IL13-Mediated Dectin-1 and Mannose Receptor …...1UMR 152 Pharma Dev, Universite de Toulouse, IRD, UPS, France. 2UMR 5623 ... tom/z650amu.Theon-columninjectedvolumewas0.1mL.The sourcetemperaturewas230

Research Article

IL13-Mediated Dectin-1 and Mannose ReceptorOverexpression PromotesMacrophageAntitumorActivities through Recognition of SialylatedTumor CellsMohamad Alaeddine1, M�elissa Prat1, V�er�ena Poinsot2, Val�erie Gouaz�e-Andersson1,H�el�ene Authier1, Etienne Meunier1, Lise Lef�evre1, Camille Alric1, Christophe Dardenne1,Jos�e Bernad1, Laurent Alric1, Bruno Segui3, Patricia Balard4, Francois Couderc2,Bettina Couderc3, Bernard Pipy1, and Agn�es Coste1

Abstract

Macrophage-mediated cytotoxicity is controlled by sur-face receptor expression and activation. Despite the numer-ous studies documenting the role of macrophage C-typelectin receptors (CLR) in pathogen elimination, little isknown about their contribution to antitumor responses.Here, we report that IL13 inhibits T-cell lymphoma andovarian adenocarcinoma development in tumor-bearingmice through the conversion of tumor-supporting macro-phages to cytotoxic effectors, characterized by a CLR signa-ture composed of dectin-1 and mannose receptor (MR). We

show that dectin-1 and MR are critical for the recognition oftumor cells through sialic acid–specific glycan structure ontheir surface and for the subsequent activation of macro-phage tumoricidal response. Finally, we validated that IL13antitumor effect mediated by dectin-1 and MR overexpres-sion on macrophages can extend to various types of humantumors. Therefore, these results identify these CLRs aspotential targets to promote macrophage antitumorresponse and represent an attractive approach to elicittumor-associated macrophage tumoricidal properties.

IntroductionMonocytes/macrophages represent a major component of

leukocyte infiltration in tumors. The beneficial and adverseeffects of macrophages on tumor progression result from theirgreat heterogeneity and plasticity (1–3). Within the tumor,macrophages are commonly termed tumor-associated macro-phages (TAMs). During early-stages tumor development,tumor-infiltrating macrophages, which promote immuneresponses and elicit tumor cell disruption, are characterizedby an IL12high and IL10low phenotype, close to M1-polarizedmacrophages. These macrophages produce cytotoxic media-tors such as proinflammatory cytokines and oxygen- andnitrogen-derived radicals, which confer strong microbicidal

and tumoricidal activity (4). In late-stage tumor progression,TAMs switch to an IL12low and IL10high phenotype with lowtumoricidal activity (5, 6). Although most reports identifythem as anti-inflammatory M2-like macrophages, activatedTAMs produce multiple proinflammatory markers character-istic of M1 phenotype (3, 7). Thus, the subset terminology M1and M2 is oversimplified and does not accurately reflect thesubpopulations of TAMs (4, 8).

Several lines of evidence suggest that the acquisition ofcytotoxic functions in macrophages depends on the expressionand activation of pattern recognition receptors (PRR). Thestimulation of Toll-like receptors (TLR) on macrophages pro-motes the activation of cytotoxic pathways, shifting tumor-supporting macrophages toward tumoricidal effectors (9, 10).Likewise, within the PRR family, C-type lectin receptors (CLR)can also modulate antitumor responses, because they canspecifically bind the tumor-related glycoforms and can affectCLR signaling and immune cell differentiation (11). The CLRsDC-SIGN and MGL expressed on dendritic cells (DC) caninteract with altered glycosylation patterns found on tumorcells and impair DC function, thereby preventing optimalpriming of tumor-specific T cells (11–14). Conversely, myeloidCLRs can deliver an activating signal to NK cells or lymphocytesin response to detection of altered self and participate to tumorelimination. This was reported for the NKG2D receptor, a CLRexpressed by NK cells that mediates the generation of CD8þ

cytotoxic lymphocytes and induces CD4þ T helper antitumorresponses (15). Similarly, dectin-1 on the surface of macro-phages is important in the recognition of tumor cells thathighly express glycan structures and in the activation of NK

1UMR 152 Pharma Dev, Universit�e de Toulouse, IRD, UPS, France. 2UMR 5623Laboratoire des IMRCP, Universit�e de Toulouse, CNRS, UPS, France. 3UMR1037Centre de recherche en canc�erologie de Toulouse (CRCT), Universit�e de Tou-louse, INSERM, UPS, France. 4Laboratoire Nutergia, Capdenac, France.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

B. Pipy and A. Coste share senior authorship of this article.

M.A. Eddine and M. Prat contributed equally to this article.

Corresponding Author: Agn�es Coste, Paul Sabatier University/IRD, Toulouse31432, France. Phone: 33-5-61-32-29-67; Fax: 33-5-61-32-22-93; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-18-0213

�2019 American Association for Cancer Research.

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cell–mediated tumor cell killing (16). Thus, myeloid CLRs haverelevant and divergent roles in tumor development.

Because the IL13-activated macrophage subset is characterizedby abundant amounts of CLRs (4, 8), they can recognize tumorcells and activate signaling pathways leading to antitumor func-tions. Here, we report that IL13 exhibited antitumor activityagainst T-cell lymphoma and ovarian adenocarcinoma throughreactive oxygen species (ROS) release and arginase-inducedL-arginine depletion.We also showed that the antitumor responseof IL13-activated macrophages was dependent upon the recog-nition of sialic acid epitopes on the surface of tumor cells by MRand dectin-1. Thus, these results, which identify these CLRs as amajor component of the antitumor response of macrophages,represent an attractive target with which to promote tumoricidalactivity of macrophages and may offer new insight into TAM-targeting anticancer treatment.

Materials and MethodsCell culture

A murine T-lymphoma cell line (EL4-luc2) was purchasedfrom Caliper in 2014 (Caliper Life Sciences). Human T-cellleukemia cells (Jurkat) were provided by Professor B. Segui in2014 (CRCT, Toulouse, France). Murine and human ovarianadenocarcinoma cells (ID8, Skov-3) and human breast adeno-carcinoma cells (MDA-MB-231), provided by Professor B.Couderc in 2015 (CRCT, Toulouse, France), were transfectedwith a vector expressing firefly luciferase (17) for biolumines-cence quantification. Cells were maintained under limitedpassage from original stocks (typically under 10). All cell lineswere tested negative for Mycoplasma (PlasmoTest; Invivogen).

MiceAll mouse experiments were performed according to proto-

cols approved by the ethic committee for animal experimenta-tion of Midi-Pyr�en�ees, France (Experimentation permit number31-067) in accordance with the European legal and institutionalguidelines (2010/63/UE) for the care and use of laboratoryanimals (approval number C 31 555 03). Il4ra�/�, Tnf�/�,Stat6�/�, and their corresponding controls were purchased fromThe Jackson Laboratory or Janvier Labs. Clec7a�/�, Mrc1�/�, andPparg�/� mice have been described earlier, and the correspond-ing floxed littermates were used as controls throughout all theexperiments (18, 19).

Preparation of mouse resident peritoneal macrophagesResident peritoneal cells were harvested from the murine

peritoneal cavity with NaCl 0.9%. Collected cells were centrifug-ed, and cell pellet was suspended in DMEM supplemented withL-glutamine, penicillin, streptomycin, and 5% heat-inactivatedfetal calf serum. Cells were allowed to adhere for 2 hours at 37�Cand 5% CO2. Nonadherent cells were then removed by severalwashing with PBS. After 2 hours of adhesion, >95% of adherentcells were macrophages as determined by F4/80 staining. The5% of contaminating cells are B cells as determined by CD19staining. Adherent murine peritoneal macrophages were pre-treated or not 24 hours with IL13 (Clinisciences, 50 ng/mL), IL4(Clinisciences, 50 ng/mL), IFNg (Invitrogen, 50 ng/mL), TNFa(Invitrogen, 50 ng/mL), IL10 (PeproTech, 50 ng/mL), IL6(PeproTech, 50 ng/mL), or LPS (Sigma-Aldrich, 100 ng/mL)before tumor cell challenge. Thirty minutes before tumor cell

challenge and during the coculture period with tumor cells,macrophages were treated or not with Z-VAD-FMK (Z-vad; Cal-biochem, 50 mmol/L), necrostatin-1 (Sigma, 1 mmol/L), N-acetylcysteine (NAC; Sigma, 5 mmol/L), L(þ)-arginine (BDH ProLabo,1 mmol/L), sialic acid (Sigma, 100 mg/mL), Lac-Nac (Sigma,0.0001–100 mg/mL), Sia-Lac-Nac (Sigma, 0.0001–100 mg/mL),Bay 61-3606 (Sigma, 10 mmol/L), MAFP (Sigma, 10 mmol/L), or15(S)-HETE (Cayman, 1 mmol/L). For all in vitro experiments,the tumor cell-to-macrophage ratio used was 1:10.

Preparation of human monocyte-derived macrophages(MDMs) and si-RNA gene silencing

Monocytes were obtained from healthy blood donors(Etablissement Francais de Sang, EFS).Written informed consentswere obtained from the donors under EFS contract number21/PVNT/TOU/IPBS01/2009-0052. According to articles L1243-4and R1243-61 of the French Public Health Code, the contractwas approved by the French Ministry of Science and Technology(agreement number AC 2009-921).

Monocytes were differentiated into IL13-MDMs by treatmentfor 72 hours with IL13 (Clinisciences, 50 ng/mL). To silencedectin-1 and MR into MDMs, lipid-based siRNA-mediated genesilencing was performed as previously described (19). Briefly,siRNA/lipid complexes are added dropwise onto MDMs. Eachwell contains a siRNA final concentration of 200 nmol/L and3.0% (vol/vol) of HiPerFect transfection reagent (Qiagen). Assoon as the complexes were added to all wells, the plates wereincubated at 37�C and 5% CO2 for 6 hours. After transfectiontime, the reactions were stopped by adding medium with thepresence of IL13 (50 ng/mL).

Extraction of T cells and quantification of extracellularmembrane carbohydrates

Mouse T cells were extracted from spleen using a negativeselection Mouse T-cell enrichment kit (Stemcell), as recom-mended by the manufacturer's protocol.

To analyze carbohydrate composition at the surface of T lym-phocytes and tumor cells, the plasma membrane of cells wasrecovered from total cell lysate by performing several centrifuga-tions steps.

Cell envelops were suspended in 200 mL buffer and 10 mL ofPNGase solution (50 U/100 mL water) were added. The incuba-tion occurred at 37�C for 3 hours. The cell envelops were thenremoved by centrifugation at 3500 rpm for 10 minutes. Super-natant was freeze-dried and submitted to a 3N MeOH/HCl(Aldrich) hydrolysis (100 mL at 80�C for 1 hour). Fatty acids werethen extracted from the medium with chloroform (100 mL).Saccharide acetylationwas performedwith 50mL acetic anhydride(Fl€uka) and 5 mL pyridine (Fl€uka). After complete evaporation,the sample was dissolved in acetonitrile (50 mL). Various mono-saccharide standards have also been prepared using the sameprotocol. All the samples were analyzed by GC-MS in order tocompare their spectra and retention times. GC-MS analyses wereperformed with a 6890N GC interfaced with 5973 MSD by usingan HP5-MS capillary column (25 m length 0.25 mm externaldiameter and 0.25 mm internal diameter). The oven program was70�C for 2 minutes, then a ramp to 300�C in 57 minutes, 10minutes at 300�C. The scanning masses ranged fromm/z 50 amutom/z 650 amu. The on-column injected volume was 0.1 mL. Thesource temperature was 230�C, the quadripole temperature set at150�C, the He flow was 0.32 m/s. Software was Chemstation.

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Quantification of tumor cell number and bindingTo evaluate tumor cell number, the pretreated or untreated

macrophages were challenged with tumor cells for 72 hours. Toevaluate the involvement of cell contact, macrophages wereseparated from EL4-luc2 cells by a transwell insert (0.4 mm poresize). For binding assays, macrophages were challenged withtumor cells for 2 hours at 4�C. Before challenge, tumor cells weretreated or not with recombinant neuraminidase A (Sigma). Non-fixed tumor cells were removed bywashing. The luciferase activitywas measured using D-luciferin (Caliper, 90 mg/mL). The bindingof EL4 tumor cells and normal T lymphocytes with untreated orIL13-treatedmacrophages was also evaluated by confocal micros-copy after CFSE labeling (Confocal ZEISS LSM780) of EL4 or Tcells. ID8-luc cell death was evaluated with the LDH cytotoxicityassay Kit (Roche).

Metabolic assays, cytokine, 15-HETE, andmRNAquantificationIL13-treated or untreated macrophages were challenged with

tumor cells for 48 hours. The arginase activity and nitrite releasewere measured as described previously (20). The arginase activitywas evaluated in cell lysates after incubation with L-arginine(0.5 mol/L), and then the urea concentration was measured at540 nm after addition of a-isonitrosopropiophenone. For nitriterelease, Griess reagent was used to quantify the concentration ofnitrite, which is a stable product of NO. The ROS production ofmacrophages was measured by chemiluminescence in the pres-ence of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol)using a thermostatically (37�C) controlled luminometer(Envision, PerkinElmer). The ROS production was monitoredfor 90 minutes (19). The cytokine releases of macrophages wereevaluated by ELISA (BD Biosciences). The 15-HETE productionof macrophages challenged with EL4-luc2 for 2 hours wasmeasured by EIA (15(S)-HETE EIA kit; Cayman).

Real-time-qPCR was performed on a LightCycler 480 systemusing LightCycler SYBR Green I Master (Roche Diagnostics). Actb(b-Actin)mRNAwas used as the invariant control. Serially dilutedsamples of pooled cDNA were used as external standards in eachrun for the quantification. Primer sequences are listed in Supple-mentary Table S1.

Flow cytometryFor cell-cycle assay, after challenge with macrophages, EL4-luc2

cells were stained with propidium iodide (PI, BD Biosciences,25mg/mL). For cell death, EL4-luc2 cellswere stainedwithAnnexinV-FITC and propidium iodide (Annexin V: FITC ApoptosisDetection Kit I, BD Biosciences). For cell proliferation, EL4-luc2cells were stained with CFSE (BD Biosciences, 10 mmol/L).

IL13-treated or untreated macrophages were challengedwith EL4-luc2 cells for 48 hours. Dectin-1, CD36, and MR weredetected, respectively, using FITC-dectin-1 mAb (Serotec),PE-CD36 mAb (Santa Cruz), or FITC-mannosylated bovineserum albumin (Sigma; ref. 21).

For immune cell infiltration and activation in ascites fromuntreated or IL13-treated EL4 tumor–bearingmice, peritoneal cellswere labeledwith the followingantibodies: F4/80-APC,MHCII-PE,CD3-PE, CD4-VioBrightFITC, CD8-VioGreen, CD25-PEVio770,NK1.1-PerCPVio700, CD19-APCVio700, CD69-PEVio770, andCD183-APC (Miltenyi Biotec).

All staining was performed in PBS supplemented with 1% fetalcalf serum. Percentages of positively labeled cells for each markerwere comparedwith an irrelevant appropriate isotype control. The

FACS analyses were done in a Becton Dickinson LSRFortessa flowcytometer with the BD FACSDiva software.

Western blot analysisIL13-treated or untreated macrophages were challenged with

EL4-luc2 cells for 48 hours. After total protein lysates extractionand transfer, membranes were incubated with a rabbit polyclonalantiphospho p47phox (Assay biotech) or a goat anti-b-actin (C11,Santa Cruz) antibodies and then with a peroxidase conjugatedsecondary antibody. The proteins were visualized with the West-ern Bright ECL (Advansta).

Arachidonic acid mobilizationAdherent peritonealmacrophageswere treated or notwith IL13

and labeled with [3H] arachidonic acid (1 mCi/well, PerkinElmer)for 24 hours. The labeled macrophages were then challenged ornot with EL4-luc2 cells for 2 hours. The [3H] arachidonic acidcontent in supernatants was quantified by measurement of theradioactivity by beta liquid scintillation counting with a 1217Wallac Rackbeta LKB 1217.

In vivo and ex vivo experimentsEL4-luc2 (1 � 106 cells/mouse) or ID8-luc (5 � 106 cells/

mouse) cells were administered intraperitoneally (i.p.) inC57BL/6, Clec7a�/� or Mrc1�/� mice (n ¼ 10 per group).For IL13 or IL4 treatments, injections of 4 mg/mouse wereperformed 3 days after tumor cell injection and then every3 days for EL4-luc2 and every 7 days for ID8-luc2. Controlgroups received saline solution. For Liposomal Clodronate(500 mg/10 g of mouse), i.p. injections were performed 1 daybefore tumor cell injection and then every 3 days. Controlgroups received control Liposome.

For adoptive transfers, peritoneal macrophages from threeNaCl or IL13 donor mice (6 � 106 macrophages) were collected,labeled with CellTrace Violet dye (Thermo Fisher Scientific), andtransferred (i.p.) into the recipient mouse (n ¼ 6) 3 days afterEL4-luc2 injection and then every 2 days. Donor mice wereinjected with IL13 or NaCl (control) 24 hours beforemacrophagecollection.

At 12 days after EL4-luc2 cells injection and 35 days afterID8-luc injection, mice were euthanized using CO2 asphyxia.Peritoneal cells were removed aseptically and the supernatants(ascite fluid) were collected for ELISA cytokine titration. Theluciferase activity was measured on peritoneal cell samplesand on each organ (spleen, lungs, and lymph nodes) with aluminometer (Envision, PerkinElmer). For in vivo ImagingSystem (IVIS), C57BL/6 mice were injected i.p. with EL4-luc2cells (1�106 cells/mouse) and treated or not i.p. with IL13(n ¼ 10 per group). At 3, 5, 8, and 12 days after EL4-luc2 cellinjection, mice were anesthetized by isoflurane and admin-istrated with luciferin into the peritoneal cavity (150 mg/g bodyweight). Mice were imaged, 6 minutes after luciferin injectionwith 30 seconds exposure length, using the in vivo imagingsystem (IVIS, PerkinElmer). The luminescent images were ana-lyzed using Living Image4.4 software. For ID8-tumor–bearingmice, tumor progression was evaluated by quantification oftumor burden in abdomen using the in vivo imaging system, byweight gain and ascites volume.

ROS production, arginase activity, and real-time PCR werestudied after macrophage adherence. To evaluate killing activityof macrophages, they were cocultured 72 hours with EL4-luc2cells (luciferase activity).

IL13 Induces Macrophage Antitumor Phenotype through CLRs

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Statistical analysisAll the results correspond to mean � SEM of triplicates and

are representative of at least three independent experiments. Foreach experiment, the data were subjected to one-way analysis ofvariance followed by the means multiple comparison methodof Bonferroni–Dunnett using Statview software. For the survivalstudy, statistical significance was determined by a log-rank test.P < 0.05 was considered statistically significant.

ResultsIL13-activated macrophages promote EL4 tumor cell death bynecrosis

Although IL6, a Th2 cytokine, significantly promoted protumoractivity of macrophages, IL13- and IL4-activated macrophagesinhibited EL4 tumor cell number similarly to Th1-driven macro-phages (IFNg/LPS- or TNFa-stimulated macrophages; Fig. 1A).

Given that IL13 did not directly alter EL4 cell number, death,cycle, and index of proliferation (Supplementary Fig. S1A–D), we

confirmed that IL13 antitumor activity is mediated throughmacrophages. Consistently, the genetic deletion of IL13 recep-tor subunit (Il4ra) in macrophages or of Stat6, a main factorinvolved in IL13 receptor–coupled signaling, totally abolishedIL13-activated macrophages tumoricidal activity (Supplemen-tary Fig. S1E).

After 24 hours of coculture with IL13-activated macrophages,EL4 cell number diminished over time (Fig. 1B), suggesting thatIL13-activated macrophages exert a cytotoxic activity. Consistent-ly, IL13-activated macrophages led to a significant arrest of EL4proliferation following 48 hours of coculture (Fig. 1C). After 48hours of coculture with IL13-activated macrophages, EL4 cellfraction in G0–G1, S, and G2–M phases decreased (Fig. 1D). Thiswas accompanied by a corresponding increase in EL4 cell fractionin the sub-G0–G1 phase. The surviving cell population decreasedover time, demonstrating that IL13-activated macrophages causecytotoxicity in EL4 cells without cell-cycle arrest. As depictedin Fig. 1E, the increase of the percentage of cells stained withboth Annexin V/PI and IP alone following 48 and 72 hours of

Figure 1.

IL13-activatedmacrophages induce EL4 tumor cell death by necrosis. A, Number of EL4-luc2 cells cultured in the presence (black bar) or not (white bar) ofcytokine-treated macrophages. B–E, Number (B), proliferation assay (C), cell cycle (D), and cell death (E) of EL4-luc2 cells cultured in the presence of untreatedor IL13-treatedmacrophages (Mf). � , P < 0.05; �� , P < 0.01 compared with the respective untreatedmacrophages; $$, P < 0.01 compared with the IFNg-treatedmacrophages; and #, P < 0.05; ##, P < 0.01 compared with the initial EL4-luc2 cell number (T0).

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coculture with IL13-activated macrophages and the absence ofincrease of cells stained with Annexin V alone indicated that EL4cell death is mediated by necrosis. Consistently, the use of Z-vadcaspase inhibitor did not alter the reduced EL4 cell numberinduced by IL13-activated macrophages nor the percentage ofEL4 cells stained with Annexin V/PI (Supplementary Fig. S1F andS1G). Necrostatin-1, a selective inhibitor of necroptosis, did notaffect the reduced EL4 cell number (Supplementary Fig. S1H).Altogether, these results demonstrated that IL13-activated mac-rophage-induced cytotoxicity in EL4 cells is mediated by necrosisthrough the caspase-independent cell death pathway.

IL13-activated macrophage tumoricidal effects depend on ROSand arginase

The use of transwell insert culture dish (Fig. 2A) demonstratedthat physical contact between IL13-activated macrophages andEL4 tumor cells is critical to generate soluble factor(s) contribut-ing to the bystander tumoricidal effect of IL13-stimulated macro-

phages. EL4 cell number in the insert was decreased only whenIL13-stimulated macrophages were simultaneously seeded withEL4 cells in companion plates (Fig. 2B).

EL4 cell physical contact with IL13-activated macrophagesrobustly amplified the expression of standard IL13 activationmarkers such as YM1 (Chi3l3), arginase-1 (Arg1), PPAR gamma(Pparg), Cd36, MR (Mrc1), and dectin-1 (Clec7a; Supplemen-tary Fig. S2A and S2B). Concomitantly, EL4 cells also triggeredNcf1 (p47phox) expression, a cytosolic subunit of NADPHoxidase complex, phosphorylation of which is essential forROS production and IL12, TNFa, and IL1b release (Supple-mentary Fig. S2A and S2C). Thus, EL4 tumor cells constituteda second signal to shift IL13-primed macrophages towardcytotoxic phenotype characterized by an induction of proin-flammatory markers and a CLR signature composed of dectin-1and MR.

The use of TNFa-deficient (Tnf�/�) macrophages showedthat this cytokine was not involved in cytotoxic activity of

Figure 2.

ROS release and arginase areinvolved in the tumoricidal effect ofIL13-activatedmacrophages.A, Schema of the transwell culturesystem. B, Number of EL4-luc2 cellsin insert in the presence ofuntreated or IL13-treatedmacrophages cultured or not withEL4-luc2 cells in 24-wellcompanion. C,Number of EL4-luc2cells cultured with untreated orIL13-treated macrophages fromTnfþ/þ and Tnf�/�mice. D–G,Nitrite release (D), ROS production(E), phosphorylated P47phox (F),and arginase activity (G) ofuntreated or IL13-treatedmacrophages in the presence orabsence of EL4-luc2 cells. H and I,Number (H) and cell death (I) ofEL4-luc2 cultured withmacrophages stimulated or notwith IL13, treated or not with NACand/or L-arginine. � , P < 0.05;�� , P < 0.01 compared withrespective untreated macrophagesand #, P < 0.05; ##, P < 0.01compared with IL13-treatedmacrophages without EL4 cells orto IL13-treatedmacrophageswithout NAC and L-arginine.






IL13-activated macrophages (Fig. 2C). Although NO secretionremained unchanged in IL13-activated macrophages stimulatedby EL4 cells (Fig. 2D), thesemacrophages released ahigher level ofROS (Fig. 2E). In line with this, phosphorylated p47phox wasincreased (Fig. 2F). Consistent with increased Arg1mRNA expres-sion in IL13-activatedmacrophages challenged by EL4 cells (Sup-plementary Fig. S2A), arginase activity was significantly higher(Fig. 2G). Finally, the use of the ROS scavenger NAC alone or thesingle supplementation of L-arginine in the medium partly inhi-bits the EL4 cell death induced by IL13-activated macrophages,demonstrating that their cytotoxic activitywas dependent both onROS and L-arginine amounts. The combination of NAC and oflarge amount of L-arginine in the medium completely abolishedthe tumoricidal response of IL13-stimulated macrophages, dem-onstrating that ROS release and the arginase activity-induced

L-arginine deprivation are sufficient for their cytotoxicity(Fig. 2H and I).

Sialic acid binding through MR and dectin-1 is critical fortumoricidal activity

Because malignant transformation often correlates withaltered glycosylation profile on tumor cell surface, we analyzedby GC/MS the sugar composition of normal lymphocytesand EL4 tumor cells (Fig. 3A; Supplementary Fig. S4A). Wedemonstrated a difference in the sugar composition betweennormal and abnormal T cells, due to the amino sugars andnot to the usual hexoses and corresponding deoxyhexoses(Fig. 3A; Supplementary Fig. S4A). EL4 tumor cells exhibitedhigher amounts of sialic acid (NAc neuraminic acid) andNAc-Galactosamine than normal T cells.

Figure 3.

The tumoricidal effect of IL13-activated macrophages requires the tumoral sialic acid interaction with MR and dectin-1. A, Chromatograms specific for acetylatedandmethylated amino-sugars or acetylated andmethylated sugars at the surface of T lymphocytes and EL4 cells. B, Binding of EL4-luc2 cells, pretreated or notby neuraminidase (NA) or with sialic acid (SA) on untreated or IL13-treated macrophages. C,Gene-expression analysis of CLRs and Siglec-1 receptor in untreatedor IL13-treatedmacrophages in the presence of EL4-luc2 cells. D, Binding of EL4-luc2 cells on untreated or IL13-treatedMrc1�/� or Clec7a�/�macrophages. E andF, Number (E) and cell death (F) of EL4-luc2 cultured with untreated or IL13-treatedMrc1�/� or Clec7a�/�macrophages. G and H, ROS production (G) andarginase activity (H) of untreated or IL13-treatedMrc1�/� or Clec7a�/�macrophages in response to EL4-luc2 cells. I, Schema of coculture system and number ofEL4-luc2 cells in insert, in the presence of untreated or IL13-treatedMrc1�/� or Clec7a�/�macrophages, cultured with EL4-luc2 cells or sialic acid in 24-wellcompanion. �� , P < 0.01 compared with respective untreated macrophages and ##, P < 0.01 compared with IL13-treated macrophages without NA, SA, and EL4cells or to IL13-treatedWT littermate.

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IL13-activated macrophages had a greater ability to bind EL4tumor cells (Fig. 3B). The pretreatment of EL4 tumor cells withneuraminidase, which cleaves the glycosidic linkage of sialic acid(Supplementary Fig. S4B), or the preincubation of IL13-activatedmacrophages with neuraminic acid, completely abolished theircapacity to interact with EL4 tumor cells (Fig. 3B). Consistently,this binding is abrogated, in a dose-dependent manner, whenIL13-activated macrophages were preincubated with sialylatedLacNac, whereas it was not altered by the presence of nonsialy-lated LacNac (Fig. 3B; Supplementary Fig. S4C).Wedemonstratedthat the normal T cells, which contain lower amounts of sialic acidat their surface, did not bind IL13-activated macrophages (Sup-plementary Fig. S4), highlighting sialic acid at the surface of EL4cells as the critical epitope responsible for the physical interactionwith IL13-activated macrophages.

The expression of siglec-1, well known for its specificity forsialic acid–containing glycans, and of CLRs, such as SIGNR-1(Cd209b), SIGNR-3 (Cd209d), MGL (Clec10a), and Mincle(Clec4e), was not changed in IL13-activated macrophages in thepresence of EL4 tumor cells (Fig. 3C). Only MR and dectin-1mRNA expression was significantly increased (Fig. 3C). Consis-tently, deficiencies for MR (Mrc1�/�) or dectin-1 (Clec7a�/�) inIL13-activated macrophages resulted in a complete loss of theircapacity to interact with EL4 cells (Fig. 3D), supporting the role ofthese CLRs in the physical interaction of IL13-activated macro-phages with EL4 tumor cells.

Targeted deficiencies forMRor dectin-1 led to the complete lossof tumoricidal activity of IL13-activatedmacrophages (Fig. 3E andF). In line with this, the induction of ROS release and arginaseactivity in IL13-activated macrophages challenged with EL4 cellswere affected by the absence of eitherMrc1 or Clec7a (Fig. 3G andH). The robust induction of standard IL13 activation markers,Ncf1, and cytokine production in IL13-activated Mrc1þ/þ orClec7aþ/þ macrophages was significantly diminished inMrc1�/� or Clec7a�/� macrophages (Supplementary Fig. S5A–C).

We then determined whether the interaction between IL13-activated macrophages and EL4 cells occurs through the bindingof tumoral sialic acid on MR/dectin-1 macrophage receptors(Fig. 3I). Similarly to EL4 cells, the sialic acid increased tumor-icidal activity of IL13-activated Mrc1þ/þ and Clec7aþ/þ macro-phages, as reflected by the decrease of EL4 cell number in insert,whereas this tumoricidal activity was lost in IL13-activatedMrc1�/� or Clec7a�/� macrophages (Fig. 3I). Altogether, thesedata reveal that the antitumor response of IL13-activated macro-phages requires a physical contact depending on the recognitionof tumor sialic acid epitope by MR and dectin-1 receptors.

Cytotoxic function is induced by Syk/P47phox signaling andAA-HETE-PPARg

Wenext dissected the signalingdownstreamofMRanddectin-1involved in acquisition of cytotoxic functions by IL13-stimulatedmacrophages against EL4 tumor cells. The use of a selective Sykinhibitor Bay 61-3606 (Bay) revealed that IL13-activated macro-phages cytotoxic activity was partially dependent on Syk (Fig. 4A).We showed that Bay abolishedROSproduction in response toEL4cells (Fig. 4B) and did not affect arginase activity induction(Fig. 4C). The amount of phosphorylated p47phox in IL13-activated Mrc1�/� or Clec7a�/� macrophages was decreased(Fig. 4D). These data established a direct link between theSyk-coupled MR/dectin-1 signaling pathway, p47phox phosphor-ylation, and ROS release.

A direct link between MR/dectin-1 and arachidonic acid (AA)metabolism to trigger macrophage cytotoxic pathways was pre-viously established (19).We demonstrated that in the presence ofMAFP, an inhibitor of AA generation, IL13-activatedmacrophagesantitumor activity was partly affected (Fig. 4A) and the inductionof arginase was abolished (Fig. 4C). Although EL4 cell challengeresulted in the induction of AA release in IL13-activated Mrc1þ/þ

or Clec7aþ/þ macrophages, it failed to do so in IL13-activatedMrc1�/� or Clec7a�/� macrophages (Fig. 4E), demonstrating thatAA-coupledMR/dectin-1 signaling is essential for the induction ofarginase activity.

To explore the link between AA metabolism and arginaseactivity induction, we evaluated the implication of PPARg , knownto directly control arginase-1 expression (19), and to be activatedby ligands derived from AA metabolism. The impaired inductionof arginase activity in IL13-activated macrophages deficient forPPARg (Pparg�/�; Fig. 4F) and the subsequent decrease of theircytotoxic activity (Fig. 4G) support the existence of a PPARg-dependent mechanism in arginase activity amplification.

PPARg is activated by endogenous ligands such as 15-deoxy-D12,14PGJ2, metabolized through the cyclooxygenases and PGDsynthase (Pgd2s), and the 12- and 15-hydroxyeicosatrienoic acids(HETE), metabolized through 12/15 lipoxygenases (Alox15;ref. 22). Although Pgd2s mRNA was not differentially expressedin IL13-activated macrophages upon EL4 challenge, Alox15 geneexpression was significantly increased (Fig. 4H). Accordingly,15-HETE production was enhanced in IL13-activated macro-phages challenged by EL4 cells (Fig. 4I). This increased 15-HETEproduction was completely lost in IL13-activated Mrc1�/� orClec7a�/� macrophages (Fig. 4I), indicating that EL4 tumor cellchallenge with IL13-activated macrophages drives the generationof 15-HETE metabolites through the AA-coupled MR/Dectin-1signaling pathway.

Finally, the addition of exogenous 15-HETE restored arginaseactivity defect (Fig. 4J) and decreased EL4 cell number (Fig. 4K)in IL13-activated Mrc1�/� or Clec7a�/� macrophages. In IL13-activated Pparg�/� macrophages, arginase activity and EL4 cellnumber was not affected by 15-HETE treatment (Fig. 4J and K),demonstrating that PPARg is required for 15-HETE–mediatedarginase and cytotoxic activities. Altogether, these results dem-onstrate that arginase activity induction in IL13-activated macro-phages in response to EL4 tumor cells is dependent on theMR-dectin-1/AA/15-HETE/PPARg axis.

In vivo IL13 treatment promotes antitumor properties ofmacrophages

Wenext showed that in vivo IL13 treatment significantly extend-ed the survival of EL4 tumor–bearing mice (Fig. 5A). We ascribedthe late mortality of IL13-treated mice to the decreased tumorburden in the abdomen, the lymph nodes, and spleen (Fig. 5B–D). Consistently, the ascites-dependent weight gain was signifi-cantly decreased (Fig. 5C), highlighting a potent role of IL13 in theinhibition of tumor development. Accordingly, compared withperitonealmacrophages fromuntreated EL4 tumor–bearingmice,macrophages from tumor ascites of IL13-treated mice showed animprovement in their ability to kill EL4 cells (Fig. 5E), to produceROS (Fig. 5F) and presented a higher arginase activity (Fig. 5G).

The in vivo treatment with IL4, a Th2 cytokine that shares IL13signaling, increased the macrophage ability to kill EL4 cells (Sup-plementary Fig. S6B), to produce ROS (Supplementary Fig. S6C),and to exert a higher arginase activity (Supplementary Fig. S6D).






In line with these results, IL4 in vivo treatment decreased ascites-tumor burden in IL4-treated EL4 tumor–bearing mice (Supple-mentary Fig. S6A). These data confirmed that IL4 and IL13treatments exhibit similar effects in the orientation of macro-phages toward a cytotoxic and tumoricidal phenotype and onlymphoma progression.

To further explore whether IL13 treatment affects TAMs phe-notype, we first characterized peritoneal macrophages of EL4tumor–bearing mice. As expected, EL4 tumor switches peritonealmacrophage phenotype toward tumor permissive status, charac-terized by an upregulation of M2 markers and an increase ofimmune tolerance factors such as Il10, Il6, and Ido2 and ofangiogenesis such as Tgfb1 (Supplementary Fig. S6E). Thesemacrophages promoted EL4 cell growth and were less efficientto produce ROS (Supplementary Fig. S6F and S6G). Upon IL13treatment, macrophages from non–tumor-bearing mice pre-

sented an induction of standard IL13 activation markers, suchas Chi3l3, Arg1, Pparg, Cd36, Mrc1, and Clec7a. This IL13-specificpolarization signature was amplified in IL13-treated tumor-bearing mice (Supplementary Fig. S6H). Whereas the mRNAexpression of Tnf and Il1b was decreased by IL13 treatment inmacrophages from non–tumor-bearing mice, these genes wereinduced in macrophages from IL13-treated tumor-bearing mice.Ncf1 expression was increased by IL13 treatment only in macro-phages from tumor ascites. Except for Il10, of which expression isdecreased by IL13 treatment only in macrophages from tumor-bearingmice, IL13 decreased the expression of immune toleranceand angiogenesis factors (Il6, Tgfb1, and Ido2) both in non–tumorand tumor-bearing mice (Supplementary Fig. S6H). Supportingthese findings, we also observed that IL12, TNFa, and IL1bamounts in tumor ascites were elevated, whereas IL10 and IL6concentrations were significantly reduced (Supplementary

Figure 4.

MR and dectin-1 induce cytotoxic functions via Syk/p47phox and AA-HETE-PPARg axis. A, EL4-luc2 cell number cultured with macrophages stimulated or not withIL13, treated or not with Bay 61-3606 and/or MAFP. B and C, ROS production (B) and arginase activity (C) of macrophages stimulated or not with IL13, treated ornot with Bay 61-3606 and/or MAFP, in response to EL4-luc2 cells. D, E, Phosphorylated P47phox (D) and [3H]Arachidonic acid release (E) of untreated and IL13-treatedMrc1�/� or Clec7a�/�macrophages in the presence of EL4-luc2 cells. F, Arginase activity of untreated and IL13-treated Pparg�/�macrophages inresponse to EL4-luc2 cells. G, Number of EL4-luc2 cells cultured in the presence of untreated and IL13-treated Pparg�/�macrophages. H, Prostaglandin D2synthase (Pgd2s) and 12/15-lipoxygenase (Alox15) gene-expression analysis in untreated and IL13-treated macrophages in response to EL4-luc2 cells. I, 15-HETEproduction of untreated and IL13-treatedMrc1�/� or Clec7a�/�macrophages in response to EL4-luc2 cells. J, arginase activity of IL13-treatedMrc1�/�, Clec7a�/�,or Pparg�/�macrophages in response to EL4-luc2 cells, in the presence or absence of 15-HETE. K, Number of EL4-luc2 cells cultured in the presence ofIL13-treatedMrc1�/�, Clec7a�/�, or Pparg�/�macrophages, in the presence or absence of 15-HETE. �, P < 0.05; �� , P < 0.01 compared with respective untreatedmacrophages and ##, P < 0.01 compared with IL13-treated macrophages without Bay, MAFP, and EL4 cells or to IL13-treatedWT littermate.

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Fig. S6I). Altogether, these data demonstrate that IL13 in vivotreatment is associated with a macrophage phenotype character-ized by a decrease of immune tolerance and angiogenic factors, aninduction of proinflammatory markers and a Dectin-1 and MRsignature.

The capacity of IL13 treatment to orient immunosuppressiveand protumor macrophages toward tumoricidal phenotype wasreinforced by the capacity of in vitro IL13 treatment to reverse theprotumor macrophages from EL4 tumor–bearing mice towardmacrophages that inhibit EL4 cell growth (Supplementary Fig.S6J). To establish that IL13 antitumor activity is dependent onmacrophages, we performed adoptive transfers of macrophagesfrom IL13-treated mice into EL4 tumor–bearing mice. Micereceiving IL13-activated macrophages showed a significantdecrease of the tumor burden in tumor ascites, lymph nodes,spleen, and lungs, as compared with mice receiving nonactivatedmacrophages (Fig. 5H and I). Consistently, macrophages frommice receiving IL13-activated macrophages showed an improve-ment in their ability to kill EL4 tumor cells (Fig. 5J) through anincrease of their capacity to release ROS (Fig. 5K) and to exertarginase activity (Fig. 5L). The phenotypic characterization oftransferred macrophages (F4/80þ MHC IIþ Cell traceþ) and host

macrophages (F4/80þ MHC IIþ Cell trace�) demonstrated thatthese two populations share the same pattern of gene expression(Supplementary Fig. S7A), suggesting a transfer of phenotypefrom transferred macrophages toward host macrophages.

Themacrophage depletion by i.p. administration of clodronateinhibited IL13-induced antitumor activities in tumor ascites,lymph nodes, spleen, and lungs (Supplementary Fig. S7B–D),reinforcing the involvement of macrophages in IL13 antitumoractivity.

We demonstrated that clodronate treatment alone was lessefficient in reducing tumor burden than macrophage conversionto an antitumor phenotype by IL13 treatment (SupplementaryFig. S7E), suggesting that the strategy to orient TAMs toward acytotoxic phenotype to reduce tumor progression is more prom-ising than their depletion. The decreased tumor burden in theperitoneum of IL13-treated mice was totally abolished in EL4tumor–bearing Mrc1�/� and Clec7a�/� mice (Fig. 5M). In linewith these results, the tumoricidal activity of macrophages fromEL4 tumor–bearing Mrc1�/� and Clec7a�/� mice was completelylost (Fig. 5N). These results support in vivo the critical role of MRand dectin-1 recognition processes on macrophages in IL13-mediated antitumor activity. IL13 administration to EL4

Figure 5.

In vivo IL13 treatment inhibits lymphoma development through the enhancement of macrophage antitumor properties.A–C, Survival (%; A), in vivo imaging (B),and weight gain (C) of untreated and IL13-treated EL4 tumor–bearing mice. D,Dissemination of EL4-luc2 cells in lymph nodes, spleen, and lungs. E–G, Killingactivity (E), ROS production (F), and arginase activity (G) of peritoneal macrophages from untreated and IL13-treated EL4 tumor–bearing mice. H and I, Numberof EL4-luc2 cell in the peritoneum (H), in lymph nodes, spleen, and lungs (I). J–L, Killing activity (J), ROS production (K), and arginase activity (L) of peritonealmacrophages collected from EL4 tumor–bearing mice injected with macrophages harvested from untreated C57BL/6 (MK C) or IL13-treated mice (MK IL13).M, EL4-luc2 cell number in the peritoneum of untreated and IL13-treated EL4 tumor–bearingMrc1�/� or Clec7a�/�mice. N, Killing activity of peritonealmacrophages against EL4 cells. � , P < 0.05 and �� , P < 0.01 compared with respective untreated EL4 tumor–bearing mice or to MK C, and ##, P < 0.01 comparedwith IL13-treatedWT littermate.






tumor–bearingmice did not affect the tumor adaptive immunity,because therewere no detected changes in the percentage of CD4þ

and CD8þ T cells, Tregs, B cells, and NK cells, or in the activationstate of T cells (Supplementary Fig. S8).

IL13-activated macrophages have tumoricidal activity againstID8 cells

To investigate whether IL13-activated macrophage-inducedcytotoxicity can be extended to another type of tumor, we eval-uated the impact of IL13 treatment on murine ovarian adeno-carcinoma cells (ID8). Although IL13 did not directly alter ID8cell number, IL13-activated macrophages significantly inhibitedtheir number over time (Fig. 6A andB). The increased LDH releaseby ID8 in the presence of IL13-activated macrophages suggestedthat the ID8 cell death is mediated by necrosis (Fig. 6C). Con-sistent with this finding, the use of Z-vad caspase inhibitor did notalter the reduced ID8 cell number induced by IL13-activatedmacrophages (Fig. 6D). IL13-activated macrophages released agreater amount of ROS (Fig. 6E) and exhibited significantly higherarginase activity after challenge with ID8 (Fig. 6F), suggesting thatROS and L-arginine are involved in their cytotoxic activity againstID8 cells.

ID8 tumor cells, which express great amounts of sialic acid(NeuNac) at their surface (Fig. 6G), interactedwith IL13-activatedmacrophages through sialic acid because the pretreatment of ID8

tumor cellswithneuraminidase completely abolished their capac-ity to bind with ID8 (Fig. 6H). IL13-activated macrophagesdeleted for MR or dectin-1 failed to bind ID8 cells (Fig. 6I). Theseresults demonstrate that the binding of sialic acid present at thesurface of ID8 tumor cells throughMR and dectin-1 is essential inthe tumoricidal activity of IL13-activated macrophages. In vivoIL13 treatment of ovarian adenocarcinoma-bearing micedecreased tumor burden in the abdomen and ascites-dependentweight gain, highlighting a potent role of IL13 in the inhibition ofovarian adenocarcinoma growth (Fig. 6J). Altogether, these datasupport the extension of cytotoxic activity of IL13-activatedmacrophages to various tumor cells.

IL13 promotes tumoricidal activity in human MDMsWe evaluated the impact of humanMDMs activated by IL13 on

human T-cell leukemia cells (Jurkat cell line). Although theexposure of Jurkat tumor cells with IL13 did not directly altertheir number (Fig. 7A), IL13-activated humanMDMs significantlydecreased Jurkat tumor cell number (Fig. 7B). The use of NAC andthe L-arginine medium supplementation demonstrated that thecytotoxic effect of IL13-activated human MDMs is completelydependent on ROS production and arginase activity (Fig. 7B).Consistently, IL13-activated human MDMs released largeamounts of ROS in response to Jurkat tumor cells (Fig. 7C), andtheir arginase activity was also significantly increased (Fig. 7D).

Figure 6.

IL13 promotes tumoricidal activity of macrophages against ovarian adenocarcinoma cells (ID8). A, ID8-luc cell number cultured in the presence or absence ofIL13-activatedmacrophages. B, ID8-luc cell number cultured with untreated or IL13-treated macrophages during 24, 48, or 72 hours. C, ID8-luc cell death in thepresence of untreated or IL13-treated macrophages. D, Number of ID8-luc cells in the presence of macrophages stimulated or not with IL13, treated or not withZ-vad. E–F, ROS production (E) arginase activity (F) of untreated or IL13-treated macrophages in the presence or absence of ID8-luc cells. G, Chromatogramspecific for acetylated andmethylated amino sugars at the surface of ID8-luc cells. Numbers are intensities�104. H, Binding of ID8-luc cells, pretreated or not byneuraminidase (NA), on untreated or IL13-treated macrophages. I, Binding of ID8-luc cells on untreated or IL13-treatedMrc1�/� or Clec7a�/�macrophages.J, In vivo imaging, weight gain, and ascites volume were determined in untreated and IL13-treated ID8 tumor–bearing mice. � , P < 0.05; �� , P < 0.01 comparedwith respective untreated macrophages and #, P < 0.05; ##, P < 0.01 compared with without macrophages, to IL13-treated macrophages without NA andID8 cells or to IL13-treatedWT littermate.

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Jurkat tumor cells contain high amounts of sialic acid at theirsurface (Fig. 7E), and the pretreatment with neuraminidasecompletely abolished their capacity to bind with IL13-activatedmacrophages (Fig. 7F). IL13-activated human MDMs lost theirtumoricidal activity against Jurkat tumor cells after silencing ofMR (Mrc1) or dectin-1 (Clec7a; Fig. 7G), underscoring the impor-tance of these CLRs on IL13-activated macrophages in the sialicacid recognition on human tumor cell and hence in the acqui-sition of antitumor phenotype.

To investigate whether IL13-activated human MDM-inducedcytotoxicity can extend on other tumor types, we evaluated theimpact of IL13 treatment on human breast adenocarcinoma(MDA-MB-231) cells. Similarly to Jurkat cells, the number of

MDA-MB-231 cells was significantly decreased by IL13-activatedhuman MDMs (Fig. 7H), which ROS amounts were induced by2-fold (Fig. 7I), and exhibited higher arginase activity in responseto MDA-MB-231 cells (Fig. 7J). These tumor cells interacted withIL13-activatedmacrophages through sialic acid (NeuNac; Fig. 7K)and expressed great amounts of this glycan epitope at their surface(Fig. 7L). Inversely, thedata on another tumor cell line, thehumanovarian carcinoma cells (Skov-3), revealed that antitumor activityof IL13-activated human MDMs is concealed by the direct pro-tumor effect of IL13 on these tumor cells (Fig. 7M). This directprotumor activity of IL13 on Skov-3 tumor cells is accompaniedby surface expression of IL13Ra2 (IL13RA2; Fig. 7N), a receptorknown topromote cancer invasion andproliferation (23, 24). The

Figure 7.

IL13 modulates human MDMs to exert antitumor activity against various types of tumor cells. A,Number of Jurkat-luc cells treated or not with IL13. B, Number ofJurkat-luc cells cultured in the presence of human MDMs stimulated or not with IL13 and treated or not with NAC and/or L-arginine. C–D, ROS production (C)arginase activity (D) of untreated or IL13-treated human MDMs challenged or not with Jurkat-luc cells. E, Chromatogram specific for acetylated andmethylatedamino sugars at the surface of Jurkat cells. Numbers are intensities�104. F, Binding of Jurkat-luc cells, pretreated or not by neuraminidase (NA), on untreated orIL13-treated human MDMs. G,Number of Jurkat-luc cells cultured in the presence of untreated or IL13-treated human MDMs transfected with siRNAsnontargeting (control) or targetingMRC1 or CLEC7a. ##, P < 0.01 of siRNA target compared with siRNA control. H, Number of MDA-MB-231-luc cells in thepresence or absence of human MDMs treated or not with IL13. I–J, ROS production (I) and arginase activity (J) of untreated or IL13-treated human MDMschallenged or not with MDA-MB-231-luc cells. K, Binding of MDA-MB-231-luc cells, pretreated or not by NA, on untreated or IL13-treated human MDMs. L,Chromatogram specific for acetylated andmethylated amino sugars at the surface of MDA-MB-231 cells. Numbers are intensities�104.M,Number of Skov-3-luccells in the presence or absence of human MDMs treated or not with IL13.N, IL13RA2mRNA expression in Jurkat-luc, Skov-3-luc, and MDA-MB-231-luc cells.� , P < 0.05; ��, P < 0.01 compared with respective untreated human MDMs or to untreated tumor cells and #, P < 0.05; ##, P < 0.01 compared withIL13-treated human MDMs without NAC, L-arginine, NA, Jurkat cells, and MDA-MB-231 cells or to respective IL13-treated tumor cells.






absence of the direct protumor activity of IL13 on Jurkat andMDA-MB-231 tumor cells is reinforced by the lack of IL13Ra2expression on their surfaces (Fig. 7N). Altogether, these resultsestablish that the macrophage antitumor effect mediated via therecognition of tumor sialic acid epitope by MR and dectin-1 canextend to various types of human tumors. They also highlight therestrictive use of IL13 to induce these CLRs due to the expressionof IL13Ra2 on some tumor cells.

DiscussionBecause TAMs are subject to local levels of many factors that

lead to protumorigenic macrophages, their education within thetumor toward tumoricidal macrophages appears to be a potentialstrategy for cancer therapy. Several lines of evidence suggest thatthe acquisition of cytotoxic functions inmacrophages depends onthe expression and activation of CLRs (16, 25), which are highlyexpressed in the Th2 microenvironment. In this context, wedetermined how IL13 modulates tumoricidal properties ofmacrophages during T-cell lymphoma and ovarian adenocarci-noma development. We demonstrated here that IL13 treatmentinhibits T-cell lymphoma and ovarian adenocarcinoma develop-ment in tumor-bearing mice through the activation of antitumorproperties of macrophages. This is consistent with a decrease ofthe tumor burden in tumor ascites, lymph nodes, spleen, andlungs after adoptive transfer of IL13-activated macrophages inEL4-bearing mice. Our results provide mechanistic insight intothe antitumor effect of IL13 and corroborate previous findingsshowing that IL13 reduced the tumorigenicity of B16F1 mel-anoma and MethA fibrosarcoma cells (26, 27). Local admin-istration of IL13 at the site of transplanted tumor cells in vivohad potent inhibitory effects on tumor growth, probably result-ing from pleiotropic effects, such as the recruitment of non-specific cells like monocytes, macrophages, and neutrophilsinto the tumor (26, 27). The advantageous effect of IL13 wassupported by an inverted correlation between the serum level ofIL13 and factors reflecting tumor progression (28).

The antitumor effect of IL13 is associated with a macrophagephenotype characterized by a significant decrease of immunetolerance and angiogenic factors. Results also demonstrate aninduction of IL13 standard markers, in particular Dectin-1, MR,and Arginase, associated with the expression of atypical markersof IL13 activation. Thus, the tumormicroenvironment can inducechanges in gene expression with the appearance of atypicalmarkers in macrophages, which phenotype has already beenoriented by IL13 treatment.

Despite the growing knowledge of the role of CLRs in pathogenelimination, little is known about their contribution to antitumorresponses. Here, we demonstrated that the tumoricidal propertiesof macrophages from IL13-treated EL4-bearing mice were abol-ished in mice deleted for MR or dectin-1, supporting the criticalrole of these receptors in IL13-mediated antitumor activity. IL13-activatedmacrophages are able to shift toward a tumoricidal stateupon the "triggering" signal provided by tumor cell recognitionthrough MR and dectin-1. We identified sialic acid as a criticalepitope at the surface of tumor cells responsible for their inter-actionwithMR and dectin-1, highlighting the glycan specificity ofthe cooperation between dectin-1 and MR for sialic acid. Thesedata support that heterodimeric complex formation among dif-ferent CLRs diversifies host PRR repertoire and hence expand theligand spectrum (29, 30).

The binding of tumor sialic acid through MR and dectin-1 isessential in the tumoricidal activity of IL13-activated macro-phages against tumor cells. Indeed, dectin-1 at the surface ofmacrophages is critical to recognize tumor cells that highly expressglycan structures and to activate NK-mediated tumor cell kill-ing (16). In line with this, the expression on tumor cells of ligandsfor the NKG2D receptor, a CLR expressed by NK cells, can alsoaugment T-cell responses, demonstrating that this CLR mediatesbeneficial immune responses against tumors (15). Here, thespecific contribution of dectin-1 and MR is reinforced by a lackof induction of other macrophage lectin-like receptors, such asDC-SIGN, MGL, Mincle, and Siglec-1, known to be involved inimmune escape (18). The identification of MR and dectin-1 asextracellular sensors for tumoricidal function promotion repre-sents amajor breakthrough in the host–tumor cell interaction andhost-mediated tumor cell elimination. Altogether, these datasupport that the CLRs on macrophages may be an adequatesystem to orchestrate an antitumor innate immune response forearly detection and removal of tumor cells with aberrant glyco-sylation, similar to their role in microbial glycan recognition andpathogen elimination.

The investigation of the signaling downstream of MR anddectin-1 identified ROS and arginase as factors involved in thecytotoxic effect of IL13-activated macrophages against tumorcells. Consistent with the involvement of MR and dectin-1 in theinduction of ROS production during infections through a signal-ing cascade requiring Syk (18, 19, 31), we demonstrated, in thecontext of the tumor, a direct link between the Syk-coupledMR/dectin-1 signaling pathway, p47phox phosphorylation, andROS release. We showed that the induction of arginase activityrequires MR/dectin-1–coupled AA mobilization, 15-HETE gener-ation, and the subsequent PPARg activation. This was supportedby the fact that the increase of CLRs favors arginase activity inresponse to parasites (25, 32, 33) and that MR/Dectin-1 triggersthe AA pathway during Leishmania infection (19).

This study also validated that IL13-activated human macro-phages display similar contributions against human T-cell leuke-mia cells.We revealed that IL13 antitumor effectiveness in humanmacrophages can extend to other tumor types as demonstrated ina human breast adenocarcinoma cell line. However, we alsoevidenced that the gene-expression signature of IL13Ra2 receptoron tumor cells, a receptor known to promote cancer invasion andproliferation (23, 24), is critical and determines the use and theefficacy of IL13.

The positive or negative roles of IL4 and IL13 cytokines intumor immunity are closely associated with their sources.Although endogenous IL4 and IL13 were reported to promotetumor growth, exogenous IL4 and IL13 delivered as recombinantprotein into the host often suppress tumor development (34, 35).Several studies of Th2 cytokine neutralization, using IL4 or IL13knockout mice or IL4 or IL13-specific antibodies, demonstrated adecrease of tumor growth and metastasis related to an enhancedTh1 response (34, 35). Coussens and colleagues demonstratedthat the significant diminution of mammary adenocarcinomametastasis in mice models of immune depletion of CD4þ T cellsor of IL4wasmediated by a suppression of protumor properties ofTAMs (36).However, several experiments using the inoculationofexogenous IL4 or IL13 showed these cytokines to be potentantitumor agents, as they induce tumor rejection in differenttypes of tumors (26, 34, 35). These tumor-inhibiting effects ofIL4 and IL13 were associated with the maturation of myeloid

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precursor cells toward inflammatory effectors and were alsodependent on tumor-infiltrating eosinophils (37). In addition,IL4-activated tumor-infiltrating dendritic cells were described topromote tumor-specific cytotoxic T-cell responses (34, 35). Con-sistentwith a Th2-driven antitumor effect through the stimulationof innate immune system, we demonstrated in this study thatexogenous IL13 treatment decreased tumor burden by enhancingthe antitumor properties of TAMs. Altogether, these results estab-lished that both Th1 and Th2 responses can be involved inantitumor immunity, suggesting that the most effective cancerimmunotherapiesmay be those that can simultaneously associatemultiple Th1 and Th2 effector mechanisms that can cooperate formaximal systemic antitumor response (38).

Although the inhibition of IL13 has received a great deal ofattention as a new therapeutic strategy for tumor diseases, IL13can promote tumoricidal activities of TAMs and inhibit cancercell proliferation (39–41), supporting that this cytokine plays acomplex role in tumor development according to the type ofmalignancy, and therefore its therapeutic control is critical andshould be considered with caution. In this context, our findingsrevealing that the antitumor effects are similar when we admin-istrate IL13 directly or IL13-polarized macrophages offer a newstrategy to avoid the possible deleterious effects of the directadministration of IL13. Our results showing that IL13 and IL4treatments exhibit similar effects in the orientation of macro-phages toward a cytotoxic and tumoricidal phenotype and onlymphoma progression provide additional data to overcomethe detrimental effects of IL13 when the tumor cells expressIL13Ra2 on their surface. On the basis that clodronate treat-ment alone is less efficient in reducing tumor burden than IL13treatment, the strategy to orient TAMs toward a cytotoxicphenotype to reduce tumor progression seems more promisingthan the depletion of tumor macrophages.

In conclusion, we have shown that IL13 exhibits efficientactivity against T-cell lymphoma and ovarian adenocarcinomathrough the enhancement of macrophage antitumor properties.Our findings suggest that IL13 strengthens tumor immunosur-veillance by increasing expression of CLRs, enabling early detec-tion and removal of tumor cells with aberrant glycosylation.

Finally, these results identify MR and dectin-1 as main compo-nents of macrophage antitumoral response and offer attractivetargets to promote TAMs' tumoricidal properties.

Disclosure of Potential Conflicts of InterestP. Balard reports receiving a commercial research grant from Nutergia. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M. Alaeddine, M. Prat, P. Balard, B. Pipy, A. CosteDevelopment of methodology: M. Alaeddine, M. Prat, V. Poinsot, V. Gouaz�e-Andersson, C. Alric, J. Bernad, L. Alric, B. Pipy, A. CosteAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Alaeddine, M. Prat, V. Poinsot, H. Authier,E. Meunier, L. Lef�evre, C. Alric, L. Alric, B. Segui, P. Balard, B. Couderc, A. CosteAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Alaeddine, M. Prat, V. Poinsot, E. Meunier,C. Alric, J. Bernad, L. Alric, F. Couderc, B. Pipy, A. CosteWriting, review, and/or revision of the manuscript: M. Alaeddine, M. Prat,V. Poinsot, C. Alric, L. Alric, P. Balard, B. Couderc, A. CosteAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Alaeddine, M. Prat, H. Authier, E. Meunier,C. Alric, C. Dardenne, J. Bernad, A. CosteStudy supervision: M. Alaeddine, P. Balard, B. Pipy, A. Coste

AcknowledgmentsWe thank Philippe Batigne and B�en�edicte Bertrand from Universit�e Paul

Sabatier for excellent technical support, Alexia Zakaroff-Girard and ChristianeP�echer (TRI imaging platform, IFR150/I2MC) for flow cytometry technicalassistance, and Pierre Cordelier and Hubert Lulka for in vivo imaging systemtechnical assistance. This research was supported by a grant from CIFRE-Association Nationale de la Recherche et de la Technologie (ANRT) awardedto M. Alaeddine (2012/1416) and from the Nutergia Laboratory (to A. Costeand B. Pipy).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 3, 2018; revised August 7, 2018; accepted December 31, 2018;published first January 4, 2019.

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Alaeddine et al.




Published OnlineFirst January 4, 2019.Cancer Immunol Res Mohamad Alaeddine, Mélissa Prat, Véréna Poinsot, et al. through Recognition of Sialylated Tumor CellsOverexpression Promotes Macrophage Antitumor Activities IL13-Mediated Dectin-1 and Mannose Receptor

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