1 were extremely low compared with most other showed a foamy, macrophage-like morphology characterized by Wnts. Only mRNA expression of Wnt6 was increased significantly a large cytoplasm and prominent nuclei (Fig. 2C, insert), suggesting at days 21 and 42 p.i. (Fig. 1B). macrophages as main cellular source. In mice aerogenically infected In parallel, the expression of the inducible NO synthase (NOS2) with 100 CFU M. tuberculosis, the size of lung granulomatous and known Wnt target genes were analyzed to correlate the induction lesions was considerably smaller; however, Wnt6-expressing cells of inflammatory pathways to the activation of the Wnt/b-catenin were easily identified at day 42 p.i. (Supplemental Fig. 1), whereas pathway (shown in Fig. 1C). NOS2 expression was strongly in- stainings of mouse lungs at day 21 were still negative (data not creased 21 d and also 42 d p.i. The expression of the well-described shown). This indicates that the intensity of Wnt6 expression is Wnt/b-catenin pathway target gene Axin2 (32) was reduced to related to the number of M. tuberculosis CFU and the extent of ∼50% at days 21 and 42 p.i. The Wnt/b-catenin target gene c-Myc M. tuberculosis–induced inflammation present in the infected (33) showed no change in expression levels. mouse lung. In an independent study of murine M. avium infec- tion, immunohistochemical analysis of infected mouse lungs also Wnt6 expression in the lung of M. tuberculosis–infected mice identified macrophage-like cells to be positive for Wnt6 (data not is localized exclusively in macrophage clusters within shown). To further characterize the identity of Wnt6+ cells, we granulomatous lesions double stained frozen sections of M. tuberculosis–infected lung To determine the cellular source of Wnt6 expression in the M. tissue for Wnt6 and the macrophage marker CD68, and analyzed tuberculosis–infected lung, we analyzed formalin-fixed lung sec- by fluorescence microscopy (Fig. 2E). CD68+ cells were found in tions by immunohistochemistry using an anti-Wnt6 Ab (Fig. 2A– large numbers in regions with high cellular density (as determined Downloaded from D). No Wnt6+ cells were detected in uninfected control lungs (Fig. by DAPI staining). Wnt6+ cells were again found exclusively in 2A). When mice were infected with 1000 CFU, lungs showed dense cellular clusters within these infiltrates. The staining for Wnt6 large areas of cellular infiltrates primarily around the bronchi at 21 was spotlike, reminiscent of a vesicular distribution. All Wnt6+ cells and 42 d p.i. (Fig. 2B–D). Demarcated clusters of Wnt6+ cells were also positive for CD68, indicating a macrophage-restricted http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Expression of Wnt6 in the lung of M. tuberculosis–infected mice. (A–D) For- malin-fixed lung sections (2 mm) of uninfected (A)andM. tuberculosis H37Rv–infected C57BL/ 6 mice [1000 CFU; (B): day 21 p.i.; (C): day 42 p.i.] were stained with an Ab against Wnt6 and an appropriate peroxidase-conjugated secondary Ab, and visualized using 3,39-diaminobenzidine. Sections were counterstained with hematoxylin. (D) As control, a serial section of day 21 p.i. in- cubated with the secondary Ab alone is shown. (E and F) Frozen sections of infected lungs (day 42 p.i., 5 mm) were analyzed by fluorescence microscopy for the expression of Wnt6 and the macrophage marker CD68 (E), and lipid droplet staining agent BODIPY 493/503 (F). Sections were incubated sequentially with the primary and appropriate fluorescence-labeled secondary Abs and counterstained with DAPI. 5186 WNT6 IN M. TUBERCULOSIS INFECTION production of Wnt6; but not all CD68+ cells contained Ags re- Wnt6 expression is induced in murine BMDMs by active with the Wnt6-targeted reagent, indicating that only a sub- mycobacteria and conserved microbial structures + set of CD68 cells were producers of Wnt6. To characterize Wnt6 mRNA expression upon microbial stimula- To address whether Wnt6 expression may be indeed increased in tion, we infected murine BMDMs of C57BL/6 origin with M. macrophages with a foamy macrophage-like morphology in vivo, tuberculosis or M. avium for 4 and 24 h. In parallel, cells were we stained frozen sections of lungs of M. tuberculosis–infected stimulated with the TLR agonists LPS (TLR4) or the synthetic mice for Wnt6 and BODIPY 493/503, a dye that identifies the lipopeptide Pam3CSK4 (TLR1/TLR2). The expression levels of all presence of neutral lipids (29). As shown in Fig. 2F, the majority 19 Wnt homologs were analyzed by RT-qPCR. Expression of of Wnt6-expressing cells was also positive for the BODIPY stain- Wnt3a, Wnt7a, Wnt7b, Wnt8a, and Wnt8b was not detectable at all ing, which demonstrates that a staining with Wnt6 mainly identifies under any of the conditions. In addition, Wnt2, Wnt3, Wnt9b, and cells carrying lipid droplets, a characteristic feature of foamy mac- Wnt16 were detected only at very low levels and not in all of the rophages. However, not all lipid droplet-carrying cells were positive samples, precluding qualitative or even quantitative conclusions for Wnt6, illustrating the heterogeneity of the foamy macrophage (data not shown). The expression of the remaining Wnt genes is population in vivo. shown in Fig. 4A (only time point 4 h): the heat map shows that To visualize mycobacteria and Wnt6 in the same section, we the family members Wnt1, Wnt10a, Wnt5b, Wnt6, Wnt11, Wnt2b, prestained lung sections of infected mice (day 42 p.i.) by ZN staining. Wnt9a, Wnt5a, and Wnt10b were detected in BMDMs. With regard In the secondary Ab control (processed without addition of the anti- to an upregulation of Wnt genes in BMDMs by mycobacteria and conserved bacterial structures, Wnt1 expression was enhanced after

Wnt6 Ab), large mycobacteria-containing areas within the granu- Downloaded from 4 h by all four stimuli, but expression levels were extremely low. lomatous lesions were detected (Fig. 3A). Using a higher magnifi- Only Wnt6 showed a comparably high and enhanced expression in cation, it became apparent that primarily clusters of macrophage- response to all four stimuli, including M. tuberculosis, analyzed at like cells contained ZN-stained, often aggregated mycobacteria (Fig. 4 and 24 h p.i. Several other Wnt genes showed slightly enhanced 3B, 3C). Analysis of ZN and Wnt6 double-stained sections revealed expression in response to LPS (Wnt2b), LPS and Pam3CSK4 that ZN+ areas were generally also positive for Wnt6 (Fig. 3D). It (Wnt10b), or LPS, Pam3CSK4, and M. avium (Wnt10a) after 4 h. appeared that most cells harboring mycobacteria were clearly pos- Wnt10a http://www.jimmunol.org/ 2 After 24 h, a slightly elevated expression was detected for itive for Wnt6, whereas ZN neighboring cells showed only a faint in response to LPS and for Wnt5b and Wnt11 in response to or no staining for Wnt6 (Fig. 3E, 3F). In particular, huge cells M. tuberculosis. harboring larger aggregates of mycobacteria frequently showed Gene expression levels of Wnt6 were analyzed in more detail a very intense staining for Wnt6. by means of dose–response experiments (see Fig. 4B). After 4 h, by guest on September 27, 2021

FIGURE 3. Detection of mycobacteria and Wnt6 in the lung of M. tuberculosis–infected mice. Formalin-fixed lung sections (2 mm) of M. tuberculosis H37Rv–infected C57BL/6 mice (1000 CFU, day 42, p.i.) were stained for acid- fast bacilli by the ZN protocol (pink) and for Wnt6 (dark brown) as described in Fig. 2. (A–C) ZN-stained secondary Ab control (without anti- Wnt6 Ab). (D–F) Wnt6 and ZN-stained section. Sections were counterstained with hematoxylin. Scale bars, 100 mm(A, D); 10 mm(B, C, E, F). The Journal of Immunology 5187 Downloaded from http://www.jimmunol.org/

FIGURE 4. Expression of Wnt homologs in murine BMDMs treated with microbial structures and mycobacteria. Murine BMDMs were challenged with various doses of LPS, Pam3CSK4, M. avium SE01, and M. tuberculosis H37Rv for 4 and 24 h, and analyzed by RT-qPCR and Western blot. (A) Heat map of

relative expression levels of all detectable Wnt homologs in murine unstimulated BMDMs and the change of expression levels in response to LPS (30 ng/ by guest on September 27, 2021 ml), Pam3CSK4 (30 ng/ml), M. avium (MOI 3), and M. tuberculosis (MOI 3) for 4 h. Shown is the mean of four independent experiments. (B) Detailed analysis of Wnt6 mRNA expression in murine BMDMs in response to different concentrations of stimuli after 4 and 24 h. Shown is the mean 6 SEM of four independent experiments. For statistical analysis, data were log-transformed and groups were compared by use of the one-way ANOVA with Tukey’s test as post hoc test (*p , 0.05, **p , 0.01, ***p , 0.001). (C) Human monocyte-derived macrophages were infected with M. tuberculosis H37Rv (MOI 3:1) for 24 h and analyzed by RT-qPCR for mRNA expression of Wnt6. Shown is the mean 6 SEM of four independent experiments. For statistical analysis, data were log-transformed and groups were compared by Student two-tailed t test (*p , 0.05). (D) Protein levels of Wnt6 were analyzed by

Western blot (LPS: 10 ng/ml; Pam3CSK4: 100 ng/ml; M. avium, M. tuberculosis: MOI 3:1). NIH3T3 cells stably transfected with a construct coding for Wnt6 or LacZ served as control. Equal loading was confirmed by staining for GAPDH. (E) Detection of Wnt6 in macrophage cultures (BMDM) infected with GFP- expressing M. tuberculosis (MOI 1) by fluorescence microscopy. Twenty-four hours p.i., cells were fixed and stained for Wnt6. Nuclei were stained with DAPI.

Wnt6 was induced in BMDMs ∼2- to 4-fold in response to differ- BMDMs were infected with M. tuberculosis overexpressing GFP ent doses of LPS, M. avium, and M. tuberculosis, and 6- to 8-fold for 24 h and stained for Wnt6. Only cells infected with M. tu- + in response to Pam3CSK4. However, no clear dose dependency berculosis showed positive signals for Wnt6. Again, the Wnt6 was observed for any of the stimuli at this time point. After 24 h, signals appeared to be spotlike, comparable with the staining all stimuli induced an even stronger expression of Wnt6 (LPS: 10- pattern observed in M. tuberculosis–infected lungs (Fig. 2E). to 16-fold, Pam CSK : 4- to 10-fold, M. avium: 8- to 25-fold, M. 3 4 k tuberculosis: 2- to 14-fold) in a strictly dose-dependent manner. Induction of Wnt6 depends on TLR–MyD88–NF- B signaling a We also addressed the expression of Wnt6 in M. tuberculosis– but is independent of TNF- production infected human primary cells and infected human monocyte-derived The induction of Wnt6 expression by TLR ligands and myco- macrophages of four healthy blood donors with M. tuberculosis bacteria as described earlier suggests a mechanism operative via H37Rv (multiplicity of infection [MOI] 3:1) and observed a signif- TLR, MyD88, and NF-kB. Dependency on TLR-MyD88 signaling icant increase of Wnt6 mRNA expression p.i. with M. tuberculosis could be shown using BMDMs derived from TLR2- and MyD88- (Fig. 4C), suggesting a mycobacteria-induced upregulation of this deficient mice (Fig. 5A, 5B). In TLR22/2 and MyD882/2 macro- Wnt homolog also in myeloid cells of human origin. phages, Wnt6 mRNA expression levels were significantly reduced Total cellular protein levels of Wnt6 in murine macrophages in response to M. tuberculosis and Pam3CSK4 when compared were analyzed by Western blot 4 and 24 h after stimulation (Fig. with wild type macrophages. Similar data were obtained when 4D). In good accordance with the data obtained by RT-qPCR, cells were infected with M. avium (data not shown). In response elevated levels of Wnt6 were detected 4 and 24 h after stimula- to LPS, Wnt6 mRNA expression was unchanged in case of TLR2 2/2 tion with LPS, Pam3CSK4, M. avium,andM. tuberculosis.In deficiency, but clearly reduced in MyD88 macrophages, pointing addition, Wnt6 protein expression was analyzed in M. tubercu- to a role for MyD88-dependent signaling also in case of TLR4 losis–infected macrophages by fluorescence microscopy (Fig. 4E). stimulation. NF-kB dependency was analyzed using a small molecule 5188 WNT6 IN M. TUBERCULOSIS INFECTION

inhibitor of IkB-a phosphorylation (Bay 11-7082). Preincubation with this inhibitor of NF-kB signaling for 30 min dose depen- dently reduced the expression of Wnt6 in response to M. avium and Pam3CSK4 (Fig. 5C). The influence of TNF-a was analyzed using macrophages from TNF-a–deficient mice (Fig. 5D). There was no apparent difference observed in Wnt6 expression in TNF2/2 +/+ versus TNF macrophages, when stimulated with LPS, Pam3CSK4, or M. avium, nor was there an induction of Wnt6 expression ob- served after stimulation with rTNF-a in either TNF2/2 or TNF+/+ macrophages, demonstrating TLR-induced Wnt6 induction to be TNF-a independent. Exogenous Wnt6 induces expression of c-Myc in a b-catenin signaling–independent manner To investigate Wnt6-induced signaling in macrophages, we used Wnt6 CM produced by Wnt6-overexpressiong NIH3T3 cells (28). RT-qPCR analysis of Wnt6-treated murine macrophages revealed a differential induction of the Wnt/b-catenin target genes Axin2

and c-Myc. Whereas Wnt6 CM induced c-Myc expression ∼9- Downloaded from fold, no difference was observed in Axin2 expression compared with cells treated with CCM (Fig. 6A). Wnt3a as well as the GSK- 3b inhibitor SB216763 induced Axin2 but not c-Myc expression in macrophages (data not shown). Western blot analysis corrob- orated a b-catenin–independent induction of c-Myc expression be- b

cause there was no accumulation of -catenin observed in mac- http://www.jimmunol.org/ rophage lysates after stimulation of the cells with Wnt6 CM (Fig. 6B). The GSK3 inhibitor SB216763 (34) was used as a positive control to mimic the activation of Wnt/b-catenin signaling and leading to accumulation of b-catenin. Wnt6 induces phosphorylation of PKC, p38, and ERK Aside from Wnt/b-catenin signaling, several other Wnt-induced signaling pathways have been described in macrophages. Re-

cently, we reported a Wnt-mediated activation of NF-kBand by guest on September 27, 2021 MAPKs (4). In addition, Pereira et al. (10) suggested an activation of Wnt/Ca2+ signaling by Wnt5a. Thus, we analyzed a potential Wnt6-mediated activation of the NF-kB and MAPK pathways using phospho-specific Abs against p65–NF-kB and the MAPKs JNK1/2, p38, and ERK1/2, respectively. Activation of Wnt/Ca2+ signaling was monitored using a pan-phospho-PKC Ab because PKC was described as a downstream target of Wnt/Ca2+ signaling (reviewed in Ref. 35). Western blot analysis revealed a fast but transient increase in ERK and p38 phosphorylation induced by Wnt6 CM (Fig. 6B). An activation of the MAPK JNK by Wnt6 CM was not observed. A strong signal for phospho-PKC isoforms at around 80 kDa was detected already in control (CCM-treated) macrophages (Fig. 6B). Treatment with Wnt6 CM led to detection of an additional band with slightly higher m.w. that may corre- spond to phosphorylation of an additional PKC isoform. This effect showed similar kinetics compared with ERK and p38 phosphorylation, and was also apparent in LPS-treated macro- phages. Wnt-mediated activation of Ca2+ signaling was previously linked to NF-kB activation (10, 15). However, NF-kB p65 phos- phorylation was not observed in response to Wnt6 CM.

FIGURE 5. The role of TLR2, Myd88, NF-kB, and TNF-a in micro- bially induced Wnt6 mRNA expression of murine macrophages. BMDMs were preincubated with indicated concentrations of the NF-kB inhibitor were challenged with various stimuli, and Wnt6 expression was analyzed Bay 11-7082 for 30 min and subsequently treated with Pam3CSK4 (30 ng/ by RT-qPCR. (A) BMDMs from C57BL/6 and TLR22/2 mice were stim- ml) or M. avium SE01 (MOI 3:1) for 4 h. (D) BMDMs from C57BL/6 and 2/2 ulated with LPS (TLR4 ; 10 ng/ml), Pam3CSK4 (TLR1/TLR2 li- TNF-a mice were stimulated with LPS (10 ng/ml), Pam3CSK4 (30 ng/ gand; 100 ng/ml), M. avium SE01 (MOI 3:1), or M. tuberculosis H37Rv ml), M. avium SE01 (MOI 3:1), or rTNF-a (50 ng/ml) for 4 h. Depicted is 2 2 (MOI 3:1 or 10:1) for 24 h. (B) BMDMs from C57BL/6 and Myd88 / the mean 6 SEM of three independent experiments. For statistical anal- mice were stimulated with LPS (10 ng/ml), Pam3CSK4 (100ng/ml), or M. ysis, data were log-transformed and indicated groups were compared by tuberculosis (MOI 3:1 or 10:1) for 24 h. (C) BMDMs of C57BL/6 mice Student two-tailed t test. **p , 0.01. n.s., Not significant. The Journal of Immunology 5189 Downloaded from http://www.jimmunol.org/

FIGURE 7. Sensitivity of Wnt6-induced gene expression and signal transduction to PTX and inhibitors of MAPK signaling. BMDMs were preincubated for 2 h with PTX (A) and for 30 min with the MAPK inhibitors U-0126 or SB203580 (B). Subsequently, Wnt6 CM or CCM was added for another 4 h, and the expression of c-Myc was analyzed by RT- qPCR. (A and B) Depicted is the mean 6 SEM of three independent FIGURE 6. Wnt6 induced gene expression and signal transduction in by guest on September 27, 2021 murine macrophages. BMDMs were stimulated for 4 h with Wnt6 CM or experiments. (C) BMDMs pretreated with 50 ng/ml PTX were incubated CCM and analyzed by RT-qPCR for the expression of the Wnt/b-catenin with Wnt6 CM or CCM for 10 min and analyzed by Western blot for PKC target genes c-Myc and Axin2 (A). Depicted is the mean 6 SEM of three and ERK phosphorylation. Equal loading was confirmed by staining for independent experiments. (B) Western blot analysis of BMDMs incubated GAPDH. ERK phosphorylation was quantified by the ratio of ERK to with Wnt6 CM, CCM, or positive controls (LPS: 100 ng/ml; GSK3 in- GAPDH signal intensity of two independent experiments. hibitor SB216763: 30 mM) for 10–120 min. Equal loading was confirmed by staining for GAPDH. Shown is one representative experiment out of k two (NF- B phospho-p65 and phospho-JNK) or three performed (all Wnt6 modulates the inflammatory response of others). mycobacteria-infected macrophages To address a potential regulatory role of Wnt6 in mycobacterial Wnt6-induced c-Myc expression is sensitive to PTX and ERK infections, we used murine BMDMs and analyzed the inflamma- inhibition tory response to mycobacteria in the presence or absence of Several Wnt-mediated effects depend on heterotrimeric G proteins Wnt6: we infected macrophages with M. tuberculosis (Fig. 8) and (reviewed in Ref. 36). To investigate the participation of hetero- M. avium (Supplemental Fig. 2). Wnt6 CM–treated M. tuberculosis– trimeric G proteins, we analyzed the sensitivity of Wnt6 CM– infected cells showed a 30% decrease in TNF-a expression after induced signaling events to PTX, an inhibitor of the a subunit of 4 h of incubation (Fig. 8A). The mRNA expression level of NOS2, Gi, Go and Gt. As shown in Fig. 7A, Wnt6 CM–induced expression which encodes a key enzyme of the antimycobacterial response of of c-Myc in murine BMDMs is reduced in a dose-dependent manner murine macrophages, was slightly reduced in Wnt6 CM–treated by preincubation with PTX. With regard to Wnt6-induced signaling M. tuberculosis–infected cells, although this did not reach statis- in BMDMs, we observed PTX-sensitive and -insensitive effects. tical significance (Fig. 8B). At the same time, we observed that Whereas the Wnt6 CM–induced phosphorylation of PKC remained expression of Arginase-1 (Arg-1), a marker for murine alterna- unchanged in the presence of PTX, ERK phosphorylation was re- tively activated macrophages (38, 39), was strongly upregulated duced ∼30% as determined by quantification of Western blots (Fig. 8C). Although Wnt6 CM alone did not alter Arg-1 expression (Fig. 7C). To further elucidate a possible role for MAPKs in the significantly, it showed a synergistic effect with M. tuberculosis Wnt6-driven c-Myc expression, we carried out experiments with infection, resulting in an ∼4-fold increase compared with infected small molecule MAPK inhibitors (37). As shown in Fig. 7B, Wnt6 cells treated with CCM. Similarly, Wnt6 CM induced enhanced CM–induced c-Myc expression was sensitive only to inhibition mRNA levels of mannose receptor 1 (MRC-1), another well-known by U0126, which has been described as an inhibitor of the ERK marker of alternative macrophage activation (39) (Fig. 8C). Also, pathway. Incubation with SB203580, an inhibitor of the p38 MAPK BCL-2, a protein with antiapoptotic activities (reviewed in Ref. 40), pathway, had no effect. was induced by Wnt6 CM in uninfected macrophages (Fig. 8D). 5190 WNT6 IN M. TUBERCULOSIS INFECTION

FIGURE 8. Exogenous Wnt6 modulates the M. tu- berculosis–induced inflammatory response of macro- phages. BMDMs derived from C57BL/6 mice were incubated with Wnt6 CM or CCM and infected with M. tuberculosis with a MOI of 1:1 or 3:1. mRNA expres- sion was measured for TNF-a (A), NOS-2 (B), Arg-1 (C), MRC-1 (C), and BCL-2 (D). TNF-a and BCL-2 mRNA expression was measured 4 h p.i., whereas in- ducible NOS2, Arg-1, and MRC-1 mRNA expression were determined by RT-qPCR 24 h p.i. Depicted is the mean 6 SEM of three to four independent experiments. For statistical analysis, data were log-transformed and indicated groups were compared by Student two-tailed t test (*p , 0.05). n.s., Not significant. Downloaded from http://www.jimmunol.org/

These data suggested that exogenously added Wnt6 actively mod- whether also endogenously formed Wnt6 would influence mac- ulates the inflammatory response of mycobacteria-infected macro- rophage c-Myc expression: already basal levels of c-Myc expres- phages. sion were significantly lower in Wnt62/2 BMDMs (data not shown) To further substantiate these findings, we used macrophages and peritoneal macrophages (Fig. 9F), when compared with from Wnt6-heterozygous and Wnt6-deficient mice and infected Wnt6+/+ macrophages. In addition, we observed a significant re- them with M. tuberculosis: Fig. 9A demonstrates that Wnt6-het- duction in M. tuberculosis–induced c-Myc mRNA expression in erozygous mice show ∼50% of the expression of WT mice, Wnt6-deficient macrophages when compared with control cells. whereas Wnt6 mRNA was completely absent in Wnt6-deficient This data set complements our findings obtained with exogenously by guest on September 27, 2021 mice at all time points analyzed. The analysis of TNF mRNA added Wnt6 and independently points to a link between Wnt6 and expression and release showed that Wnt62/2 BMDMs revealed the function of c-Myc in macrophages. An enhanced expression of a significantly enhanced formation of this antimycobacterial me- c-Myc has previously been associated with promotion of the cell diator (Fig. 9B). Similar data were obtained when cultures of cycle also in macrophages (41). Thus, we monitored a potential resident peritoneal cells, mainly consisting of in vivo differenti- effect of Wnt6 on macrophage proliferation, using both c-Myc ated macrophages (pMF), were used (Fig. 9F). These data sets mRNA expression and analysis of the proliferation marker Ki-67 demonstrate a proinflammatory phenotype of Wnt62/2 macro- as readout systems. RT-qPCR analysis revealed an enhanced Ki-67 phages, when infected with M. tuberculosis. We also observed mRNA expression in Wnt6 CM–stimulated BMDMs after 24 h that M. tuberculosis–induced NOS2 mRNA levels were signifi- (Figs. 10A, 11). Analysis of Ki-67 protein expression by fluo- cantly reduced in Wnt62/2 cells (Fig. 9C). However, the magnitude rescence microscopy identified an enhanced number of Ki-67+ of nitrite formation of Wnt6+/+ and Wnt62/2 macrophages, re- nuclei in Wnt6 CM–treated macrophage cultures (Fig. 10B). A flecting the total NO formation of the two cell types, was compa- similar effect was observed when macrophages were treated with rable, although the overall level of nitrite formation was very low. CSF-1, used as a positive control. These data indicate that Wnt6 Notably, NO formation of LPS-stimulated Wnt62/2 cells was sig- may promote proliferation of murine macrophages. To further sup- nificantly decreased compared with Wnt6 +/+ macrophages (data not port these data, we monitored macrophage cell proliferation using shown), suggesting a reduced activity of NOS-2 in response to this the xCELLigence system (Roche Applied Science). This real-time TLR4 agonist. At the same time, M. tuberculosis–induced Arg-1 system allows measurements of the electrical impedance across mRNA expression in Wnt62/2 mice was reduced by .75–90%, the bottom of the cell-culture plate (Cell Index) and has been when compared with Wnt6+/+ cells (Fig. 9D). Similarly, MRC-1 shown to be equivalent to assays measuring metabolic activity mRNA expression was significantly decreased in Wnt6-deficient, (42). Fig. 10C documents that Wnt6 CM–treated cells showed a uninfected macrophages (Fig. 9D). Notably, the analysis of steeper ascent and a higher overall level of the Cell Index com- MRC-1 gene expression in M. tuberculosis–infected cells did not paredwithCCM-treatedcells,suggestinganenhancedprolif- lead to differences between the two cell types (data not shown). eration and higher cell numbers after 3 d. Restimulation by BCL-2 mRNA levels were found to be dose dependently induced addition of fresh CM after 3 d resulted in a further rapid increase by M. tuberculosis infection of Wnt6+/+ but not Wnt62/2 BMDMs of the Cell Index in Wnt6 CM–treated cells. An increase of the (Fig. 9E). Cell Index was also observed in CSF-1–treated macrophage cultures, although different kinetics were observed: the Cell Index Wnt6 induces proliferation in macrophages rose slowly but continuously to a level comparable with that obtained Having observed that exogenous Wnt6 drives c-Myc expression in with Wnt6 CM–treated cells after 6 d. To independently confirm macrophages, we used Wnt6-deficient macrophages to address our real-time proliferation data, we performed [3H]thymidine in- The Journal of Immunology 5191

rophages, again indicating proliferation-inducing effects of Wnt6 (Fig. 10D).

Discussion Wnt signaling has been primarily described as a master regulatory pathway during ontogeny and homeostatic processes. There is now growing evidence demonstrating a regulatory role for Wnt sig- naling also in inflammatory and infectious diseases. In this study, we monitored the expression of all 19 Wnt homologs in mice experimentally infected with M. tuberculosis via the aerosol route. We identified Wnt6 to be induced and localized its expression exclusively to macrophage clusters within granulomatous lesions. In vitro experiments using BMDMs uncovered a TLR-, MyD88-, and NF-kB–dependent but TNF-a–independent induction of Wnt6 gene expression. Analysis of Wnt6-induced signaling using Wnt6 CM suggests a PTX-sensitive and ERK-dependent, but b-catenin– independent, induction of the known Wnt target gene c-Myc.Further studies on Wnt6 function identified a new role for this largely

unexplored member of the Wnt family in murine macrophage Downloaded from proliferation and polarization, suggesting a distinct immunomod- ulatory potential. We recently reported that most receptors of the Fzd family and the Wnt/b-catenin target gene Axin2 are significantly downregu- lated in the infected lung of M. tuberculosis–infected mice (20).

This study further corroborates the notion that Wnt signaling is http://www.jimmunol.org/ broadly repressed during the course of infection in the lung: we observed that mRNA expression levels of the majority of Wnt homologs (Wnt2, Wnt2b, Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt8a, and Wnt10b) were also significantly reduced during M. tubercu- losis infection. The potential physiological relevance of this counter-regulation of inflammatory versus Wnt/b-catenin–driven anti-inflammatory and homeostatic processes has been discussed only recently (4, 20). A growing body of experimental data from independent laboratories underscores the anti-inflammatory po- by guest on September 27, 2021 tential of Wnt/b-catenin signaling by: 1) reduction of proinflam- matory cytokine formation in macrophages (4, 20) and also septic shock models (18, 19, 43), 2) inhibition of transendothelial mono- cyte migration (16), and 3) induction of tolerance by reprogram- ming dendritic cells (17). Therefore, our discovery that some Wnts (Wnt1, Wnt10a, and Wnt6) are induced in an inflammatory environment such as the lung of M. tuberculosis–infected mice is of particular interest. We identified CD68+ cells with a foamy macrophage-like morphology within granulomatous lesions as the main cellular source of Wnt6. Our observation that Wnt6+ cells contain lipid vesicles as shown by BODIPY 493/503 staining supports the notion that Wnt6 in- deed stains a subset of foamy macrophages in vivo. These clusters of foamy macrophages within large cellular infiltrates of primarily FIGURE 9. Wnt6 deficiency affects the effector functions of in vitro lymphoid origin have been proposed to be the habitat of M. tu- and in vivo differentiated macrophages in response to M. tuberculosis. berculosis in experimental mouse models and also in human BMDMs (A–E) or peritoneal macrophages (pMF)(F)fromNMRIWnt6+/+ + 2/+ 2/2 disease (44, 45). Therefore, Wnt6 cells may represent a specific (wt), Wnt6 and Wnt6 mice were infected with M. tuberculosis subset of macrophages facilitating bacterial persistence rather H37Rv as indicated. mRNA expression was determined by RT-qPCR: (A) than eradicating the pathogen. This line of argument is further Wnt6, 4 h and 24 h; (B) TNF-a,24h,(C) NOS2,24h;(D) Arg-1,24h and MRC-1, 4 h; (E) BCL-2, 24 h and (F) c-Myc, 24 h. The release of supported by our data obtained from in vitro experiments using TNF-a (B, F) and Nitrite (C) were measured as described in Materials and murine BMDMs: a systematic expression screen of macrophages Methods. Depicted is the mean 6 SEM of three to five independent treated with pathogenic mycobacteria and conserved bacterial experiments. For statistical analysis data were log-transformed and indi- structures revealed Wnt6 and Wnt1 as only Wnt ligands that are cated groups were compared by Student two-tailed t test. *p , 0.05, **p , induced .2-fold by all stimuli used, including M. tuberculosis. 0.01. n.s., Not significant. Analysis of M. tuberculosis–infected cell cultures by fluorescence microscopy suggests that only infected macrophages, but not by- corporation assays. After 4 d of treatment with CM, BMDMs were stander cells, contain detectable amounts of Wnt6. This may indi- pulsed with [3H]TdR for a total time of 12 h. BMDM lysates of cate a mechanism requiring direct contact with microbial structures cells stimulated with Wnt6 CM showed a 25-fold increased rather than a paracrine induction by secreted mediators such as [3H]TdR incorporation when compared with CCM-treated mac- inflammatory cytokines. 5192 WNT6 IN M. TUBERCULOSIS INFECTION

The induction of Wnts has been only poorly described in in- flammatory settings: to date, an upregulation of Wnt6 has been almost exclusively observed in microarray-based studies, for ex- ample, in M. tuberculosis–infected murine macrophages (46), in the bladder of mice after stimulation with LPS, in a rat renal al- lograft model, in coloscopic biopsies of ulcerative colitis patients, and most recently also in the lung of asthma patients (14, 47–49). Of note, Liu et al. (50) describe an induction of Wnt6 mRNA by Salmonella typhimurium infection of murine intestinal epithelia cells ex vivo. However, none of these studies defined the mode of induction or assigned a function of Wnt6. We now identified Wnt6 mRNA expression induced in BMDMs by microbial stimuli to be dependent on TLR-, Myd88-, and NF-kB signaling, but inde- pendent of TNF-a. Regarding the well-known property of TNF-a to activate NF-kB and to boost TLR-driven, NF-kB–mediated gene transcription, it can only be assumed that activation of NF- kB may be necessary but not sufficient to induce Wnt6 expression. These data also support the notion that Wnt6 expression is induced

locally in macrophages that have direct contact to or are infected Downloaded from by mycobacteria. The ZN/Wnt6 double staining, which reveals that in vivo most Wnt6+ cells are infected, further strengthens this interpretation of our data. If this were to apply also to M. tuber- culosis–infected macrophages within the granulomatous lesion, Wnt6 may become a useful biomarker.

Although this study mainly focuses on cells of the murine im- http://www.jimmunol.org/ mune system, we also demonstrate a significant increase of Wnt6 mRNA expression in human monocyte-derived macrophages in- fected with M. tuberculosis H37Rv, indicating that this Wnt ho- molog may be subject to a similar regulation in human macrophages. Based on this study, which shows that immunohistochemical de- tection of Wnt6 protein is related to the number of CFU in a given mouse lung, and our recent observation that M. tuberculosis clin- ical isolates differ in their virulence patterns (26), detailed im- munohistochemical studies are now needed to characterize the cells by guest on September 27, 2021 and the conditions leading to the expression of the Wnt6 protein during pulmonary tuberculosis in humans. Secreted Wnt proteins are proposed to act predominantly, al- though not entirely, as short-range mediators (reviewed in Ref. 51). In fundamental work by Shimizu et al. (52), different Wnt homologs were characterized by their ability to morphologically transform C57MG epithelia cells and their potential to activate Wnt/b-catenin signaling. Wnts today classified as so-called ca- nonical Wnts (e.g., Wnt1, Wnt3, and Wnt3a) induced strong morphological changes that could be correlated with their ability to induce b-catenin stabilization. Wnt6 was shown to induce only slight morphological changes and only in an autocrine but not in a paracrine manner. An activation of Wnt/b-catenin signaling was not observed. This was later corroborated by Gazit and col- leagues (53), describing that again Wnt1, Wnt3, and Wnt3a, but not Wnt6, induced Wnt/b-catenin signaling in Fzd1-overexpressing HEK293T cells. We therefore investigated whether Wnt6 may influence macrophage-specific effector functions in primary cells and during infection. Wnt6 CM did not induce Wnt/b-catenin sig- naling in primary murine macrophages as determined by analysis of b-catenin protein levels and Axin2 mRNA expression. Remarkably, FIGURE 10. Wnt6 has proliferation-inducing effects on murine mac- another dedicated Wnt/b-catenin target gene, c-Myc (33), turned out rophages. BMDM derived from C57BL/6 mice were incubated with Wnt6 CM, CCM, or rCSF-1 (50 ng/ml) for 1–6 d with a restimulation at day 3. (A) Ki-67 expression after 24 h was determined by RT-qPCR. Data were log-transformed and groups compared using a paired Student two-tailed t (expressed as cell index) of 12,500 cells/well was determined by real-time test. Depicted is the mean 6 SEM of four independent experiments. *p , impedance measurement using the xCELLigence system (Roche). (D) Cells 0.05. (B) Cells were stained for Ki-67 after 24 h and analyzed by fluo- were seeded as described for (C). At day 4, cells were pulsed with [3H]- rescence microscopy. Nuclei were stained with DAPI. Shown are repre- thymidine for a period of 12 h. Depicted is mean 6 SEM of the relative sentative pictures from one out of two independent experiments. Origi- [3H]-thymidine incorporation (counts per minute/culture normalized to nal magnification 3250. (C) Over a period of 6 d the cellular growth CCM treated control) of two independent experiments. The Journal of Immunology 5193 to be induced by Wnt6 CM in a b-catenin–independent manner. phages (M2) based on gene expression induced in response to This finding is further complemented by the observation that c-Myc pathogen- and cytokine-derived stimulation (38, 56). In this study, expression is significantly reduced in Wnt62/2 macrophages we show that Wnt6 induces the expression of Arg-1 and reduces the infected with M. tuberculosis. It is also well in line with our ob- formation of TNF-a in M. tuberculosis–infected macrophages, servation that Axin2, but not c-Myc expression, is reduced in the which supports the notion that Wnt6 shifts macrophage polarization lung during the course of M. tuberculosis infection, suggesting toward an M2-like phenotype. El Kasmi et al. (57) demonstrated a Wnt/b-catenin–independent expression of c-Myc also in vivo. that mycobacteria induce Arg-1 in a TLR- and Myd88-dependent Mechanistic studies in primary macrophages revealed that ex- but STAT6-independent manner. Our data are in line with these pression of c-Myc is dependent on activation of the MAPK ERK. findings because we observe that Wnt6 expression is also TLR and The transcription factor c-Myc is an important regulator of cell Myd88 dependent. Thus, we may have identified Wnt6 as a so far proliferation in various cell types, including macrophages (reviewed unknown but important regulatory factor in this signaling chain in Ref. 41). But as a global regulator of chromatin, it exerts func- driving macrophage M2 differentiation. The almost complete loss tions in virtually all cellular processes, for example, in cell me- of Arg-1 mRNA levels in Wnt62/2 macrophages is accompanied tabolism, differentiation, apoptosis, and many more (reviewed in by a significant, albeit incomplete reduction of c-Myc levels in Ref. 54). these cells. Whether this correlation is indeed functionally related, In-depth analysis of Wnt6-mediated signaling revealed that phos- for example, in the sense that the lack of c-Myc is causal for the phorylation of ERK and the induction of c-Myc expression is sen- reduced Arg1 expression, needs to be addressed in further analy- sitive to PTX, implying an involvement of heterotrimeric G pro- ses. This is not unlikely, because it was shown that c-Myc drives teins that has been discussed for several signaling events mediated the expression of C/EBPb (58), a transactivating factor essential Downloaded from by G protein–coupled receptors of the Frizzled family (reviewed for Arg-1 induction (57). in Ref. 36). It is currently not known whether a single receptor or A recent study showed that alternative differentiation of hu- multiple receptors mediate Wnt6-induced signaling. Notably, the man macrophages into a M2 phenotype requires c-Myc (59), Wnt6-induced change in PKC phosphorylation was not altered a transcription factor that we find significantly induced also in by pretreatment of BMDM with PTX, indicating that different murine macrophages by Wnt6. This prompted us to investigate

receptors and downstream signaling systems may be involved. The a potential proliferation-inducing potential of Wnt6. Indeed, http://www.jimmunol.org/ m.w. of the additional PKC isoform that was detectable in Wnt6- treatment of murine BMDMs with Wnt6 CM turned out to have treated macrophages could correspond to PKCε (55). a cell-cycle–promoting effect as shown by the analysis of Ki67 In recent years, the concept of macrophage polarization has and thymidine incorporation assays in vitro. A more detailed been developed and further sophisticated: macrophages are now analysis is needed to validate whether Wnt6 may support mac- seen as a spectrum of phenotypic subtypes ranging from classically rophage growth also in vivo. It is well-known that Wnt proteins activated macrophages (M1) to the alternatively activated macro- promote cellular growth, even in inflammatory settings (60). With by guest on September 27, 2021

FIGURE 11. Model of Wnt6 induction and Wnt6-induced signaling events in murine macrophages. Wnt6 and proinflammatory cytokines are induced in macrophages by microbial structures like LPS, lipopeptides (LP), or pathogenic mycobacteria (M. tuberculosis, M. avium) in a TLR–Myd88–NF-kB– dependent manner (1). Secreted Wnt6 acts on macrophages in an autocrine and paracrine manner (e.g., macrophage clusters in the granulomatous lesion in the M. tuberculosis–infected lung) (2). Wnt6 attenuates the inflammatory response of macrophages by reducing TNF-a formation and drives c-Myc, Arg-1, BCL-2, and Ki-67 gene expression; c-Myc expression is mediated by G protein–coupled receptors and the MAPK ERK (3). Thereby a milieu favoring survival and proliferation of macrophages within the site of infection is created. 5194 WNT6 IN M. TUBERCULOSIS INFECTION regard to the M. tuberculosis–infected lung, there is currently no 11. Reischl, J., S. Schwenke, J. M. Beekman, U. Mrowietz, S. Stu¨rzebecher, and J. F. Heubach. 2007. Increased expression of Wnt5a in psoriatic plaques. J. In- evidence supporting a potential Wnt6-induced proliferative effect of vest. Dermatol. 127: 163–169. macrophages within the granulomatous lesion, a microenvironment 12. Christman, M. A., II, D. J. Goetz, E. Dickerson, K. D. McCall, C. J. Lewis, regarded as a classical Th1 setting. However, the local mediator F. Benencia, M. J. Silver, L. D. Kohn, and R. Malgor. 2008. Wnt5a is expressed in murine and human atherosclerotic lesions. Am. J. Physiol. Heart Circ. Physiol. milieu may be decisive whether macrophages actively proliferate 294: H2864–H2870. also in inflammatory conditions. Jenkins et al. (61) recently dem- 13. Ouchi, N., A. Higuchi, K. Ohashi, Y. Oshima, N. Gokce, R. Shibata, Y. Akasaki, onstrated the proliferation of tissue-resident macrophages during A. Shimono, and K. Walsh. 2010. Sfrp5 is an anti-inflammatory adipokine that modulates metabolic dysfunction in obesity. 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