Chronic helminth infection and helminth-derived egg antigens promote adipose tissue M2 macrophages...

The FASEB Journal • Research Communication

Chronic helminth infection and helminth-derived eggantigens promote adipose tissue M2 macrophages andimprove insulin sensitivity in obese mice

Leonie Hussaarts,* Noemı Garcıa-Tardon,* Lianne van Beek,† Mattijs M. Heemskerk,†

Simone Haeberlein,* Gerard C. van der Zon,‡ Arifa Ozir-Fazalalikhan,* Jimmy F. P. Berbee,§

Ko Willems van Dijk,† Vanessa van Harmelen,† Maria Yazdanbakhsh,* and Bruno Guigas*,‡,1

Departments of *Parasitology, †Human Genetics, ‡Molecular Cell Biology, and §Endocrinology andMetabolic Diseases, Einthoven Laboratory for Experimental Vascular Medicine, Leiden UniversityMedical Center, Leiden, The Netherlands

ABSTRACT Chronic low-grade inflammation associ-ated with obesity contributes to insulin resistance and type2 diabetes. Helminth parasites are the strongest naturalinducers of type 2 immune responses, and short-lived in-fection with rodent nematodes was reported to improveglucose tolerance in obesemice. Here, we investigated theeffects of chronic infection (12 weeks) with Schistosomamansoni, a helminth that infects millions of humansworldwide, on whole-body metabolic homeostasis andwhite adipose tissue (WAT) immune cell composition inhigh-fat diet-inducedobeseC57BL/6malemice.Ourdataindicate that chronic helminth infection reduced bodyweight gain (262%), fatmass (289%), and adipocyte size;lowered whole-body insulin resistance (223%) and glucoseintolerance (216%); and improved peripheral glucoseuptake (+25%) and WAT insulin sensitivity. Analysis of im-mune cell composition by flow cytometry and quantitativePCR (qPCR) revealed that S. mansoni promoted strongincreases in WAT eosinophils and alternatively activated(M2)macrophages. Importantly, injections with S. mansoni-soluble egg antigens (SEA) recapitulated the beneficial ef-fect of parasite infection on whole-body metabolic ho-meostasis and induced type 2 immune responses in WATand liver. Taken together, we provide novel data suggest-ing that chronic helminth infection and helminth-derivedmolecules protect against metabolic disorders by pro-motingaThelper 2 (Th2) response, eosinophilia, andWATM2 polarization.—Hussaarts, L., Garcıa-Tardon, N., vanBeek, L., Heemskerk, M. M., Haeberlein, S., van der Zon,G. C., Ozir-Fazalalikhan, A., Berbee, J. F. P., Willems vanDijk, K., van Harmelen, V., Yazdanbakhsh, M., Guigas, B.Chronic helminth infection and helminth-derived eggantigens promote adipose tissue M2 macrophages andimprove insulin sensitivity in obese mice. FASEB J.29, 000–000 (2015). www.fasebj.org

Key Words: obesity • immunometabolism • obesity • eosinophils •

type 2 inflammation • Schistosoma mansoni

THE OBESITY EPIDEMIC REPRESENTS A growing threat to publichealth, not only in industrialized countries but also in urbancentersofdevelopingcountries.Obesity significantly increasesthe risk for thedevelopmentof type2diabetes, cardiovasculardiseases, and eventually, cancer (1, 2) and is often associatedwith a state of chronic, low-grade inflammation, which con-tributes to tissue-specific insulin resistance and whole-bodymetabolic dysfunction (3). Among the underlyingmolecularmechanisms, classically activated (M1) macrophages wereshown to accumulate in WAT from obese mice, where theysecrete proinflammatory cytokines, such as IL-1b and TNF-a(4–6). These cytokines interfere with insulin signaling (7, 8)and induce lipolysis (9,10), thereby increasingcirculating freefatty acids that promote peripheral insulin resistance (11).Other immune cell types, including neutrophils (12), mastcells (13), B cells (14, 15), and CD8+ T cells (16), have alsobeen shown to mediate insulin resistance.

By contrast, M2 macrophages prevail in lean WAT andare involved in the maintenance of adipose tissue insulinsensitivity, partly through secretionof the anti-inflammatorycytokine IL-10 (6, 17). The M2 phenotype is promoted byTh2-type cytokines, such as IL-4, secreted by WAT eosino-phils (18), and IL-5 and IL-13, released from WAT innatelymphoid type 2 cells (ILC2s) (19). In addition, Th2 andregulatory T cell responses, as well as administration of IL-4,have been associated with protection against insulin re-sistance (20–22). Together, these studies illustrate that type2 and anti-inflammatory responses are beneficial for theexpanding adipose tissue environment and the mainte-nance of tissue-specific insulin sensitivity and whole-bodyglucose homeostasis.

Helminth parasites are the strongest natural inducers oftype 2 inflammatory responses, and epidemiologic studies

Abbreviations: Arg1, arginase 1; AUC, area under the curve;Chil3, chitinase-like 3; Clec4f, C-type lectin domain family 4,member F; Emr1, epidermal growth factor-like module-containingmucin-like hormone receptor-like 1; F, forward; FCS, fetal calfserum; GTT, glucose tolerance test, HFD, high-fat diet;HOMA-IR, HOmeostasis Model Assessment of Insulin Resistance;

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1 Correspondence: Departments of Parasitology and Mo-lecular Cell Biology, Leiden University Medical Center, P.O.Box 9600, Postal Zone L4-Q, 2300 RC Leiden, The Nether-lands. E-mail: b.g.a.guigas@lumc.nldoi: 10.1096/fj.14-266239This article includes supplemental data. Please visit http://

www.fasebj.org to obtain this information.

0892-6638/15/0029-0001 © FASEB 1

The FASEB Journal article fj.14-266239. Published online April 7, 2015.

in India and rural China revealed that helminth infectionsinversely correlate with metabolic syndrome (23–25). Inaddition, seminal papers recently reported that the rodentnematode Nippostrongylus brasiliensis, which is spontane-ously clearedwithin 2 weeks of infection, improves glucosetolerance in diet-induced obese mice (18, 26) associatedwith WAT eosinophilia (18) or increased M2 gene ex-pression (26). Furthermore, SEA could protect againstatherosclerosis in hyperlipidemic LDL receptor knockoutmice (27). These studies suggest that manipulation of theimmune system by helminths or their molecules might bebeneficial for metabolic homeostasis. However, it remainsunclear which aspects of whole-body energy metabolismare affected by the worms, and the immunologic changesthat take place in WAT have not yet been characterized atthe cellular level.

Furthermore, asmost helminth infections inhumans arechronic in nature, it would be important to test whether thebeneficial effect on metabolic homeostasis also occurs ina model of chronic infection. Of the various helminthspecies, schistosomes are among the most prevalent andchronically infect millions of people worldwide (28).Whereas infection with N. brasiliensis induces a strong Th2response thatmediatesparasite rejectionwithin2weeksafterinfection, the Th2 response in schistosomiasis emerges af-ter 5–6 weeks of infection, with the onset of egg productionthat also triggers the development of M2 macrophages(29). Inmost individuals, infection often reaches a chronicstage, characterized by a decline in Th2 inflammation andthe presence of regulatory B and T cells (30, 31). In thepresent study, therefore, we investigated the impact ofS. mansoni infection for 12 weeks on whole-body metabolichomeostasis, WAT insulin sensitivity, and WAT immunecell composition inmice fed a low- or high-fat diet (LFD orHFD). Next, to study the impact of helminth-derived mol-ecules onmetabolicdisorders inapathogen-free setting,wetreated HFD-fed mice with SEA for 4 weeks and assessedwhole-bodyglucose toleranceand insulin sensitivity and theimmune cell composition of WAT and liver.

MATERIALS AND METHODS

Animals, diet, and S. mansoni infection

All mouse experiments were performed in accordance with theGuide for theCare andUseof LaboratoryAnimals of the InstituteforLaboratoryAnimalResearchandhave received approval fromthe university Ethical Review Boards (DEC2189; Leiden Univer-sity Medical Center, Leiden, The Netherlands). Male C57BL/6Jmice (8–10 weeks old; Charles River, L’Arbresle Cedex, France)were housed in a temperature-controlled room with a 12-hourlight-dark cycle. Throughout the experiment, food and tap water

were available ad libitum. Mice were fed a HFD (45% energy de-rived from fat; D12451; Research Diets, Wijk bij Duurstede, TheNetherlands) or a LFD (10% energy derived from fat; D12450B;ResearchDiets), which were similar in composition in all respectsapart fromthe total fat content.After 6wk,micewere randomizedaccording to body weight and fasting plasma glucose and insulinlevels andpercutaneously infectedwith 36S.mansoni cercariae, asdescribed previously (32).Miceweremonitored for 12 additionalweeks. The effects of chronic infection were assessed in 2 in-dependent experiments. Before SEA injections, mice were feda LFD or HFD for 12 weeks, after which, they were randomizedaccording to body weight, fasting plasma glucose and insulinlevels, and fat mass. SEA (50 mg) was injected intraperitoneallyonce every 3 days for a period of 4 weeks. The effects of SEAtreatment were assessed in 2 independent experiments.

Plasma analysis

Blood samples were collected from the tail tip of 4-hour unfedmice (food removed at 9 AM) by use of chilled capillaries. Bloodglucose level was determined by use of a glucometer (Accu-Chek;Roche Diagnostics, Almere, The Netherlands), and plasma in-sulin level was measured by use of a commercial kit, according tothe instructionsof themanufacturer (Millipore, Amsterdam,TheNetherlands).

Glucose and insulin tolerance tests

The effect of chronic S. mansoni infection on glucose tolerancewas assessed by intravenous glucose tolerance test (GTT)at weeks5 and 11 postinfection.Mice were unfed for 6 hours, and the testswere carried out at 2 PM. After an initial blood collection (t = 0),a glucose load {2 g D-glucose/kg total body weight, of which 50%of the glucose was [6,6-2H2]glucose (Sigma-Aldrich, Zwijndrecht,The Netherlands)] was administered in conscious mice via in-jection in the tail vein. Blood sampling was performed by tailbleeding at 2.5, 15, 30, 60, 90, and 120 minutes: 5–10 mL wholeblood was spotted on sample carrier paper (Sartorius Stedim,Goettingen, Germany), and an additional drop was used tomeasure glucose by use of a glucometer (Accu-Chek; RocheDiagnostics). To analyze peripheral glucose uptake, blood spotglucose enrichmentwasmeasured by extracting glucose from thefilter paper with 75 ml water (Aqua B. Braun, Oss, The Nether-lands) and 1mlmethanol. The extracted glucose was derivatizedto aldonitrile penta-acetate and reconstituted in 100 ml ethylacetate, of which 1 mL was injected for gas chromatography(HP6890II)/mass spectrometry (HP5973; Hewlett-Packard, PaloAlto, CA, USA), as described previously (33). Mass-over-chargeratios of 187, 188, and 189 were monitored in selective ion mon-itoring mode, from which the percentage of unlabeled and la-beled glucose was calculated based on theoretical isotopicdistribution. Concentrations of labeled glucose were calculatedbased on the plasma glucose levels and values were natural, logtransformed, afterwhich adecay curvewasfitted. Individual decaycurves were calculated, of which the slope represents the periph-eral glucose uptake. The effect of SEA treatment on glucose tol-erance was assessed by an intraperitoneal GTT (2 g D-glucose/kgtotal body weight) in 6-hour unfedmice at week 3 postinjections.

Whole-body insulin sensitivity was determined by an intra-peritoneal insulin intolerance test (ITT) at 5 and 11 weekspostinfection or at week 3 post-SEA treatment. Mice were fastedfor 4 hours, and the tests were carried out at 1 PM. After an initialblood collection (t = 0), an i.p. bolus of insulin (1U/kg lean bodymass; NovoRapid; Novo Nordisk, Alphen aan den Rijn, TheNetherlands) was administered to the mice. Blood glucose wasmeasured by tail bleeding at 15, 30, 60, and 120 minutes afterinsulin administration by use of a glucometer.

(continued from previous page)ILC, innate lymphoid cell; IRb, insulin receptor b; Itgax,integrin, aX (complement component 3 receptor 4 sub-unit); ITT, insulin tolerance test; LFD, low-fat diet; M1,classically activated; M2, alternatively activated; PMA, phor-bol myristate acetate; qRT-PCR, quantitative RT-PCR; R,reverse; Retnla, resistin like a; RER, respiratory exchangeratio; RplP0, ribosomal protein, large, P0; SEA, Schistosomamansoni-soluble egg antigens, SVF, stromal vascular fraction;Th, T helper, WAT, white adipose tissue

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Body composition and indirect calorimetry

Body composition was measured by MRI by use of an EchoMRI(Echo Medical Systems, Houston, TX, USA). Groups of 8 micewith free access to food and water were subjected to individualindirect calorimetric measurements at week 11 postinfection fora period of 7 consecutive days by use of a Comprehensive Labo-ratory Animal Monitoring System (Columbus Instruments, Co-lumbus, OH, USA). Before the start of the measurements, theanimals were acclimated to the cages and the single housing fora period of 48 hours. Feeding behavior was assessed by real-timefood intake. Spontaneous locomotor activity was determined bythe measurement of beam breaks. Oxygen consumption andcarbon dioxide production weremeasured at 15-minute intervalsand normalized for body-surface area (kg0.75). Respiratory ex-change ratio (RER) and energy expenditure were calculated, asdescribed previously (34).

Isolation of adipocytes and stromal vascular fraction fromadipose tissue

Gonadal (epididymal), visceral (mesenteric), and s.c. (flank)adipose tissueswere collected frominfectedanduninfectedmice,minced, and digested for 1 hour at 37°C in HEPES buffer (pH7.4) containing 0.5 g/l type 1 collagenase from Clostridium histo-lyticum (Sigma-Aldrich) and 2% (w/v) dialyzed bovine serum al-bumin (Fraction V; Sigma-Aldrich). The disaggregated adiposetissue was filtered through a 236 mm nylon mesh. Mature adipo-cytes were isolated from the surface of the filtrate and washedseveral times with PBS. Cell size was determined by use of animaging technique implemented inMatlab (MathWorks, Natick,MA, USA), which automatically determines size of isolated adi-pocytes from microscopic pictures (;1000 cells/fat tissue sam-ple). The adipocyte size distribution, mean adipocyte diameterand volume, and adipocyte number/fat pad were calculated, asdescribedpreviously (35).The residueof thegonadal andvisceraladipose tissuefiltratewas used for the isolationof stromal vascularcells for flow cytometry. In brief, after centrifugation (350 g,10 min, room temperature), the supernatant was discarded, andthe pellet was treated with erythrocyte lysis buffer. The cells werewashed twice with PBS and counted manually or by use of anautomated cell counter (TC10; Bio-Rad Laboratories, Hercules,CA, USA). Following SEA injections, stromal vascular cells fromgonadal adipose tissue were isolated, as described above, with theexception that the disaggregated adipose tissue was passedthrough a 100 mm cell strainer that was washed with PBS andsupplementedwith 2.5mMEDTAand 5% fetal calf serum(FCS).

Isolation of CD45+ cells from liver tissue

Livers were minced and digested for 45 minutes at 37°C inRPMI 1640 + GlutaMAX (Life Technologies, Bleiswijk, TheNetherlands) containing 1 mg/mL collagenase type IV fromC. histolyticum, 2000 U/mL DNase (both Sigma-Aldrich), and1 mM CaCl2. The digested liver tissues were passed through100 mm cell strainers that were washed with PBS, supple-mented with 2.5 mM EDTA and 5% FCS. Following centri-fugation (530 g, 10 minutes, 4°C), the supernatant of thefiltrate was discarded, after which the pellet was resuspendedin PBS + 2.5 mM EDTA and 5% FCS and centrifuged at 50 gto remove hepatocytes (3 minutes, 4°C). Next, supernatantswere collected and pelleted (530 g, 10 minutes, 4°C). Thepellet was treated with erythrocyte lysis buffer, and the cellswere washed once more with PBS + 2.5 mM EDTA and 5%FCS. CD45+ cells were isolated by use of LS columns andCD45 MicroBeads (35 mL/liver; Miltenyi Biotec, BergischGladbach, Germany), according to themanufacturer’s protocol.

Isolated CD45+ cells were counted and processed, as describedfor the stromal vascular fraction (SVF).

Processing of isolated cells for flow cytometry

For analysis of macrophage and lymphocyte subsets, iso-lated stromal vascular cells and CD45+ cells from liverwere stained with the live/dead marker Aqua (Invitrogen,Carlsbad, CA, USA), after which they were fixed with 1.9%paraformaldehyde (Sigma-Aldrich) and stored in FACS buffer(PBS, 0.02% sodium azide, 0.5% FCS) at 4°C in the dark untilsubsequent analysis. For analysis of cytokine production, isolatedcells were cultured for 4 hours in culturemedium in the presenceof 100 ng/mL phorbol myristate acetate (PMA), 1 mg/mL ion-omycin, and 10 mg/mL Brefeldin A (all Sigma-Aldrich). Afterculture, cells were washed with PBS, stained with Aqua, and fixedas described above.

Flow cytometry

For analysis of lymphocyte subsets, SVF cells were stained withantibodies against CD4 (GK1.5), CD3 (17A2), B220 (RA3-6B2),or CD19 (1D3; all eBioscience, San Diego, CA, USA); CD8 (53-6.7) andCD45 (104; bothBioLegend, SanDiego, CA, USA); andNK1.1 (PK136; eBioscience, San Diego, CA, USA, or BD Bio-sciences, San Jose, CA, USA). Following SEA injections, whenILC2s were analyzed, additional antibodies were includedagainst Thy1.2 (52-2.1) and CD11b (M1/7; both eBioscience)and CD11c (HL3) and GR-1 (RB6-8C5; both BD Biosciences)to gate on lineage-negative Thy1.2+ cells. For analysis of mac-rophages and eosinophils, cells were permeabilized with 0.5%saponin (Sigma-Aldrich), in which they were also stained.Cells were incubated with an antibody against Ym1 conjugatedto biotin (R&D Systems, Minneapolis, MN, USA), washed, andstained with streptavidin-peridinin chlorophyll protein com-plex (BD Biosciences) and antibodies directed against CD45,CD11b, CD11c, and F4/80 (BM8; eBioscience); SiglecF (E50-2440; BD Biosciences); and following SEA injections, Ly6C(HK1.4; BioLegend). Cytokine production of Th2 cells andILC2s was analyzed following permeabilization, as describedabove, by use of antibodies against CD11b, CD11c, GR-1, B220,NK1.1, CD3, CD45, CD4, Thy1.2, IL-4 (11b11; eBioscience),IL-13 (eBio13A; eBioscience), and IL-5 (TRFK5; BioLegend).Flow cytometry was performed by use of a FACSCanto (BDBiosciences), and gates were set according to fluorescenceminus one controls. Representative gating schemes are shownin Supplemental Fig. 1.

In vivo insulin signaling

Mice were unfed for 4 hours and subjected to an intraperitonealinjection of human recombinant insulin (1 U/kg body weight;NovoRapid; Novo Nordisk) at 1 PM. Mice were then killed after15minutes, andgonadal andvisceral adipose tissueswere isolatedand snap frozen immediately. Subsequently, the tissue samples(;30 mg) were lysed in ice-cold buffer containing 50mMHepes(pH 7.6), 50 mM NaF, 50 mM KCl, 5 mM NaPPi, 1 mM EDTA,1 mM EGTA, 1 mM DTT, 5 mM b-glycerophosphate, 1 mM so-dium vanadate, 1%Nonidet P-40, and protease inhibitor cocktail(Complete; Roche Diagnostics). Western blots were performedby use of phospho-specific (Thr308-PKB; Cell Signaling Tech-nology, Leiden, The Netherlands) or total primary antibodies(tubulin from Cell Signaling Technology; insulin receptor b(IRb) from Santa Cruz Biotechnology, Dallas, TX, USA), as de-scribed previously (36). Bands were visualized by enhanced

SCHISTOSOMA MANSONI IMPROVES INSULIN SENSITIVITY 3

chemiluminescence and quantified by use of ImageJ (NationalInstitutes of Health, Bethesda, MD, USA).

RNA purification and qRT-PCR

RNAwasextractedfromsnap-frozenadiposetissuesamples(;20mg)by use of TriPure RNA isolation reagent (Roche Diagnostics).Total RNA (1mg) was reverse transcribed, and qRT-PCRwas thenperformedwith theSYBRGreenCoreKitonaMyiQthermalcycler(Bio-Rad Laboratories) by use of specific primers sets: 59-GCCAC-CAACCCTTCCTGGCTG-39 [integrin, aX (complement compo-nent 3 receptor 4 subunit; Itgax)-reverse (R)], 59-TTGGACACTC-CTGCTGTGCAGTTG-39 [Itgax-forward (F)], 59-GTCCCCAAA-GGGATGAGAAG-39 (Tnfa-R), 59-CACTTGGTGGTTTGCTACGA-39(Tnfa-F),59-TCCTGGACATTACGACCCCT-39(Nos2-R),59-CTC-TGAGGGCTGACACAAGG-39 (Nos2-F), 59-TCAGCCAGATGCAG-TTAACGCCC-39 (Ccl2-R), 59-GCTTCTTTGGGACACCTGCTGCT-39(Ccl2-F),59-CCTGCCCTGCTGGGATGACT-39[resistinlikea (Retnla)-R],59-GGGCAGTGGTCCAGTCAACGA-39(Retnla-F),59-ACAATTAG-TACTGGCCCACCAGGAA-39 [chitinase-3-like protein 3 (Chil3)-R],59-TCCTTGAGCCACTGAGCCTTCA-39 (Chil3-F), 59-GACCACGGG-GACCTGGCCTT-39 [arginase 1 (Arg1)-R], 59-ACTGCCAGACTG-TGGTCTCCACC-39 (Arg1-F), 59-CCTCACAGCAACGAAGAACA-39(Il4-R),59-ATCGAAAAGCCCGAAAGAGT-39(Il4-F),59-TGGGGGTAC-TGTGGAAATGC-39 (Il5-R), 59-CCACACTTCTCTTTTTGGCGG-39(Il5-F),59-CCCTGGATTCCCTGACCAAC-39(Il13-R),59-GGAGGCTGGA-GACCGTAGT-39(Il13-F),59-CTTTGGCTATGGGCTTCCAGTC-39[epidermal growth factor-like module-containing mucin-like hor-monereceptor-like1(Emr1)-R], 59-GCAAGGAGGACAGAGTTTA-TCGTG-39 (Emr1-F), 59-ACTGAAGTACCAAATGGACAATGTTA-GT-39 [C-type lectin domain family 4, member F (Clec4f)-R],59-GTCAGCATTCACATCCTCCAGA-39 (Clec4f-F).mRNA expres-sion was normalized to ribosomal protein, large, P0 (RplP0)mRNAcontent and expressed as fold change compared with noninfected,LFD-fedmice by use of theΔΔcomparative thresholdmethod.

Statistical analysis

All data are presented as means 6 SEM. Statistical analysis wasperformed by use of GraphPad Prism version 6.00 for Windows(GraphPad Software, La Jolla, CA, USA) with 2-tailed unpairedStudent’s t test. Differences between groups were consideredstatistically significant at P , 0.05. For repeated measurements,data were analyzed assuming the same scatter to increase power.

RESULTS

Chronic S. mansoni infection reduces fat mass indiet-induced, obese mice

To study theeffect of chronichelminth infectiononwhole-body energy homeostasis, C57BL/6 male mice were feda LFD or HFD for 6 weeks before infection with S. mansonifor 12 additional weeks. HFD induced a time-dependentincrease in bodyweight (Fig. 1A), fatmass (Fig. 1B,C), andmean adipocyte volume (Fig. 1D) when compared withLFD-fedmice. In response to S.mansoni infection,HFD-fedmice gained significantly less weight (Fig. 1A), an effectexclusively resulting from a reduction in body-fatmass without affecting lean body mass (Fig. 1B, C).Morphometric analysis of various WATs revealed thatchronic S. mansoni infection reduced HFD-inducedadipocyte hypertrophy (Fig. 1D), whereas cell numbersremained unaffected (data not shown). In LFD-fedanimals, S. mansoni induced a small but significant de-crease in gonadal adipocyte mean volume but did notaffect body weight and fat mass. Next, by use of meta-bolic cages, we found that food intake and spontaneouslocomotor activity were not affected by chronic infection(Fig. 1E, F), and indirect calorimetry also revealed that

Figure 1. Chronic S. mansoni infection reduces body-weight gain, fat mass, and adipocyte size in diet-induced, obese mice. Micewere fed a LFD or a HFD for 6 wk before infection with S. mansoni cercaria or sham infection for 12 weeks. Body weight wasmonitored throughout the experimental period (A). The change (D) in body composition from the start of infection (B), weightof different fat pads (C), and adipocyte mean volume (D) were measured at week 12 postinfection. Sub. cut., s.c. Food intake (E),spontaneous locomotor activity (F), RER (G), and energy expenditure (EE; H) were assessed by use of fully automated, single-housed metabolic cages during week 11. Results are expressed as means 6 SEM. *P , 0.05 HFD vs. LFD; #P , 0.05 helminth- vs.sham-infected group (n = 4–11 animals/group in B and D–H and 12–19 animals/group in A and C).

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infection did not affect the RER (Fig. 1G) or energy ex-penditure (Fig. 1H) in HFD-fed animals.

Chronic S. mansoni infection improves whole-bodyglucose tolerance and insulin sensitivity indiet-induced, obese mice

We next investigated the effect of chronic S. mansoni in-fection on whole-body metabolic homeostasis in lean anddiet-induced, obese mice. As expected, HFD increasedfasting plasma glucose and insulin levels (Fig. 2A) andhomeostasis model assessment of insulin resistance

(HOMA-IR; Fig. 2B). Furthermore, HFD impaired whole-body glucose tolerance (Fig. 2C, D), peripheral glucoseuptake (Fig. 2E, F), and insulin sensitivity (Fig. 2G, H).Chronic S. mansoni infection restored fasting blood glucoseand insulin levels in mice on HFD (Fig. 2A), resulting ina time-dependent reduction in HOMA-IR (Fig. 2B). Fur-thermore, chronic infection restored HFD-induced whole-body glucose tolerance (Fig. 2C, D), improved peripheralglucose uptake (Fig. 2E, F), and promoted whole-body in-sulin sensitivity (Fig. 2G,H). Of note, except for a slight butsignificant decrease in fasting plasma insulin level,S. mansoni infection did not affect any metabolic param-eters in LFD-fed mice. Overall, these data indicate that

Figure 2. Chronic S. mansoni infection improves whole-body glucose tolerance and insulin sensitivity in diet-induced, obese mice.Mice were fed a LFD or HFD and were infected with S. mansoni, as described in the legend of Fig. 1. Plasma glucose and insulinlevels (A) were determined in 4-hour unfed mice at week 12 postinfection. HOMA-IR was calculated throughout theexperimental period (B). An intravenous GTT (2 g D-glucose with 50% [6,6-2H2]glucose/kg body weight) was performed in6-hour unfed mice at week 11. Blood glucose levels were measured at the indicated time-points (C), and the area under the curve(AUC) of the glucose excursion curve was calculated as a measure for glucose tolerance (D). Blood was also collected fordetermination of the time-dependent change in [6,6-2H2]glucose concentration by gas chromatography/mass spectrometry (E).For each mouse, a decay curve was fitted, of which the slope represents peripheral glucose uptake (F). An intraperitoneal ITT(1 U/kg lean body mass) was performed in 4-hour unfed mice at week 11. Blood glucose levels were measured at the indicated time-points, (G) and the AUC of the glucose excursion curve was calculated as a measure for insulin resistance (H). After 12 weeks ofinfection, 4-hour unfec mice received an intravenous injection of insulin (1 U/kg lean body mass) and were killed by cervicaldislocation after 15 minutes. Gonadal and visceral adipose tissues were collected and snap frozen immediately. The proteinexpression of IRb (I) and the phosphorylation state of PKB-Thr308 (pPKB; J) were assessed by Western blot and quantifiedby densitometry analysis. Tubulin expression was used as internal housekeeping protein. Representative Western blots are shown(K). Results are expressed as means 6 SEM. *P , 0.05 HFD vs. LFD; #P , 0.05 helminth- vs. sham-infected group. C, E, and G)Statistical significance of HFD sham vs. LFD sham and HFD infected vs. HFD sham is shown (n = 12–19 animals/group in A and Band 3–11 animals/group in C–J).

SCHISTOSOMA MANSONI IMPROVES INSULIN SENSITIVITY 5

S. mansoni improves whole-body glucose tolerance andinsulin resistance in diet-induced, obese mice.

Chronic S. mansoni infection improves adipose tissueinsulin sensitivity in diet-induced, obese mice

To study the effect of chronic S.mansoni infectiononWAT-specific insulin sensitivity, mice were subjected to an acuteintraperitoneal insulin injection. The expression of IRband insulin-inducedphosphorylationof PKBwere assessedbyWestern blot in gonadal and visceralWAT. As expected,HFD reduced IRb protein expression (Fig. 2I, K) and im-pairedPKBphosphorylation in response to insulin (Fig. 2J,K) in gonadal and visceral WAT, indicating tissue-specificinsulin resistance. Chronic S. mansoni infection restoredIRb protein expression and insulin-induced PKB phos-phorylation in WAT of HFD-fed mice (Fig. 2I–K), sug-gesting that the beneficial effect of helminths observed atthe systemic level might be secondary to improved tissue-specific insulin sensitivity.

Chronic S. mansoni infection increases adipose tissueeosinophils and M2 macrophages

The immune cell composition of WAT, specifically the eo-sinophil content and the balance between M1 and M2macrophages, has been shown to play a crucial role in themaintenanceofadipocyte insulin sensitivity andwhole-bodymetabolic homeostasis (6). To investigate whether chronicS. mansoni infection affects the immune-cell composition inWAT, the SVF was isolated from gonadal and visceral WATof sham- and helminth-infected mice, and the cell compo-sition was analyzed by flow cytometry (see SupplementalFig. 1 for gating strategy). We found that HFD significantlyreduced B cell, NK cell, and NKT cell numbers/gram ofgonadalWAT(SupplementalFig. 2A).S.mansonipromotedinfiltration of leukocytes into gonadal WAT, resulting inincreased numbers of all lymphocyte subsets studied inLFD- andHFD-fedmice (Supplemental Fig. 2A). Nomajoreffect of diet or infection was observed on lymphocytes invisceral WAT (Supplemental Fig. 2B).

Subsequent analysis of eosinophils, identified by CD45and Siglec F expression, revealed a trend for a decrease inWAT eosinophil numbers in HFD-fed mice (Fig. 3A, B) inline with a previous report (18). Chronic S. mansoni in-fection induced a strong increase in eosinophil infiltrationinto gonadal and visceral WATs from LFD- and HFD-fedmice (Fig. 3A, B).

Finally, theexpressionofCD11candYm1inCD11b+F4/80+

cells allowed us to discriminate between M1 and M2macrophages, respectively (17) (Fig. 3C). HFD promoteda significant increase in M1 macrophages in gonadal WAT(Fig. 3D) and a decrease in M2 macrophages in visceralWAT (Fig. 3E). Analysis of the adipose tissue gene expres-sionconfirmed thatM1markerswere increased inHFD-fedmice, an effect particularly clear in gonadalWAT, althoughM2-associated genes were not down-regulated significantly(Fig. 3G, H). Chronic S. mansoni infection had a marginaleffect on WAT M1 macrophage counts, as determined byflow cytometry analysis, but strongly increased M2 macro-phagenumbers ingonadal andvisceralWATfromLFD-and

HFD-fedmice (Fig. 3D, E), shifting theM2/M1 ratio towardM2 (Fig. 3F). In line with these results, infection induceda strongup-regulationofM2-associated genes in gonadal andvisceral WAT from LFD- and HFD-fed mice, whereas the ex-pression ofM1-related genes was barely affected (Fig. 3G,H).Lastly, mRNA expression of the type 2-associated cytokinesIL-4 and IL-5 were also up-regulated significantly in WATfromhelminth-infectedmice (Fig. 3G,H). Taken together,these results suggest that chronic S. mansoni infection pro-motes WAT type 2 inflammation characterized by adiposetissue eosinophilia and accumulation of M2 macrophages.

SEA improve whole-body metabolic homeostasis andpromote a type 2 immune response in WAT and liverfrom obese mice

Toexclude that thebeneficial effects of helminth infectiononmetabolic homeostasis are simply a result of parasitism,we next investigated whether S. mansoni-derivedmoleculescan alleviate diet-induced metabolic disorders. For thispurpose, HFD-fed mice were subjected to repetitive in-traperitoneal injections with SEA, which were shown topromote a strong Th2 response in vitro (37) and in vivo(38). Importantly, treatment with SEA for 4 weeks neitheraffectedbodyweightnor leanor fat bodymass (Fig. 4A)butimproved fastingplasmaglucose and insulin levels (Fig. 4B,C), HOMA-IR (Fig. 4C), and whole-body glucose tolerance(Fig. 4E) and insulin sensitivity (Fig. 4F).

Like the chronic parasite infection, SEA exposure pro-moted WAT eosinophilia (Fig. 5A), associated with accu-mulationofM2macrophages (Fig. 5B), leading to a shift inthe M2/M1 ratio toward M2 polarization (Fig. 5C). As weshowed that helminth infection promoted gene expres-sion of type 2-associated cytokines inWAT (Fig. 3), we nextdetermined the numbers of CD4+ T cells and ILCs(lineage-negative Thy1.2+) in gonadal WAT (see Supple-mental Fig. 1 for gating strategy) and analyzed cytokineexpression by these lymphocyte subsets following stimula-tion with PMA and ionomycin (gating strategy shown inFig. 5E). Compared with LFD, HFD decreased the totalnumber of CD4+ T cells and ILCs (Fig. 5D) and the per-centage of CD4+ T cells expressing IL-4 but not IL-5 or IL-13 (Fig. 5F) and did not significantly affect cytokine pro-ductionby ILCs(Fig. 5G).TreatmentofHFD-fedmicewithSEA strongly enhanced the percentage of IL-4-, IL-5-, andIL-13-expressing CD4+ T cells in gonadal WAT (Fig. 5F)and slightly increased IL-5 production by ILCs (Fig. 5G).These findings were confirmed by qPCR, which showedthat SEA promotes gene expression of M2-associatedmarkers and type 2 cytokines (Fig. 5H).

As classic activation of liver macrophages has also beenobserved in diet-induced obesity (39), we determined theeffect of SEA treatment on the hepatic immune cell com-position. Analysis of myeloid cells (Fig. 6A) showed thatHFD did not affect liver eosinophil numbers (Fig. 6A).Assessment of CD45+SiglecF2CD11bloF4/80hi macro-phages (Fig. 6B), which were identified previously asKupffer cells (40), showed that HFD promoted CD11cexpression in these cells with no effect on Ym1expression(Fig. 6C), thereby strongly decreasing the Ym1/CD11cratio (Fig. 6D). HFD also decreased the numbers of CD4+

T cells and ILCs (Fig. 6E), although analysis of cytokine

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Figure 3. Chronic S. mansoni infection increases adipose tissue eosinophils and M2 macrophages. Mice were fed a LFD or HFDand infected with S. mansoni, as described in the legend of Fig. 1. At the end of the experiment (week 12), various adipose tissueswere collected. Small tissue pieces were snap-frozen for qPCR analysis, and the remaining tissue pieces were used for SVFisolation. Following fixation and permeabilization, SVF cells were stained and analyzed by flow cytometry. The complete gatingscheme is shown in Supplemental Fig. 1. Representative flow cytometry plots from gonadal adipose tissue show the percentage ofeosinophils based on Siglec F expression in Aqua2CD45+ cells (A). FSC, Forward-scatter. The numbers of eosinophils/gram of

(continued on next page)

SCHISTOSOMA MANSONI IMPROVES INSULIN SENSITIVITY 7

production (Fig. 6F) showed that HFD did not affectexpression of Th2 cytokines by T cells (Fig. 6G). HFDreduced the frequency of IL-5- and IL-13-expressing ILCs(Fig. 6G). SEA injections promoted eosinophil in-filtration (Fig. 6A) but did not affect macrophage po-larization (Fig. 6B,C), which was also confirmed by qPCRanalysis (Fig. 6I). In line with our findings in WAT, SEAstrongly increased expression of IL-4, IL-5, and IL-13 byCD4+T cells in the liver (Fig. 6G), without an effect on theexpression of type 2 cytokines by ILCs (Fig. 6H). Takentogether, these findings indicate that helminth-derivedmolecules improve metabolic homeostasis associatedwith the induction of eosinophils and Th2 cells in WATand liver and M2 macrophage polarization in WAT.

DISCUSSION

Over the past decade, it has become increasingly clear thatmultiple facets of the Th2-associated immune responsepromote metabolic homeostasis (6). Landmark studies

have shown that infection of diet-induced, obesemice withthe rodent nematodeN. brasiliensis ameliorateswhole-bodyinsulin sensitivity and glucose tolerance (18, 26). In thepresent study, we analyzed which aspects of whole-bodyenergy metabolism and WAT immune-cell compositionare affected by helminths by use of a model of chronicS. mansoni infection. Unlike N. brasiliensis, which givesa strong Th2 response that mediates parasite clearancewithin 2 wk, S. mansoni establishes a chronic infection,characterized by the presence of Th2 cells, M2 macro-phages, anda regulatorynetwork (29).To study theeffect ofTh2-inducing conditions in a pathogen-free setting, wenextexposed mice on a HFD to repetitive injections with SEA.

We report that chronic exposure to S. mansoni inducesa type 2 immune response in adipose tissue and improvesinsulin sensitivity and glucose tolerance. These findingsindicate that the beneficial effect of chronic S. mansoniinfection on whole-body metabolic homeostasis, as repor-ted previously for short-lived infection with N. brasiliensis(18, 26), is a hallmarkof helminth infection.Unique toourstudy, we have performed in-depth metabolic profiling,

tissue were determined (B). Macrophages were identified as Aqua2CD45+SiglecF-CD11b+F4/80+ cells. Representative flow cytometryplots from gonadal adipose tissue show the percentage of CD11c+ (M1) and Ym1+ (M2) macrophages (C). The numbers of M1 and M2macrophages/gram tissue in gonadal (D) and visceral (E) WAT were determined, and the M2/M1 ratios were calculated (F). mRNAexpression of the indicated genes in gonadal (G) and visceral (H) adipose tissues was quantified by RT-PCR relative to the RplP0 gene andexpressed as fold difference compared with the noninfected LFD-fed mice. Itgax encodes CD11c; Retnla encodes Fizz1; Chil3 encodes YM1.Results are expressed as means 6 SEM. *P , 0.05 HFD vs. LFD; #P , 0.05 helminth- vs. sham-infected group (n = 8–16 animals/group).

Figure 4. SEA improves whole-body metabolic homeostasis in HFD-induced, obese animals. Mice were fed a LFD or HFD for 12 weeks,after which they were treated intraperitoneally with PBS or 50 mg SEA, once every 3 days for a period of 4 weeks. Body weight and bodycomposition were analyzed after 4 weeks of treatment (A). Plasma glucose (B) and insulin (C) levels were determined in 4-hour unfedmice after 4 weeks of treatment, and HOMA-IR was calculated (D). An intraperitonal GTT (2 g/kg body weight) was performed in 6-hour unfed mice at week 3. Blood glucose levels were measured at the indicated time-points, and the AUC of the glucose excursioncurve was calculated as a measure for glucose tolerance (E). An intraperitoneal ITT (1 U/kg lean body mass) was performed in 4-hourunfed mice at week 3. Blood glucose levels were measured at the indicated time-points, and the AUC of the glucose excursion curvewas calculated as a measure for insulin resistance (F). Results are expressed as means6 SEM. *P, 0.05 HFD vs. LFD; #P, 0.05 PBS vs.SEA (n = 11–13 animals/group in A–E and 3–6 animals/group in F).

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which showed that helminth infection specifically reducedfat mass and dampened HFD-induced adipocyte hyper-trophy.However, surprisingly, we did notfind any changesin food intake or energy expenditure in our experimentalconditions, leading us to speculate that chronic S. mansoniinfection may affect nutrient efficiency by impairing in-testinal lipid absorption. Another possibility is that dietaryfat could be incorporated into eggs by the worms and nextexcreted through the feces. Further studies are required to

clarify this specific point. Remarkably, infection improvedfasting plasma glucose and insulin levels, whole-body glu-cose tolerance, and insulin sensitivity in HFD-fed mice.Among the possible underlying mechanisms, we foundthat S. mansoni reversed HFD-induced inhibition of pe-ripheral glucose uptake, whichmay be secondary to tissue-specific improvement of insulin sensitivity, as S. mansoni-infected, HFD-fed mice exhibited higher insulin-inducedPKB phosphorylation in WAT than uninfected controls.

Figure 5. SEA treatment promotes adipose tissue Th2 polarization and accumulation of eosinophils and M2 macrophages. Mice werefed a LFD or HFD for 12 weeks, after which they were treated intraperitoneally with PBS or 50 mg SEA, once every 3 days for a period of4 weeks. At the end of the experiment (week 4), gonadal adipose tissue was collected and processed as described in the legend of Fig. 3.Following fixation, SVF cells were stained and analyzed by flow cytometry. Gating schemes for ex vivo analysis of lymphocyte subsets areshown in Supplemental Fig. 1. The numbers of eosinophils (A) and M1 and M2 macrophages (B)/gram of tissue were determined asdescribed in the legend of Fig. 3, and the M2/M1 ratio was calculated (C). The numbers of CD4+ T cells and ILCs were determined(D). Intracellular cytokine production was analyzed after 4 hours stimulation with PMA and ionomycin in the presence of Brefeldin A.CD4+ T cells were identified as Aqua2CD45+Thy1.2+Lineage+CD4+ cells, and ILCs were identified as Aqua2CD45+Lineage2Thy1.2+

cells, in which the lineage cocktail included antibodies against CD11b, CD11c, B220, GR-1, NK1.1, CD3, and CD4. Representative flowcytometry plots show the gating strategy for analysis of cytokine-expressing CD4+ T cells and ILCs (E). The frequencies of cytokine-expressing T cells (F) and ILCs (G) were determined. mRNA expression of the indicated genes was analyzed as described in the legendof Fig. 3 (H). Retnla encodes Fizz1; Chil3 encodes YM1. Results are expressed as means6 SEM. *P, 0.05 HFD vs. LFD; #P, 0.05 PBS vs.SEA [n = 9–13 animals/group for all measurements except for intracellular analysis of IL-5 (n = 3–7)].

SCHISTOSOMA MANSONI IMPROVES INSULIN SENSITIVITY 9

Except for small effects on adipocyte volume, fasting in-sulin, and energy expenditure, none of the metabolicparameters analyzed were affected by S. mansoni infection inmice on LFD, suggesting that helminths improve metabolichomeostasis independently of putative S. mansoni-inducedpathologies. We cannot exclude the possibility that part ofthe effect may be secondary to body-weight loss or in-creased glucose/lipid metabolism by the helminths them-selves.However, sustainedexposure toSEAdidnotaffectbodyweight significantly but improved HOMA-IR, whole-body glu-cose tolerance, and insulin sensitivity, in line with a previousreport (41). Taken together, these findings suggest that thebeneficial effects of helminths on metabolic homeostasis arenot secondary to parasitism on host metabolism but likelya result of a direct effect onmetabolic tissues or immune cells.

We demonstrate that S. mansoni infection reduced diet-induced body-weight gain and improved HOMA-IR once in-fection was established beyond 6 weeks. Glucose tolerance

and peripheral glucose uptake were also improved after 11but not 5 weeks of infection (Supplemental Fig. 3). As eggproductionby adultworms triggers thedevelopmentof a type2 response;6 weeks after infection (42), it is therefore likelythat the beneficial effect of helminths on metabolic homeo-stasis may be mediated by the presence of eggs. This is sup-portedfurtherbyourdatashowingthatS.mansoniegg-derivedsoluble molecules improve glucose tolerance and insulinsensitivity in HFD-fed mice. Interestingly, improvements inmetabolic homeostasis were also found in obesemice treatedwith the LewisX-containing glycan lacto-N-fucopentaose III(41). Of note, we have demonstrated previously that v-1,aLewisX-containing immunomodulatoryRNase isolatedfromSEA, skews strong Th2 responses (43, 44). Whether v-1 con-tributes to the beneficial effect of helminth infection or SEAinjection on energy homeostasis requires further study.

It is well established that adipocyte hypertrophy, inresponse to HFD, induces cell necrosis, leading to the

Figure 6. SEA treatment promotes accumulation of eosinophils and Th2 polarization in liver. Mice were fed a LFD or a HFD for 12weeks, after which they were treated intraperitoneally with PBS or 50 mg SEA, once every 3 days for a period of 4 weeks. At the end of theexperiment, livers were collected, and a small piece was snap-frozen for qPCR analysis. From the remaining liver tissue, CD45+ cells wereisolated and analyzed by flow cytometry. Gating schemes for ex vivo analysis of lymphocyte subsets are shown in Supplemental Fig. 1. Thenumbers of eosinophils (A) were determined. Kupffer cells were gated based on CD11b and F4/80 expression in Aqua2CD45+SiglecF2

cells (B). The number of CD11c+ and YM1+ Kupffer cells (C) per gram liver were determined, and the YM1+/ CD11c+ ratio wascalculated (D). The numbers of CD4+ T cells and ILCs were determined (E) Intracellular cytokine expression was analyzed as describedin the legend of Fig. 5. The gating strategy for analysis of cytokine-expressing CD4+ T cells and ILCs is shown (F ). The frequencies ofcytokine-expressing CD4 T cells (G) and ILCs (H) were determined. mRNA expression of the indicated genes was analyzed as describedin the legend of Fig. 3 (I). Emr1 encodes F4/80; Itgax encodes CD11c; Retnla encodes Fizz1; Chil3 encodes YM1. Results are expressed asmeans 6 SEM. *P , 0.05 HFD vs. LFD; #P , 0.05 wild-type vs. mannose receptor, C type 1 (Mrc1; n = 11–13 animals/group).

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infiltration of M1 macrophages, which later form crown-like structures around the necrotic cells (45, 46). ChronicS. mansoni infection did not reduceM1 gene expression orcell numbers, suggesting that M1 macrophages continueto reside in WAT, even though glucose homeostasis im-proves. In line with this, SEA injections did not affect M1cell numbers.Thesefindingsdiffer froma studybyFujisakaet al. (47), in which the antidiabetic drug pioglitazone re-duced expression of the CD11c-encoding gene and low-ered M1 cell numbers in gonadal WAT in HFD-fed mice.In addition, infection with N. brasiliensis and pioglitazonetreatment reduced total WAT macrophages (18, 47),whereas chronic S. mansoni infection or SEA administra-tion increased WAT macrophage numbers, as a result ofan increase inM2macrophage numbers. Taken together,these findings indicate that the M2/M1 ratio, rather thanan increaseordecrease inaparticularmacrophage subset,might be critical for metabolic homeostasis.

Mechanistically, the rise inWATM2numbers, followingchronic S. mansoni infection or repeated SEA administra-tion, may be a result of local macrophage proliferation,which has been reported in the pleural cavity upon in-fection with the filarial helminth Litomosoides sigmodontis inresponse to IL-4 (48). An alternative explanation could bethat S. mansoni or SEA inducesWATmonocyte infiltrationand differentiation into M2 macrophages, which is plausi-ble, as we observed a significant increase in bloodCD11b+Ly6C+ inflammatory Ym1-expressing monocytesduring chronic infection (data not shown). Of note, it israther unlikely that helminth-derived molecules triggerM2 polarization by interacting directly withmacrophages,as it has been described that SEA treatment does not in-duce expression of Chil3 (encoding Ym1),Mrc1, and Arg1by bone marrow-derived macrophages in vitro (27).

Importantly, it was demonstrated recently that mainte-nanceofM2macrophages inWATdependson thepresenceof IL-4-secreting eosinophils (18), which are sustained by IL-5- and IL-13-producing ILC2s (19). In our study, we showedthat chronic S. mansoni infection and SEA treatment pro-moted eosinophil accumulation in WAT, consistent withprevious reports on N. brasiliensis infection (18, 19), and in-creasedthemRNAlevelsof the type2cytokines IL-4andIL-5.By analyzing intracellular IL-4, IL-5, and IL-13 cytokine pro-duction by lymphocytes isolated from gonadal WAT of SEA-treated mice, we found that CD4+ T cells but not ILCs pro-duced increased levels of these type 2 cytokines. Recent lit-eraturedescribedthat IL-13productionby ILC2s inresponseto S. mansoni egg challenge peaks after 7 days of challengeand is reduced to baseline by Day 21 (49). As we analyzecytokine responses after 4 weeks of SEA administration, wespeculate that ILC2 cytokine production has already di-minished. Therefore, it is still possible that S. mansoni-induced ILC2s may be the first trigger for eosinophilia andaccumulation of M2 macrophages in WAT. Then, at a laterstage of infection or after long-term helminth antigen ex-posure,Th2-derived cytokines, suchas IL-4,maymediateM2proliferation (48). Of note, we analyzed expression of a va-riety of eosinophil-attracting chemokines and eotaxins inWATbut foundnoeffectofSEAtreatment(datanot shown).Taken together, the interaction amongthe different celltypes involved, as well as their relative contribution to thebeneficial effect of helminths onWAT insulin sensitivity andwhole-body metabolic homeostasis, requires further studies.

In addition to profound effects of SEA treatment onimmune cells in WAT, we observed increased eosinophiliaandTh2cytokineexpression in the liver, suggesting that theadipose tissue is not an exclusive target of SEA. Helminthmolecules may indeed work as a double-edged sword, act-ing on inflammatory and metabolic pathways in WAT andliver. Interestingly, it has been reported that IL-4 and IL-13may contribute to glucose homeostasis by directly regulat-ing hepatic insulin sensitivity and glucose production, re-spectively (21, 50).Theexact contributionof the liver to thewhole-body metabolic beneficial phenotype observed inresponse to SEA treatment remains to be clarified.

In conclusion, our work has revealed that chronic in-fection with S. mansoni, as well as SEA treatment, protectsagainst metabolic disorders in a mouse model of diet-induced obesity. We have established that S. mansonireduces adipocyte size and promotes peripheral glucoseuptake and WAT-specific insulin sensitivity. Throughanalysis of immune cell composition at the cellular level,we show that SEA injections strongly increase eosinophilsand Th2 cells in WAT and liver, although they only pro-mote M2 polarization in WAT. Collectively, our dataidentify antigens derived from S.mansoni eggs as attractiveagents for therapeutic manipulation of the immune sys-tem in the context of metabolic disorders. Several clinicaltrials are currently registered to assess the safety or efficacyof helminth therapy for the treatment of various in-flammatory diseases inhumans. As helminth infections caninduce pathologic conditions, studies are now focusing onhelminth-derived molecules (51, 52). The identification ofsingle active molecules and studying the mechanisms bywhich they improvewhole-bodymetabolichomeostasismayoffer new insights toward the development of novel thera-peutics for the treatment of metabolic syndrome.

The authors thank Bart Everts (Leiden University MedicalCenter, Leiden, The Netherlands) for critically reading themanuscript. This work was supported by an EFSD/Lilly ResearchGrant Fellowship from the European Federation for the Study ofDiabetes (to B.G.), a Scientific Programme Indonesia-Netherlands-The Royal Netherlands Academy of Sciences Grant (SPIN-KNAW;KNAW-57-SPIN3-JRP; to M.Y. and B.G.), the EU-funded projectIDEA (HEALTH-F3-2009-241642; to M.Y.), a ZonMW TOP Grantfrom the Dutch Organization for Scientific Research (91214131;to M.Y. and B.G.), and a grant from the Board of Directors of theLeiden University Medical Center (to V.v.H.).

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Received for publication October 24, 2014.Accepted for publication March 9, 2015.

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