Arginase I Suppresses IL-12/IL-23p40-Driven Intestinal Inflammation during Acute Schistosomiasis

The Journal of Immunology

Arginase I Suppresses IL-12/IL-23p40–Driven IntestinalInflammation during Acute Schistosomiasis

De’Broski R. Herbert,*,† Tatyana Orekov,*,† Amanda Roloson,*,† Monica Ilies,‡

Charles Perkins,*,† William O’Brien,x Stephen Cederbaum,{ David W. Christianson,‡

Nives Zimmermann,‖ Marc E. Rothenberg,‖ and Fred D. Finkelman*,†,#

Alternatively activated macrophages prevent lethal intestinal pathology caused by worm ova in mice infected with the human

parasite Schistosoma mansoni through mechanisms that are currently unclear. This study demonstrates that arginase I (Arg I),

a major product of IL-4– and IL-13–induced alternatively activated macrophages, prevents cachexia, neutrophilia, and endotox-

emia during acute schistosomiasis. Specifically, Arg I-positive macrophages promote TGF-b production and Foxp3 expression,

suppress Ag-specific T cell proliferation, and limit Th17 differentiation. S. mansoni-infected Arg I-deficient bone marrow chi-

meras develop a marked accumulation of worm ova within the ileum but impaired fecal egg excretion compared with infected

wild-type bone marrow chimeras. Worm ova accumulation in the intestines of Arg I-deficient bone marrow chimeras was

associated with intestinal hemorrhage and production of molecules associated with classical macrophage activation (increased

production of IL-6, NO, and IL-12/IL-23p40), but whereas inhibition of NO synthase-2 has marginal effects, IL-12/IL-23p40

neutralization abrogates both cachexia and intestinal inflammation and reduces the number of ova within the gut. Thus,

macrophage-derived Arg I protects hosts against excessive tissue injury caused by worm eggs during acute schistosomiasis by

suppressing IL-12/IL-23p40 production and maintaining the Treg/Th17 balance within the intestinal mucosa. The Journal of

Immunology, 2010, 184: 6438–6446.

Interleukin-4 and IL-13 protect hosts against a variety of para-sitic helminths by signaling through IL-4Ra–chain (IL-4Ra) onboth bone marrow (BM)- and non–BM-derived cells (1). Mac-

rophages (Mfs) activated through IL-4Ra suppress lethal in-flammation caused by worm ova produced by Schistosoma mansoni,a tropical parasitic helminth that causes lung, liver, and intestinal fi-brotic granulomas in 250 million people worldwide (2, 3). IL-4Ra–dependent alternatively activated Mfs (AAMfs) may downregulateS. mansoni-induced inflammation during acute schistosomiasis (7–9wk postinfection) through production of immunosuppressive cyto-kines such as IL-10 and TGF-b that antagonize IL-17–associatedneutrophilia and tissue injury (4). AAMfs could also inhibit thedifferentiation of classically activatedMfs (CAMfs), which secreteproinflammatory cytokines such as IL-6, IL-12/IL-23p40, and pro-duce reactive nitrogen and oxygen intermediates that cause oxidativetissue damage (5). In contrast, IL-4Ra–mediated signaling induces

debilitating liver fibrosis during chronic S. mansoni infection (.12wk postinfection) (6), which suggests that the Th2 response providesa selective advantage for the host that prevents rapid lethality at theexpense of chronic morbidity.Arginase I (Arg I) is an enzyme postulated to downregulate in-

flammatory responses and facilitate tissue remodeling, because itcompetes with inducible NO synthase-2 (NOS-2) for L-arginine andinitiates the pathways that lead to synthesis of proline and polyamines(7). MfArg I production is strongly stimulated by IL-4/IL-13 duringhelminth infections, although it can alsobe induced to a limited extentby unicellular pathogens through aSTAT-6–independent pathway (8).Mf-derived Arg I has recently been shown to decrease chronic im-munopathology in S. mansoni-infected mice by suppressing Th2 cellexpansion and reducing chronic liver fibrosis (9). However, becauseIL-4/IL-13–induced Arg I may antagonize classical Mf activationduring acute infection with S. mansoni (10), we asked whether Arg Ialso protects against acute immunopathology initiated by worm ovadeposition in host tissues. Indeed, S. mansoni-infected chimeric micelacking Arg I in BM-derived cells develop IL-12/IL-23p40–dependent neutrophil-associated gut pathology, intestinal hemor-rhage, and endotoxemia. This suggests thatAAMf production ofArgI is a host protective mechanism that restricts tissue damage inducedby worm ova during their excretion from blood vessels into the in-testinal lumen by downregulating production of IL-12 and/or IL-23that would otherwise cause lethal intestinal inflammation.

Materials and MethodsMice and S. mansoni infection

Sex-matched 6- to 8-wk-old BALB/c male wild-type (WT) and IL-4Ra–deficient mice were purchased from Taconic Farms (Germantown, NY)and used for BM chimera generation and experiments that involvedtreatment with arginase or NO synthetase antagonists. Efficiency of donorchimerism was determined by evaluation of IL-4Ra surface staining byflow cytometry as described previously (11). WT C57BL/6 (CD45.1) mice

*Research Service, Cincinnati Veterans Administration Medical Center, Cincinnati,OH 45220; †Division of Immunology, University of Cincinnati College of Medicine,Cincinnati, OH 45267; ‡Department of Chemistry, University of Pennsylvania, Phil-adelphia, PA 19104; xDepartment of Molecular and Human Genetics, Baylor Collegeof Medicine, Houston, TX 77030; {Mental Retardation Research Center, Universityof California Los Angeles, Los Angeles, CA 90095; and ‖Division of Allergy andImmunology and #Division of Immunobiology, Cincinnati Children’s Hospital Med-ical Center, Cincinnati, OH 45229

Received for publication June 24, 2009. Accepted for publication March 26, 2010.

This work was supported by the U.S. Department of Veterans Affairs and NationalInstitutes of Health Grant RO1GM083204.

Address correspondence and reprint requests to Dr. De’Broski R. Herbert, Division ofImmunobiology, Cincinnati Children’s Research Foundation, 3333 Burnet Avenue,Cincinnati, OH 45229. E-mail address: [email protected]

Abbreviations used in this paper: AAMf, alternatively activated macrophage; Arg I,arginase I; AST, aspartate aminotransferase; BEC, S-(2-boronoethyl)-L-cysteine; BM,bone marrow; CAMf, classically activated Mf; IL-4Ra, IL-4Ra–chain; Mf, mac-rophage; MFI, mean fluorescence intensity; MPO, myeloperoxidase; NOS-2, NOsynthase-2; PEC, peritoneal exudate cell; RELMa, resistin-like molecule a; Treg,regulatory T cell; WT, wild-type.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902009

mailto:[email protected]

were purchased from The Jackson Laboratory (Bar Harbor, ME), and sex-matched animals were used as recipients of BM isolated from Arg I2/2 orheterozygous Arg I+/2 (postnatal days 9–12) mice (CD45.2). Recipientswere sublethally irradiated (1175 rad of [137Cs]) and reconstituted with13 106 total BM cells, and donor chimerism was evaluated 10 wk later viaflow cytometry using splenocytes stained with allotype-specific Abs thatrecognized CD45.1 or CD45.2 (BD Pharmingen, San Diego, CA). Micethat showed ,95% donor chimerism were excluded from further experi-mental analysis. Mice were anesthetized and percutaneously infected with50–70 S. mansoni cercariae (11) from snails that were provided by theNational Institute of Allergy and Infectious Diseases SchistosomiasisResource Center through NIAID Contract N0-AI-30026.

Determination of chimerism via flow cytometry

PBMCs or splenocytes were isolated by Ficoll gradient, after whichsplenocytes were washed in FACS buffer (HBSS, 1% FCS, and 0.2% so-dium azide) and incubated with anti-FcgRII/RIII mAb (2.4G2). PBMCswere stained with fluorochrome-labeled rat mAbs to mouse CD11b (M1/70) or the allotypic markers CD45.1 and CD45.2 (BD Pharmingen). Cellswere then stained with the mAbs and analyzed with BD FACSCalibur andCellQuest software. Percent donor cells were calculated as describedpreviously (11).

T cell proliferation via CFSE dilution

Naive CD4+ cells were isolated from single-cell suspensions of lymphnode and spleen from 6- to 8-wk-old OVA-specific TCR transgenic mice(OTII), using an anti–CD4-labeled magnetic microbead (clone L3T4)-based MACS separator system (Miltenyi Biotec, Bergisch Gladbach,Germany). Purity was ∼90% as determined by flow cytometry. IsolatedCD4+ cells were incubated with 10 mM CFSE (Invitrogen, Carlsbad, CA)in sterile Dulbecco’s PBS at 37˚C for 10 min, quenched with 3% BSA/PBS, and used for Mf coculture experiments.

Mf-T cell coculture

Mfs were derived from BM by culturing in DMEM with 10% FCS andL-glutamine, supplementedwith supernatant from anM-CSF–producing cellline (CMG, 1/32 dilution of supernatant). On day 3, differentiatedMfs wereharvested with 0.05% trypsin, and 0.02% EDTA and M-CSF–containingmediumwere replacedwith freshD-10mediumwithM-CSF. On day 5,Mfs(1 3 105/well) were either left untreated or pulsed with 50 mg/well OVA(Sigma-Aldrich, St. Louis, MO) overnight, washed several times, and co-cultured with purified naive OT-II CD4+ cells at a ratio of 10:1 (CD4+/Mf)for 96 h. Cell supernatants were removed at 96 h and stored at280˚C prior toanalysis by ELISA.Cells were harvested, stainedwithAPC-conjugated anti-CD4 (clone GK1.5) (eBioscience, San Diego, CA) and 7-aminoactinomycinD (Sigma-Aldrich), and evaluated for CFSE mean fluorescence intensity ofevents within the CD4+ T cell gate.

Determination of S. mansoni tissue egg burden, granulomameasurement, and immunohistochemistry

Biopsies of liver and intestine (ileum) were collected from each mouse,weighed, and digested in 5% KOH at 37˚C for 16 h, and eggs were countedat 340 magnification. Data are expressed as eggs per gram of tissue. Forgranuloma size determination, 10% formalin-fixed and paraffin-embeddedorgans were histologically processed into sections of liver or intestinaltissue that were cut at 5 mm and stained with H&E. Individual granulomaareas were measured on coded slides; only granulomas that possesseda central egg were evaluated. Quantitation of area was performed using anSPT Diagnostics imaging system and Simple PCI C-Imaging systems soft-ware (SDR Clinical Technology, Cranberry Township, PA) for granulomaarea measurement. Data shown are mean 6 SE of 150 granulomas/groupfrom two independent experiments. Arg I immunohistochemistry wasperformed on paraffin-embedded sections. Citric acid-based Ag retrievalwas used, and sections were incubated with the mouse on mouse blockingkit and an avidin/biotin complex staining kit (Vector Laboratories, Bur-lingame, CA) used for diaminobenzidine-based detection.

Fecal egg quantification

Fecal egg number was determined as described previously (10). In brief,fecal pellets from individual mice at 8 wk postinfection were collectedand weighed. Feces were homogenized in cold 23 PBS and centrifuged at500 3 g at 4˚C. Pellets were reconstituted in fresh 23 PBS, after whicha fecal slurry was spread over a petri dish, and eggs were counted usinga stereomicroscope at 310 magnification. Data are expressed as eggs pergram of feces.

Real-time PCR

RNA was obtained from intestinal tissue and DNAse I-treated cDNA wasgeneratedusingSuperScript IIReverseTranscriptase (Invitrogen).Real-timePCRwas carried out on aGeneAmp7500 instrument (PEBiosystems, FosterCity, CA) with the Sybr Green detection reagent. Cycle threshold values forgenes evaluated were determined, and fold induction was compared withvimentin using the 1/Dct method. The sequences are as follows: vimentin,59-GTG CGC CAG CAG TAT GAA AG-39 (forward) and 59-GCA TCGTTG TTC CGG GTT GG-39 (reverse); Arg I, 59-CAG AAG AAT GGAAGAGTC AG-39 (forward) and 59-CAG ATATGC AGG GAG TCACC-39(reverse); resistin-like molecule a (RELMa), 59-AGA TGG GCC TCCTGC CCT GCT GGG-39 (forward) and 59-ACC TGG TGA CGG GCGACG ACG GTT-39 (reverse); TGF-b1, 59-CCG CAACAACGC CAT CTATG-39 (forward) and 59-AGC CCT GTA TTC CGT CTC CT-39 (reverse);IL-17A, 59-ACTACC TCA ACC GTT CCA CG-39 (forward) and 59-CACACC CAC CAG CAT CTT CT-39 (reverse); IL-12/IL-23p40, 59-TGT GGAATG GCG TCT CTG T-39 (forward) and 59-GGG TCT GGT TTG ATGATG TCC-39 (reverse); IL-10, 59-AAG GGT TAC TTG GGT TGC CA-39(forward) and 59-TCA CTC TTC ACC TGC TCC AC-39 (reverse); IL-22,59-TGC TTC TCATTG CCC TGT G-39 (forward) and 59-TGG ATG TTCTGG TCG TCA CC-39 (reverse); and IL-6, 59-TTC ACA AGT CGG AGGCTT-39 (forward) and 59-CA GTT TGG TAG CAT CCAT-39 (reverse).

Evaluation of cytokine production and morbidity

IL-12/IL-23p40, TGF-b, IL-17A, IFN-g, and IL-6 levels were measuredusing commercially available ELISA kits (eBioscience). In some instances,production of IL-4, TNF, and IFN-g was measured in sera by in vivo cy-tokine capture assay (11). Serum LPS concentration was measured usinga commercially available LAL kit (BioWhittaker, Walkersville, MD) asdescribed previously (11). Mouse myeloperoxidase (MPO) was measuredby a commercially available ELISA (Cell Sciences, Canton, MA). Nitritewas measured by Griess reaction (Promega, Madison, WI). Ileal punchbiopsy specimens were cultured in RPMI 1640 plus 10% FCS for 24 h, andsupernatants were stored at280˚C prior to ELISA. Determination of serumlevels of aspartate transaminase was performed at the Cincinnati VeteransAdministration Medical Center Clinical Pathology Laboratory (Cincinnati,OH) as described previously (11). Peritoneal exudate cells (PECs) wereisolated following peritoneal lavage of WT and Arg I2/2 BM chimeras at7 wk postinfection with RPMI 1640 plus 10% FCS, allowed to adhere toplastic for 1 h, washed, trypsinized, and replated at 2.53 104/ml. Cells wereleft untreated or exposed to LPS (1 mg/ml) for 16 h, and supernatants wereharvested and stored at 280˚C prior to analysis.

Pharmacological inhibitors

For arginase inhibition, mice were provided drinking water that contained0.2% S-(2-boronoethyl)-L-cysteine (BEC), which was synthesized andpurified as described previously (12). Some mice were injected biweeklywith 2 mg of the NOS-2 antagonists 1400W (Sigma-Aldrich) or NG-monomethyl-L-arginine (Sigma-Aldrich). Rat-anti-mouse IL-12/IL-23p40(C17.8) (Wistar Institute Philadelphia, PA) or an IgG2a isotype control(GL117) was produced in Pristane-primed (Acros Organics) athymic nudemice as ascites and purified by ammonium sulfate precipitation and diethyl-amino-ethyl-cellulose column chromatography as described previously (4).

Statistical analysis

Statistical significance was assessed by either one-tailed Students t test (twogroups) or ANOVA for multiple groups with a posthoc Bonferroni’s test todetermine significance; all were performed using PrismGraph Pad software.

ResultsArg I expression is dependent on IL-4Ra expression inBM-derived cells

Duringmurine schistosomiasis, the nature of L-argininemetabolismis closely linked to disease outcome. Whereas IFN-g–driven NOS-2 is associated with CAMfs and increased mortality, IL-4/IL-13–driven Arg I production is associated with AAMfs and long-termsurvival (7, 10). Consistent with this, S. mansoni-infected chimericmice that express IL-4Ra only on BM-derived cells survive betterthan infected mice that lack IL-4Ra on these cells (11), and in-testinal Arg I mRNA is increased 8- to 9-fold in chimeras thatexpress IL-4Ra only on BM-derived cells (non–BMIL-4Ra2/2)compared with chimeras that express IL-4Ra only on non–BM-

The Journal of Immunology 6439

derived cells (BM IL-4Ra2/2) (Fig. 1A). Intestinal Arg I mRNAlevels are also ∼3-fold higher in S. mansoni-infected non–BMIL-4Ra2/2 than in infected WT mice, suggesting that increased Arg Iproduction by IL-4/IL-13–responsive BM-derived cells may com-pensate for the failure of non–BM-derived cells to respond to IL-4and IL-13. Furthermore, BM-derived IL-4Ra is required for Arg Iexpression by inflammatory cells that encapsulate worm ova withinintestinal granulomas during acute schistosomiasis (Fig. 1B).

Arg I protects S. mansoni-infected mice from cachexia andmortality during acute infection

We predicted that if Arg I production by AAMfs reduces tissueinjury, impairment of arginase activity should exacerbate infection-induced immunopathology caused by worm ova that lodge in hostorgans. Two approaches were used to test this hypothesis: 1) S.mansoni-infected WT BALB/c mice were treated with BEC,a competitive inhibitor that specifically blocks Arg I and II function(12, 13); and 2) BM chimeric mice, generated by reconstitutinglethally irradiated C57BL/6 WT mice (Ly5.1+) with BM fromLy5.2+ Arg I-deficient (Arg I2/2 BM) or Ly5.2+ WT (WT BM)mice, were infected with S. mansoni. Results of studies with thesetwomodels were remarkably similar and were consistent with thoserecently reported in Pesce et al. (9). S. mansoni-infection causedsevere weight loss (Fig. 2A) and ∼20–30% mortality (Fig. 2B) inmice provided with 0.2% BEC in drinking water, but these did notoccur in BEC-treated uninfected mice or in infected mice that were

not treated with BEC. Similarly, severe weight loss (Fig. 2C) and30–35% mortality (Fig. 2D) developed in infected Arg I2/2 BMmice but not infected WT BMmice. The monocyte/Mf population(as defined by CD11b+ staining) was consistently .95% donorderived in both chimeric groups (data not shown). However, wenoted that most worm-infected mice began to recover ∼9 wkpostworm inoculation, regardless of whether mice were BEC-treated or Arg I BM2/2 chimeras (data not shown). This is distinctfrom S. mansoni-infected Mf-specific IL-4Ra–deficient mice,which experience 100% mortality by 9 wk postinoculation (10).Thus, Arg I production appears to be an important mechanism butnot the only mechanism by which myeloid cell IL-4Ra expressioninhibits lethal inflammation.

BM-derived Arg I protects against worm ova-induced damageto the intestine

S. mansoni adult worms produce large numbers of parasite ova(size range, 60–100 mm) starting 5.5 wk postinfection, which causegranulomas to form in liver and intestine. Arg I2/2 BM chimerasdid not show any signs of excessive liver injury (Fig. 3A) or alteredgranuloma size (Fig. 3B), but these animals developed numeroushemorrhagic areas within the gut mucosa by 7.5 wk postinfectionthat were not observed in WT BM chimeras (Fig. 3C). These ab-normalities were accompanied by neutrophil infiltration (Fig. 3D)

FIGURE 1. Arg I production in S. mansoni granulomas requires IL-4Ra

expression only on BM-derived cells. A, Intestinal mRNA transcripts for

Arg I were quantitated by real-time PCR 7.4 wk postinoculation in

S. mansoni-infected BM chimeras that expressed IL-4Ra on all cells (WT),

lacked IL-4Ra expression on all cells (IL-4Ra2/2), lacked IL-4Ra ex-

pression on BM-derived cells (BMIL-4Ra2/2), or lacked IL-4Ra ex-

pression on non–BM-derived cells (non–BMIL-4Ra2/2). Transcript levels

are expressed as fold increase compared with naive WT tissue. Experiment

performed three times with similar results. n = 6–8 mice/group; pp, 0.05;

ppp , 0.01 compared with WT. B, Immunohistochemical staining for Arg

I was performed 7.4 wk postinfection on intestinal granulomas from WT

(top left panel), IL-4Ra2/2 (top right panel), BMIL-4Ra2/2 (bottom left

panel), and BMIL-4Ra2/2 (bottom right panel) mice. Original magnifi-

cation 3400. Representative photos from 150 granulomas examined per

group. Positive staining indicated by dark brown color.

FIGURE 2. Mice treated with an arginase antagonist or chimeras lacking

BM-derived Arg I develop cachexia and increased mortality during acute

schistosomiasis. Weight change (A) and survival (B) of naive or S. mansoni-

infectedmice thatwere administered normal drinkingwater or drinkingwater

containingBEC.Weight change (C) and survival (D) ofWTandArg I2/2BM

chimeras following inoculation with 60–70 S. mansoni cercariae. Repre-

sentative of three independent experimentswith 8–10mice/group.pp, 0.05;

ppp, 0.01; pppp, 0.001 compared with WT-infected group.

6440 Arg I SUPPRESSES WORM-INDUCED IMMUNOPATHOLOGY

and greatly increased serum levels of endotoxin (Fig. 3E). Arg I2/2

BM chimeras had significantly fewer worm ova within the feces(Fig. 3F) but increased numbers of worm ova in the terminal ileumcompared with WT controls (Fig. 3G). A remarkably similarphenotype was noted in BEC-treated WT mice analyzed between 7and 8 wk postinfection (data not shown). In contrast, we neverobserved increased numbers of hepatic ova in S. mansoni-infectedArg I2/2 BM chimeras (Fig. 3H) or BEC-treated WT mice (datanot shown). These data suggest that the rate in which parasite ovaare excreted from the bowel wall was markedly impaired in micelacking BM-derived Arg I. Thus, Arg I deficiency preferentially

exacerbated intestinal injury but only caused marginal effects onS. mansoni-induced liver pathology.

Arg I-deficient chimeras produce molecules associated withCAMf

By 7.5 wk postinfection, BM Arg I deficiency did not impair IL-4(Fig. 4A) or IL-13 (data not shown) production but resulted ina significant increase of IL-12/IL-23p40, IFN-g and IL-6 (Fig. 4B–D), as compared with WT controls. Similar abnormalities in cy-tokine profile were observed in S. mansoni-infected IL-4Ra2/2,IL-42/2, and Mf-specific IL-4Ra2/2 mice (10, 14, 15); therefore,we hypothesized that BM Arg I deficiency during S. mansoni in-fection predisposes Mfs to express proinflammatory moleculesassociated with CAMfs. To address this issue, adherent PECs fromArg12/2 BM or WT chimeras were stimulated with LPS to mimicendotoxin exposure as worm ova pass from the small intestine intothe fecal stream, a process that normally occurs during infection(16). Indeed, LPS-stimulated significantly more IL-12/IL-23p40(Fig. 4E) and nitrite (Fig. 4F) from Arg I2/2 adherent peritonealexudates, compared with WT cells.To directly assess whether Mf-specific Arg I expression influ-

ences T cell proliferation or cytokine production, in vitro cocultureexperiments were performed that usedWTor Arg I2/2BMMfs andCFSE-labeled CD4+ T cells purified from OVA-specific TCRtransgenic mice (OT-II). As expected, marginal dilution of CFSEwas observed when WT Mfs were incubated with OVA-specificT cells in the absence of Ag (Fig. 5A); however, Arg I2/2 Mfsstimulated a moderate amount of CFSE dilution (indicative of pro-liferation) (Fig. 5B). In the presence ofOVA,WTMf-inducedCFSEdilution in OT-II cells was 3-fold less than Arg I2/2 Mf (Fig. 5C,

FIGURE 3. BM Arg I deficiency causes increased tissue injury, ova

accumulation, and neutrophilic inflammation in the intestine during acute

schistosomiasis. Serum levels of AST (A) and liver granuloma size (B).

Representative of 150 granulomas evaluated with 8–10 mice/group. C,

Representative photographs of terminal ileum from S. mansoni-infected

WT BM or Arg I2/2 BM chimeras 7.4 wk postinoculation. Note areas of

hemorrhage (arrows). D, Representative photomicrographs of intestinal

granulomas in WT BM and Arg I2/2 BM chimeras at 7.5 wk postworm

inoculation. Arrowheads point to erythrocytes, and arrows indicate neu-

trophils. Fifty granulomas examined per group. Original magni-

fication3600. Serum endotoxin levels (E), number of parasite ova in feces

(F), intestine (G), and liver (H) at 7.4 wk postinoculation. Representative

of three independent experiments with 8–10 mice/group. pp, 0.05; ppp,0.01; pppp , 0.001 compared with WT-infected group. AST, aspartate

aminotransferase.

FIGURE 4. Arg I-deficient BM chimeras produce elevated proin-

flammatory cytokines and show evidence of classical Mf activation.

Systemic production of IL-4 (A), IL-12/IL-23p40 (B), IFN-g (C), and IL-6

(D) in WT and Arg I2/2 BM chimeras analyzed 7.4 wk postinoculation.

Adherent peritoneal cells from S. mansoni-infected WT BM or Arg I2/2

BM chimeras were stimulated with 1 mg/ml Escherichia coli endotoxin for

24 h and supernatants analyzed for levels of IL-12/IL-23p40 (E) and nitrite

(F). Representative of three independent experiments with mean 6 SE,

n = 8–10 mice/group. pp , 0.05; ppp , 0.01.


5D). Analysis of culture supernatants revealed an inverse correlationbetween the production of IL-6 and TGF-b, with increased IL-6 andsignificantly less TGF-b produced byT cell-Arg I2/2Mf coculturescomparedwith theWTMf-T cell cocultures (Fig. 5E, 5F). This wasassociated with significantly increased levels of IL-17A, but nodifference between IFN-g levels in T cell-Arg I2/2 Mf cocultures,compared with T cell-WTMf cocultures (Fig. 5G, 5H). Combined,this suggests that Arg I + Mfs suppress Th17 differentiation fromnaive Ag-specific T cells.Real-time PCR was used to determine whether the lack of Arg I +

Mf resulted in a more proinflammatory microenvironment forS. mansoni ovawithin the small intestine. Results revealed amarkeddecrease in the relative amounts of IL-10 and TGF-b (Fig. 6A, 6B)and a significant increase in IL-12/IL-23p40, IL-6, IL-22, andIL-17AmRNA transcripts (Fig. 6C–F) in the intestinal tissue of ArgI2/2BMchimeras, as comparedwithWTBMchimeras 7.5wk afterS. mansoni inoculation. This expression profile correlated with

impaired Foxp3 (forkhead box P3) and elevated retinoic acid re-ceptor-related orphan receptor (Rorgt) expression (Fig. 6G, 6H),suggesting an imbalance of regulatory T (Treg) and Th17 cells,respectively (17). As expected, S. mansoni-infected Arg I2/2 BMchimeras express much less Arg I mRNA (Fig. 6I) but similar levelsof otherAAMf-associated genes, includingRELMa (FIZZ-1) (Fig.6J), as compared with infected WT controls. Thus, lack of BM-derived Arg I (most likely in Mfs) profoundly disrupts the normalcytokine and cellularmilieu ofwormovawithin the intestine towarda more proinflammatory environment.

IL-12/IL-23p40 neutralization in Arg I-deficient BM chimerasabrogates cachexia and gut injury

To determine whether NO and/or IL-12/IL-23p40 directly contributeto intestinal pathology in S. mansoni-infected mice, pharmacologicalinhibition of NOS-2 and neutralization of IL-12/IL-23p40 was per-formed. Treatment ofArg I2/2BMchimeraswith theNOS-2–specific

FIGURE 5. Arg I-deficient macrophages drive Ag-

specific CD4+ T cell proliferation and preferentially in-

duceTh17differentiation.Contour plots showingMFIof

CFSE within OTII CD4+ T cells following coculture

with (A, C) WT or (B, D) Arg I-deficient macrophages

that were either (A,B) untreated or (C,D) pulsed with 50

mg/ml OVA. Analysis was performed at 96 h. From the

experiment shown above, supernatants were analyzed

for production of IL-6 (E), TGF-b (F), IL-17 (G), and

IFN-g (H) by ELISA. N, media alone; n, OVA. Repre-

sentative of three independent experiments withmean6SE of triplicatewells. pp, 0.05; ppp, 0.01.MFI, mean

fluorescence intensity.


antagonists 1400W (Fig. 7A) or NG-monomethyl-L-arginine (data notshown) did not reverse cachexia, but treatment with anti–IL-12/IL-23p40 mAb abrogated infection-induced weight loss (Fig. 7B). Anti–IL-12/IL-23p40 mAb treatment also significantly reduced serum en-

dotoxin levels (Fig. 8A) and the number of gut-trapped ova 8 wk afterworminoculation (Fig. 8B).Anti–IL-12/IL-23p40mAb treatment alsoreduced serum levels of MPO (an indicator of neutrophil de-granulation) (Fig. 8C), IFN-g, (Fig. 8D), IL-17A (Fig. 8E), and ex vivoIL-6 production by ileal explants (Fig. 8F). Combined, this demon-strates a critical role for IL-12/IL-23p40 production in driving theneutrophil associated intestinal injury that occurs in S. mansoni-infected Arg I2/2 BM chimeras.

DiscussionTh2-associated cytokines (IL-4/IL-13) can promote host protectionduring many parasitic and inflammatory diseases through mech-anisms that are incompletely understood. We have previouslydemonstrated that myeloid cell-specific IL-4Ra expression wascritical for induction of AAMfs, host survival, and suppression ofmultiorgan inflammation during murine schistosomiasis (10, 11).In this paper, we demonstrate that BM-derived IL-4Ra expressionis required for Arg I production at the nidus of inflammationcaused by S. mansoni ova within the intestine. Mf-derived Arg Isuppresses Ag-specific T cell proliferation and Th17 differentia-tion in vitro, and Arg I neutralization during S. mansoni infectionreduces Treg-associated cytokine production, increases Th1- andTh17- associated intestinal inflammation, and leads to 20–40%mortality. Arg I2/2 BM chimeras are rescued via neutralization ofIL-12/23p40 (essential component of IL-12 and IL-23), whichsuggests that CAMfs are largely responsible for this severe in-flammatory disease phenotype (18). Thus, IL-4/IL-13 productionsuppresses pathogenesis driven by CAMfs through the antago-nistic functions of Arg I; a conclusion that is remarkably con-cordant with demonstration that intracellular pathogens induceArg I in Mfs as an immune evasion strategy that dampens CAMfeffector function (8).AAMf-derived Arg I can promote expulsion of gastrointestinal

helminths via promoting smooth muscle contraction or decreasingworm viability (19, 20). However, host protection during schis-tosomiasis is achieved by suppression of inflammation directed

FIGURE 6. Arg I-deficient BM chimeras show de-

creased expression of immunosuppressive cytokines

and increased Th17-associated cytokines in the in-

testine during acute schistosomiasis. Intestinal mRNA

levels for murine IL-10 (A), TGF-b (B), IL-12/IL-

23p40 (C), IL-6 (D), IL-17 (E), IL-22 (F), Foxp3 (G),

Rorgt (H), ArgI (I), and RELMa (J), quantitated by

real-time PCR 7.4 wk post-S. mansoni inoculation of

BM chimeras. Representative of three independent ex-

periments with mean 6 SE, n = 8–10 mice/group. pp,0.05; ppp , 0.01.

FIGURE 7. IL-12/IL-23p40 neutralization but not NOS-2 inhibition

rescues Arg I2/2 BM chimeras from cachexia. Weight change of

S. mansoni-infected WT and Arg I2/2 BM chimeras that were adminis-

tered 2 mg biweekly injections of the NOS-2 antagonist 1400W versus

saline (vehicle) (A) or 2 mg rat anti–IL-12/IL-23p40 (C17.8) versus rat

IgG2a (GL117) isotype control mAb (B) starting 5 wk postworm in-

oculation. Representative of two independent experiments n = 8–10 mice/

group. pp , 0.05; ppp , 0.01.


against the highly immunogenic worm ova, not by elimination ofadult worms (16). Indeed, death from S. mansoni infection isdriven by granulomatous pathology that, if left unchecked, causesextensive collateral damage to liver and intestinal tissues (21).There is extensive evidence from infected humans and mice thatdemonstrates Th2 differentiation is host protective during schis-tosomiasis (13, 22). In contrast, inflammation characterized byTh1 and/or Th17 responses and neutrophil accumulation stronglycorrelates with severe hepatosplenic disease marked by cachexiaand death (23). Th2 responses generated in patients with a milderform of intestinal schistosomiasis are characterized by fecal occultblood, diarrhea, and abdominal pain, which can persist for .20 ybut is rarely fatal (3). Our data in the mouse model support a hy-pothesis that AAMf-derived Arg I drives a critical immunoreg-ulatory network that limits worm ova-driven inflam-mation within the gut mucosa through antagonizing Th1- andTh17-associated immunopathology.S. mansoni adult worm pairs livewithin themesenteric veins of the

mammalianhost.Although the largemajority ofwormova are carriedvia portal blood flow into the liver, a smaller percentage travel in theopposite direction (against blood flow) into the small- and large-bowel tissue for excretion in the feces (3, 16). Thus, S. mansoni eggsthat lodge within the liver are at a “dead end,” whereas S. mansonieggs within the intestine tissue are “en route” to the external envi-ronment. Although each worm ova can be 60–100 mm in diameter,infection ofmost immunocompetentmouse strainsdoesnot cause anyobvious sign of gut injury. However, mice lacking IL-4–responsiveMfs develop marked intestinal hemorrhage, endotoxemia, and∼100% mortality by 9 wk postinfection (10, 14, 15). DemonstratingthatBM-derivedArg I also protects against intestinal hemorrhage and

neutrophilic inflammation is consistent with an essential role forAAMfs, inasmuch as IL-4Ra expression in BM-derived cells wasessential for Arg I expression. Interestingly, Arg I2/2 chimeras har-bored significantly greater numbers of worm ova within the bowelwall, but fewer excreted ova at 7.5 wk postinfection (Fig. 3). Anthonyet al. (20) also described an IL-4R+Mf populationwithin themucosaof the small intestine that killed Heligmosomoides polygyrus larvaethrough amechanism dependent on arginase activity. Taken together,this indicates that Mf-derived Arg I confers host protection throughdirect effects on the parasite or indirect effects that modulate immuneresponses generated by the host.Arg I limits intestinal inflammation directed against worm ova

lodged in the intestine by promoting anti-inflammatory cytokineproduction and inhibiting proinflammatory cytokine production.Using an OVA transgenic TCR system, we show that coculture ofArg I-deficient Mfs with naive CD4+ T cells caused significantlygreater T cell proliferation compared with WT Mfs. More im-portantly, cultures that contained Arg I-deficient Mfs producedsignificantly more IL-6 and IL-17 and significantly less TGF-bcompared with cultures containing naive CD4+ T cells and WTMfs. The ratio of IL-6 to TGF-b is a critical determinant of Th17versus Foxp3+ Treg induction (24, 25), and high levels of TGF-bdirectly suppress Th17 differentiation (17). Consistent with this,S. mansoni-infected Arg I2/2 BM chimeras produce significantlymore intestinal mRNA transcripts for Rorgt, IL-17, and IL-22 andsignificantly less Foxp3, IL-10, and TGF-b compared with in-fected WT BM chimeras. This is also consistent with our recentdemonstration that the immunosuppressive cytokines IL-10 andTGF-b redundantly suppress lethality associated with IL-17 pro-duction and neutrophil expansion during acute schistosomiasis (4).

FIGURE 8. IL-12/IL-23p40 neutralization

abrogates intestinal inflammation and injury and

reduces S. mansoni intestinal egg accumulation

in Arg I2/2 BM chimeras. Levels of serum en-

dotoxin (A), number of parasite ova in intestine

(B), serum MPO (C), IFN-g (D), IL-17A (E),

and IL-6 (F) produced from ileal punch biopsies

and serum levels of IFN-g and IL-17A analyzed

8 wk postworm inoculation. Mean 6 SE of 8–10

mice/group. Representative of two independent

experiments. pp , 0.05; ppp , 0.01.


In contrast to the profound effects of Arg I deficiency on intestinalinflammation, we find no evidence for increased liver injury orgranulomasize in theArg I2/2chimerasduringacute schistosomiasis.This contrasts with previous studies performed with S. mansoni-infected mice that have Mf/neutrophil-specific IL-4Ra deficiencybecause thesemice fail to upregulate Arg I but also develop increasedliver damage and larger granulomas (10). The mechanistic basis forthese differences may be related to the production of other immu-noregulatory molecules by AAMfs. RELMa, an IL-4–induced,AAMf-produced molecule that suppresses S. mansoni ova-inducedlung inflammation (26), was induced in Arg I2/2 chimeras at levelsequivalent to WT chimeras.As our study was being completed, Pesce et al. (9) reported that

mice lacking Mf-derived Arg I suffer increased mortality duringchronic schistosomiasis because of increased liver fibrosis, exac-erbated liver granulomatous pathology, increased hepatic CD4+

Th2 cells, and impaired T cell suppression byAAMf. In their study,Mf/neutrophil-specific Arg I-deficient mice began to die 8–9 wkpostinfection, and hemorrhagic intestinal pathology was noted atautopsy (9). In fact, there were increased percentages of Th1 andTh17 cells isolated from affected organs of Arg I-deficient mutants,but mortality was attributed to excessive Th2-driven fibrosis andother complications that developed beyond 12 wk of infection (9).Despite these differences in interpretation, together both studiessupport the conclusion that Arg I is a critical suppressor of T cell-associated inflammation during S. mansoni infection.Mechanistically, Arg I expression could suppress inflammation

via inhibition of Mf and/or dendritic cell production of the p40component of IL-12 and IL-23, which stimulates Th1 and Th17responses, respectively (23, 27). Consistent with this hypothesis,adherent PECs from S. mansoni-infected Arg I2/2 BM chimerasrelease significantly greater amounts of IL-12/IL-23p40 than PECsfrom infected WT chimeras. Strikingly, IL-12/IL-23p40 neutrali-zation reverses nearly all of the markers of disease exacerbation inArg I2/2 chimeras such as increased IL-6, IFN-g, and IL-17 pro-duction; neutrophil degranulation; endotoxemia; numbers of wormova lodged in the ileum; and infection-induced weight loss. Thisdemonstrates that IL-12/23p40 production is largely responsiblefor severe disease in the absence of Arg I. Production of IL-12/IL-23p40 is induced by treatment of alternatively activated humanmonocytes with an arginase antagonist (28), and IL-12/IL-23p40promotes severe schistosomiasis in mouse strains that produce el-evated IL-17 in response to worm ova (29).Demonstrating that anti–IL-12/IL-23p40 mAb treatment sup-

presses both serum LPS levels and intestinal inflammation sug-gests that excessive production of IL-12 and IL-23, rather thanincreased serum LPS, is the proximal event in S. mansoni-infectedArg I-deficient mice. Consistent with this, Gobert et al. (30)demonstrated that pharmacological inhibition of arginase activityincreased proinflammatory cytokine production and exacerbatedcolitis induced by Citrobacter rodentium, and it was also shownthat neutralization of IL-12/IL-23p40 reduces the severity ofmurine arthritis (31). Therefore, our findings support the view thatArg I production from AAMfs suppress IL-12/23–driven in-flammation, which may promote disruption of the mucosal barrierand leakage of intestinal bacteria and/or LPS into the blood.Taken together, our studies of Arg I deficiency during acute

schistosomiasis and the studies of Pesce et al. Arg I about deficiencyduring chronic schistosomiasis suggest a scenario that beginswith theentry of S. mansoni ova into host organ tissues that drives a Th2 re-sponse (32). IL-4 and IL-13 then stimulateAAMf differentiation andArg I production, which in turn promotes TGF-b secretion but sup-presses production of IL-6, IL-12, and IL-23. This reduces Th1 andTh17 differentiation and increases the Treg response that promotes

increased IL-10 and TGF-b. These later cytokines limit intestinalneutrophilic inflammation caused by the passage of worm ovathrough host intestinal tissue and may promote tissue repair as wormova are passed out of the intestine and into the fecal stream throughoutthe course of disease. Increased IL-4 and IL-13 production, however,has a price: it promotes development of hepatic cirrhosis that causesmorbidity and death during the chronic phase of schistosomiasis.Finally, it is worth pointing out that this scenario is a notable

example of convergent evolution, inasmuch as it benefits the hostby preventing life-threatening intestinal inflammation during theacute phase of S. mansoni infection but also benefits the parasiteby promoting long-term dissemination of worm ova from the in-fected host. This strong selective advantage for both parasite andhost may account for the persistence of Th2 responses despitedriving fibrotic disease during chronic schistosomiasis.

AcknowledgmentsWe thank Steve Finan, Jeff Bailey, and Kathryn Niese for expert technical

assistance.

DisclosuresThe authors have no financial conflicts of interest.

References1. Urban, J. F., Jr., N. Noben-Trauth, L. Schopf, K. B. Madden, and F. D. Finkelman.

2001. Cutting edge: IL-4 receptor expression by non-bone marrow-derived cells isrequired to expel gastrointestinal nematode parasites. J. Immunol. 167: 6078–6081.

2. Chitsulo, L., P. Loverde, and D. Engels. 2004. Schistosomiasis. Nat. Rev. Mi-crobiol. 2: 12–13.

3. Gryseels, B., K. Polman, J. Clerinx, and L. Kestens. 2006. Human schistoso-miasis. Lancet 368: 1106–1118.

4. Herbert, D. R., T. Orekov, C. Perkins, and F. D. Finkelman. 2008. IL-10 andTGF-b redundantly protect against severe liver injury and mortality during acuteschistosomiasis. J. Immunol. 181: 7214–7220.

5. La Flamme, A. C., E. A. Patton, B. Bauman, and E. J. Pearce. 2001. IL-4 playsa crucial role in regulating oxidative damage in the liver during schistosomiasis.J. Immunol. 166: 1903–1911.

6. Wynn, T. A., R. W. Thompson, A. W. Cheever, and M. M. Mentink-Kane. 2004.Immunopathogenesis of schistosomiasis. Immunol. Rev. 201: 156–167.

7. Hesse, M., M. Modolell, A. C. La Flamme, M. Schito, J. M. Fuentes, A. W. Cheever,E. J. Pearce, and T. A. Wynn. 2001. Differential regulation of nitric oxide synthase-2and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shapedby the pattern of L-arginine metabolism. J. Immunol. 167: 6533–6544.

8. El Kasmi, K. C., J. E. Qualls, J. T. Pesce, A. M. Smith, R. W. Thompson,M. Henao-Tamayo, R. J. Basaraba, T. Konig, U. Schleicher, M. S. Koo, et al.2008. Toll-like receptor-induced arginase 1 in macrophages thwarts effectiveimmunity against intracellular pathogens. Nat. Immunol. 9: 1399–1406.

9. Pesce, J. T., T. R. Ramalingam, M. M. Mentink-Kane, M. S. Wilson, K. C. ElKasmi, A. M. Smith, R. W. Thompson, A. W. Cheever, P. J. Murray, andT. A. Wynn. 2009. Arginase-1–expressing macrophages suppress Th2 cytokine-driven inflammation and fibrosis. PLoS Pathog. 5: e1000371.

10. Herbert, D. R., C. Holscher, M. Mohrs, B. Arendse, A. Schwegmann,M. Radwanska, M. Leeto, R. Kirsch, P. Hall, H. Mossmann, et al. 2004. Al-ternative macrophage activation is essential for survival during schistosomiasisand downmodulates T helper 1 responses and immunopathology. Immunity 20:623–635.

11. Herbert, D. R., T. Orekov, C. Perkins, M. E. Rothenberg, and F. D. Finkelman.2008. IL-4Ra expression by bone marrow-derived cells is necessary and suffi-cient for host protection against acute schistosomiasis. J. Immunol. 180: 4948–4955.

12. Kim, N. N., J. D. Cox, R. F. Baggio, F. A. Emig, S. K. Mistry, S. L. Harper,D. W. Speicher, S. M. Morris, Jr., D. E. Ash, A. Traish, and D. W. Christianson.2001. Probing erectile function: S-(2-boronoethyl)-L-cysteine binds to arginaseas a transition state analogue and enhances smooth muscle relaxation in humanpenile corpus cavernosum. Biochemistry 40: 2678–2688.

13. Cama, E., D.M. Colleluori, F. A. Emig, H. Shin, S. W. Kim, N. N. Kim, A. M. Traish,D. E. Ash, and D. W. Christianson. 2003. Human arginase II: crystal structure andphysiological role in male and female sexual arousal. Biochemistry 42: 8445–8451.

14. Fallon, P. G., E. J. Richardson, G. J. McKenzie, and A. N. McKenzie. 2000.Schistosome infection of transgenic mice defines distinct and contrasting path-ogenic roles for IL-4 and IL-13: IL-13 is a profibrotic agent. J. Immunol. 164:2585–2591.

15. Brunet, L. R., F. D. Finkelman, A. W. Cheever, M. A. Kopf, and E. J. Pearce.1997. IL-4 protects against TNF-a–mediated cachexia and death during acuteschistosomiasis. J. Immunol. 159: 777–785.

16. Pearce, E. J., and A. S. MacDonald. 2002. The immunobiology of schistoso-miasis. Nat. Rev. Immunol. 2: 499–511.


17. Zhou, L., J. E. Lopes, M. M. Chong, I. I. Ivanov, R. Min, G. D. Victora, Y. Shen,J. Du, Y. P. Rubtsov, A. Y. Rudensky, et al. 2008. TGF-b–induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgt function. Nature 453: 236–240.

18. Mosser, D. M., and X. Zhang. 2008. Activation of murine macrophages. Curr.Protoc. Immunol. Chapter 14: Unit 14.2.

19. Zhao, A., J. F. Urban, Jr., R. M. Anthony, R. Sun, J. Stiltz, N. van Rooijen,T. A. Wynn, W. C. Gause, and T. Shea-Donohue. 2008. Th2 cytokine-inducedalterations in intestinal smooth muscle function depend on alternatively activatedmacrophages. Gastroenterology 135: 217–225.e1.

20. Anthony, R. M., J. F. Urban, Jr., F. Alem, H. A. Hamed, C. T. Rozo, J. L. Boucher,N. Van Rooijen, and W. C. Gause. 2006. Memory T(H)2 cells induce alternativelyactivated macrophages to mediate protection against nematode parasites. Nat. Med.12: 955–960.

21. Hoffmann, K. F., A. W. Cheever, and T. A. Wynn. 2000. IL-10 and the dangers ofimmune polarization: excessive type 1 and type 2 cytokine responses inducedistinct forms of lethal immunopathology in murine schistosomiasis. J. Immunol.164: 6406–6416.

22. Patton, E. A., A. C. La Flamme, J. A. Pedras-Vasoncelos, and E. J. Pearce. 2002.Central role for interleukin-4 in regulating nitric oxide-mediated inhibition ofT-cell proliferation and g interferon production in schistosomiasis. Infect. Im-mun. 70: 177–184.

23. Rutitzky, L. I., and M. J. Stadecker. 2006. CD4 T cells producing pro-inflammatory interleukin-17 mediate high pathology in schistosomiasis. Mem.Inst. Oswaldo Cruz 101(Suppl. 1): 327–330.

24. Zhou, L., I. I. Ivanov, R. Spolski, R. Min, K. Shenderov, T. Egawa, D. E. Levy,W. J. Leonard, and D. R. Littman. 2007. IL-6 programs T(H)-17 cell differen-tiation by promoting sequential engagement of the IL-21 and IL-23 pathways.Nat. Immunol. 8: 967–974.

25. Zhou, L., M. M. Chong, and D. R. Littman. 2009. Plasticity of CD4+ T celllineage differentiation. Immunity 30: 646–655.

26. Nair, M. G., Y. Du, J. G. Perrigoue, C. Zaph, J. J. Taylor, M. Goldschmidt,G. P. Swain, G. D. Yancopoulos, D. M. Valenzuela, A. Murphy, et al. 2009. Alter-natively activated macrophage-derived RELM-a is a negative regulator of type 2inflammation in the lung. J. Exp. Med. 206: 937–952.

27. Ivanov, I. I., B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille,D. J. Cua, and D. R. Littman. 2006. The orphan nuclear receptor RORgt directsthe differentiation program of proinflammatory IL-17+ T helper cells. Cell 126:1121–1133.

28. Babu, S., V. Kumaraswami, and T. B. Nutman. 2009. Alternatively activated andimmunoregulatory monocytes in human filarial infections. J. Infect. Dis. 199:1827–1837.

29. Rutitzky, L. I., J. R. Lopes da Rosa, and M. J. Stadecker. 2005. Severe CD4T cell-mediated immunopathology in murine schistosomiasis is dependent onIL-12p40 and correlates with high levels of IL-17. J. Immunol. 175: 3920–3926.

30. Gobert, A. P., Y. Cheng, M. Akhtar, B. D. Mersey, D. R. Blumberg, R. K. Cross,R. Chaturvedi, C. B. Drachenberg, J. L. Boucher, A. Hacker, et al. 2004. Pro-tective role of arginase in a mouse model of colitis. J. Immunol. 173: 2109–2117.

31. Takahashi, N., V. C. de Jager, A. Gluck, M. Letzkus, N. Hartmann, F. Staedtler,F. Ribeiro-Dias, M. Heuvelmans-Jacobs, W. B. van den Berg, and L. A. Joosten.2008. The molecular signature of oxidative metabolism and the mode of mac-rophage activation determine the shift from acute to chronic disease in experi-mental arthritis: critical role of interleukin-12p40. Arthritis Rheum. 58: 3471–3484.

32. Pearce, E. J., C. M. Kane, J. Sun, J. J. Taylor, A. S. McKee, and L. Cervi. 2004.Th2 response polarization during infection with the helminth parasite Schisto-soma mansoni. Immunol. Rev. 201: 117–126.


Documents

Arginase I Suppresses IL-12/IL-23p40-Driven Intestinal Inflammation during Acute Schistosomiasis