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Infection Distal to the GutMutes Immune Responses to Mycobacterial Chronic Gastrointestinal Nematode Infection

Maizels, Antonio Gigliotti Rothfuchs and Susanne NylénÖsterblad, Helena Helmby, James P. Hewitson, Rick M.Isabel Dellacasa-Lindberg, Cajsa Classon, Markus Katja Obieglo, Xiaogang Feng, Vishnu Priya Bollampalli,

http://www.jimmunol.org/content/196/5/2262doi: 10.4049/jimmunol.1500970January 2016;

2016; 196:2262-2271; Prepublished online 27J Immunol 

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The Journal of Immunology

Chronic Gastrointestinal Nematode Infection Mutes ImmuneResponses to Mycobacterial Infection Distal to the Gut

Katja Obieglo,*,1,2 Xiaogang Feng,*,2 Vishnu Priya Bollampalli,*,2

Isabel Dellacasa-Lindberg,* Cajsa Classon,* Markus Osterblad,* Helena Helmby,†

James P. Hewitson,‡ Rick M. Maizels,‡ Antonio Gigliotti Rothfuchs,* and

Susanne Nylen*

Helminth infections have been suggested to impair the development and outcome of Th1 responses to vaccines and intracellular

microorganisms. However, there are limited data regarding the ability of intestinal nematodes to modulate Th1 responses at sites

distal to the gut. In this study, we have investigated the effect of the intestinal nematode Heligmosomoides polygyrus bakeri on Th1

responses to Mycobacterium bovis bacillus Calmette–Guerin (BCG). We found that H. polygyrus infection localized to the gut can

mute BCG-specific CD4+ T cell priming in both the spleen and skin-draining lymph nodes. Furthermore, H. polygyrus infection

reduced the magnitude of delayed-type hypersensitivity (DTH) to PPD in the skin. Consequently, H. polygyrus–infected mice

challenged with BCG had a higher mycobacterial load in the liver compared with worm-free mice. The excretory–secretory

product from H. polygyrus (HES) was found to dampen IFN-g production by mycobacteria-specific CD4+ T cells. This inhibition

was dependent on the TGF-bR signaling activity of HES, suggesting that TGF-b signaling plays a role in the impaired Th1

responses observed coinfection with worms. Similar to results with mycobacteria, H. polygyrus–infected mice displayed an

increase in skin parasite load upon secondary infection with Leishmania major as well as a reduction in DTH responses to

Leishmania Ag. We show that a nematode confined to the gut can mute T cell responses to mycobacteria and impair control of

secondary infections distal to the gut. The ability of intestinal helminths to reduce DTH responses may have clinical implications

for the use of skin test–based diagnosis of microbial infections. The Journal of Immunology, 2016, 196: 2262–2271.

Control of mycobacteria and other intracellular infectionsof macrophages are dependent on the generation of Th1cells. Th1 cells produce IFN-g, which is required to

activate macrophages for killing the infecting organism (1). De-velopment of such responses can be measured by a delayed-typehypersensitivity (DTH) skin test reaction in both mice and hu-mans. Indeed, the Mantoux test for tuberculosis (TB) and the

Montenegro test for leishmaniasis are still used to screen for in-fection with Mycobacterium and Leishmania, respectively. Skin

test reactivity may suggest the generation of protective immuneresponses, but depending on the size of the induration, can also be

indicative of disease and warrant further examination (http://www.cdc.gov/tb/) (2).The only available vaccine against TB is infection with live at-

tenuated Mycobacterium bovis bacille Calmette–Guerin (BCG),normally given in the skin. This infection/vaccination regimen haslimited and highly variable efficacy in different parts of the world (3).Helminth infections evoke Th2 and regulatory immune re-

sponses. Both of these responses can counteract Th1 development.Accordingly, worm infection is proposed to impair immune re-sponses that control mycobacteria (4–6). Infection with worms hasalso been associated with a reduced ability to respond to BCGvaccination (7, 8). Geographically, areas of high TB incidence andpoor TB vaccine efficacy typically have a high prevalence of in-testinal helminth infections (9). However, the impact helminthshave on vaccine efficacy and other secondary infections remainsan open question. Indeed, a number of studies report a lack ofcorrelation between intestinal worms and secondary infections(10–13). In common for many of the studies describing an asso-ciation between worms and increased susceptibility to secondaryinfection, or reduced inflammatory response in experimental au-toimmune disease, is that the effects have been observed in tissue(s) in direct or close contact with the worm (14, 15). In contrast,the effects of gastrointestinal (GI) worms on infections distal tothe worm itself remain poorly characterized.The nematode Heligmosomoides polygyrus bakeri (in this paper

referred to as H. polygyrus) causes an infection strictly confined tothe gut. In resistant hosts, H. polygyrus infection stimulates astrong Th2-type response that drives the expulsion of the worm

*Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 171 77Stockholm, Sweden; †Department of Immunology and Infection, London School ofHygiene and Tropical Medicine, London WC1E 7HT, United Kingdom; and‡Institute of Immunology and Infection Research, University of Edinburgh, Edin-burgh EH9 3JT, United Kingdom

1Current address: Department of Parasitology, Leiden University Medical Center,Leiden, the Netherlands.

2K.O., X.F., and V.P.B. contributed equally to this work.

Received for publication April 27, 2015. Accepted for publication December 22,2015.

This work was supported by the Swedish Research Council, the Bill and MelindaGates Foundation Grand Challenge Award, Ake Wibergs Stiftelse, Stiftelsen ClaesGroschinskys Minne, Karolinska Institutet funds, European Union/Marie Curıe GrantFP7-MC-IRG-247684, and the Swedish Society for Medicine.

Address correspondence and reprint requests to Dr. Susanne Nylen and Dr. AntonioGigliotti Rothfuchs, Department of Microbiology, Tumor and Cell Biology, Karolin-ska Institutet, Nobelsvag 16, Box 280, Stockholm 171 77, Sweden. E-mail addresses:Susanne.Nylen@ki.se (S.N.) and Antonio.Rothfuchs@ki.se (A.G.R.)

The online version of this article contains supplemental material.

Abbreviations used in this article: BCG, bacille Calmette–Guerin; DC, dendriticcell; DTH, delayed-type hypersensitivity; HES, Heligmosomoides polygyrus bakeriexcretory–secretory; LAg, Leishmania Ag; LN, lymph node; MHC-II, MHCclass II; PPD, purified protein derivative; SWAg, soluble worm Ag; TB, tuberculosis.

This article is distributed under The American Association of Immunologists, Inc.,Reuse Terms and Conditions for Author Choice articles.

Copyright� 2016 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/16/$30.00

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(16, 17). Despite the generation of a protective Th2 response, theworm can persist and establish long-lasting infection in most labo-ratory mouse strains (reviewed in Ref. 18). This is facilitated by theregulatory responses H. polygyrus evokes. In the chronic phase ofH. polygyrus infection, there is an expansion of regulatory Foxp3+

T cells in the gut (17). These regulatory Foxp3+ T cells, driven in partby a TGF-b–like activity released from the parasite (19), dampeneffector T cell responses aiding persistent worm infection.Chronic infestation with worms is the norm in animals and

humans. Thus, H. polygyrus provides a relevant model to study theeffects a gastrointestinal nematode infection has on immune re-sponses to secondary infections. Furthermore, H. polygyrus onlycauses moderate intestinal pathology and the infection is typicallyasymptomatic in wild-type mice. Thus, secondary infections canbe delivered in animals that are seemingly healthy. We used thismodel to investigate the effect of H. polygyrus infection on theoutcome of mycobacteria-triggered Th1 responses at distal sites.Our results show that H. polygyrus infection can inhibit primingand recall responses to BCG and promote mycobacterial growthin vivo. Our data reinforce TGF-b signaling as a key component ofH. polygyrus–mediated immune suppression. On the basis of ourfindings, we also suggest caution in the use of a skin-test reactivity–based diagnostic when performed in worm-infected individuals.

Materials and MethodsMice

C57BL/6, congenic CD45.1 (Ly5.1), and P25-TCRTg RAG-12/2 (20) 3RAG-12/2 ECFP (provided by Dr. R. Germain, National Institute of Al-lergy and Infectious Diseases, Bethesda, MD) were bred and maintainedunder specific pathogen-free conditions (Department of Microbiology,Tumor, and Cell Biology, Karolinska Institutet, Stockholm, Sweden). Fe-male mice were used if not otherwise mentioned. All experiments wereconducted in accordance to ethical regulations following approval byStockholm’s Norra Djurforsoketiskanamnd.

Infections

All infections were performed in wild-type (C57BL/6 or congenic Ly5.1/CD45.1) mice.

At 4–5 wk of age, mice were infected by oral gavage with 200 H.polygyrus L3 larvae, obtained as described previously (21, 22). The worminfections were considered chronic after 28 d. At the end of each experi-ment, the worm burden was estimated by counting viable worms that hadmigrated out of the opened intestine through a fine net into a tube con-taining RPMI 1640 medium at 37˚C within 3–4 h.

M. bovis BCG strain SSI 1331 was obtained from Statens Serum In-stitute (Copenhagen, Denmark), expanded in 7H9 medium as previouslydescribed (23), and inoculated at 1 3 106 CFU in the footpad, ear pinnae,or i.v. into the tail vein. For quantification of mycobacterial load in tissue,single-cell suspensions were plated onto 7H11 agar supplemented withOADC (BD Biosciences) and cultured at 37˚C for 21 d.

Leishmania major, Freidlin (a gift from Dr. D. Sacks, National Instituteof Allergy and Infectious Diseases), was maintained in M199 supple-mented with 20% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and200 mM streptomycin. A total of 1 3 105 metacyclic promastigotes,enriched form stationary cultures using Ficoll 400 gradient separation (24),were injected into the ear dermis. Control animals were either left un-treated or injected with PBS/medium as indicated in the figure legends.

Detection of promastigote growth was done by microscopy. To determinethe burden of Leishmania parasites, tissue homogenates were prepared aspreviously described (25) and cultured in limiting dilutions in 96-wellplates using M199 medium supplemented with 20% FCS, 2 mM L-gluta-mine, 100 U/ml penicillin, and 200 mM streptomycin.

Histopathological analysis

To enumerate and measure granulomas, livers and spleens from infectedmice were fixed in 4% paraformaldehyde in PBS for $12 h, followedby dehydration and paraffin embedding. The tissue was cut into 6-mm-thick sections and stained with H&E. Processing and H&E staining ofparaformaldehyde-fixed samples was performed at the Cancer CenterKarolinska, Pathology Laboratory, Karolinska University Hospital (Stockholm,

Sweden). Analysis was performed by microscopy on 10 fields/sample at afinal magnification of 3100. To allow pooling of granuloma area, the in-dividual experiment was normalized by dividing the sample value with themean value of all samples in the experiment.

Ags

Adult worms were isolated from the small intestines by allowing the wormsto migrate through a fine mesh into a 50-ml tube with RPMI 1640 mediumwith 100 U/ml penicillin, 200 mM streptomycin, and 0.5 mg/ml gentamicinat 37˚C for 3–4 h. Collected worms were washed extensively, incubatedovernight in RPMI 1640 medium containing antibiotics, followed by fur-ther washing in the same medium. The washed worms were used forpreparation of whole soluble worm Ag (SWAg) by resuspending adultworms in 10 mM Tris-HCl and adding complete protease inhibitor(Roche), according to the manufacturer’s instructions. The worms werehomogenized by repeated freeze-thaw cycles, followed by mechanicaldisruption and pulsed sonication. After centrifugation, protein concentra-tion in the supernatant was determined by OD at 280 nm. For injection oflarval Ags, L3 larvae were collected from cultures and washed extensivelyin water containing 200 U/ml penicillin and 400 mM streptomycin. The“PPD” footpad was conditioned with 200 L3 larvae, as previously de-scribed by others (26). Control mice were injected with the water used inthe last wash of the L3 larvae preparation.

H. polygyrus excretory–secretory (HES) products were isolated fromadult worm cultures as previously described (27). Purified protein deriv-ative (PPD; Statens Serum Institute) was used to measure recall responsesin vivo by injecting 10 mg PPD in the footpad. Whole freeze-thawedLeishmania Ag (LAg) (28) was prepared from stationary-phase proma-sitgote cultures by repeated freeze-thawing cycles. Protein concentrationwas measured at 280 nm, and 50 mg Ag was injected in the footpad.

All Ags injected in the footpad were delivered in a volume of 30 ml.

Cell transfer

For in vivo assessment of Ag-specific T cells, total lymph nodes (LN) fromP25-TCRTg RAG2/2 mice were collected, and single-cell suspensionswere prepared. Isolated P25 TCRTg (P25) cells were labeled with 1 mMCFSE (Invitrogen) in PBS for 10 min at room temperature. The labelingreaction was stopped by adding FCS and cells were subsequently washedin RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin,and 200 mM streptomycin to remove any unbound dye. A total of 1 3 105

CFSE-labeled P25-TCRTg T cells (CD45.2+) were injected i.v. into the tailvein of recipient (CD45.1+) congenic mice receiving BCG. Control ani-mals received 1 3 106 cells.

In vitro restimulation

Single-cell suspension of the tissue of interest was prepared as describedpreviously (23). Isolated cells were diluted in RPMI 1640 medium sup-plemented with 10% FCS, 100 U/ml penicillin, and 200 mM streptomycinto a concentration of 2 3 106 cells/ml and stimulated in 96-well tissueculture plates with 10 mg/ml PPD, 2 mg/ml purified anti-CD3 Ab (BDBiosciences), or worm Ags as indicated in figures for 3 d at 37˚C in 5%CO2. Irradiated (30 Gy) splenocytes from naive mice were used as APCs ata ratio of 1:5. Supernatants were collected and stored at 280˚C until cy-tokines were measured by ELISA.

ELISA

Cytokine levels in culture supernatants were measured by sandwich ELISA.Immulon 2B plates (Nunc) were coated overnight, 4˚C, with capture Ab.For IFN-g, the capture Ab was diluted in carbonate buffer (0.1 M Na2CO3,0.1 M NaHCO3, and 1 mM NaN3 [pH 9.6]), all other capture Abs werediluted in PBS. The plates were then blocked for 2 h at 37˚C with 5% milkin diluent solution (1% BSA and 0.05% Tween 20 in PBS) and subsequentlyincubated overnight, 4˚C, with the sample supernatants or the respectivestandards. Bound protein was detected with biotin-labeled detection Abs for2 h at 37˚C, followed by incubation with peroxidase-labeled streptavidin(2 h, 37˚C) and development with ABTS peroxidase substrate (both KPL).Plates were read at 405 nm. The following Ab clone pairs were used: IFN-g(R4-6A2/XMG1.2), IL-5 (TRFK5/TRFK4), and IL-10 (JES5-2A5/JES5-16E3) all from BD Biosciences and Jackson ImmunoResearch Laborato-ries. For detection of IL-17 and TGF-b, mouse Quantikine kits (R&DSystems) were used according to manufacturer’s instructions.

In vitro stimulation of P25-TCRTg cells

P25-TCRTg cells were isolated and in some experiments CFSE labeled, asdescribed above. Splenic dendritic cells (DCs) were isolated from colla-

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genase IV and DNAse I–treated spleens as previously described (23), fol-lowed by magnetic enrichment of CD11c+ cells using CD11c microbeads(Miltenyi Biotec), according to manufacturer’s instructions. Splenic DCswere incubated with HES and SWAg at various concentrations for 2 h andsubsequently cocultured with P25-TCRTg at a ratio of 1:5 and stimulatedwith multiplicity of infection 1 of BCG 1331. In some experiments, HES andSWAg were given to DCs in the presence of 5 mM ALK5 inhibitorSB-431542 (Torics), treatment with 5 ng recombinant human TGF-b(R&D Systems) 6 SB-431542 was used as control. Positive control stim-ulations were with 2 mg/ml Ag85B240–254 peptide and 100 ng/ml LPS(Sigma-Aldrich).

Flow cytometry staining

Single-cell suspensions from tissues were incubated with various combi-nations of fluorochrome-conjugated rat anti-mouse mAbs specific for CD4(RM4-5), CD11b (M1/70), CD11c (HL3), MHC class II (MHC-II) I A/I E(M5/114.15.2), CD44 (IM7), CD45.2 (104), CD69 (H1.2F3), (BD Bio-sciences), CD326/EpCAM (G8.8), CD103 (2E7), (BioLegend), CD4(RM4-5), B220 (RA3-6B2), and latency-associated peptide (TW7-16B4)(eBioscience), for 45 min in FACS buffer (2% FCS in 5 mM EDTA and0.1% azide) containing 0.5 mg/ml anti-mouse FcgIII/IIR (2.4G2) (BDBiosciences). Live-dead staining was done using live-dead dye (LifeTechnologies). For analysis of intracellular cytokine production, cells werestimulated ex vivo for 6 h with 10 mM Ag85B240–254 peptide in thepresence of 10 mg/ml brefeldin A (Sigma-Aldrich) prior to surface stain-ing, followed by fixation in 2% paraformaldehyde (Electron MicroscopySciences) and permeabilization with 1% saponin (Sigma-Aldrich) andstaining with anti–IFN-g (XMG1.2) (BD Biosciences). For staining of Foxp3,we first surface stained cells and then prepared cells for intranuclear/-cellularstaining using eBioscience Foxp3 staining set (FJK-16S), according to themanufacturer’s instructions. Irrelevant isotype-matched Abs were used todetermine levels of nonspecific binding. Cell proliferation was measuredby CFSE dilution.

To track cell migration from the skin to the draining LN 20 ml 0.5 mMCFSE in PBS were injected into the same footpad in which BCG vacci-nation had been delivered 48 h earlier, as described previously (29). Thepopliteal lymph node (pLN) was collected 24 h after CFSE injection.Single-cell suspensions of pLN were stained for expression of CD11c,CD11b, MHC-II, CD103 and CD326. Detection and phenotypic charac-terization of CFSEhi cells were done by FACS. FACS acquisition wasperformed using CyAn (Beckman Coulter), LSRII or FACSCanto (BDBiosciences). Analysis was done on single cells gated as lymphocytes byforward-side scatter using FlowJo software (Tree Star).

Gene expression and real-time PCR

Total mRNA was isolated from tissue using TRIzol (Sigma-Aldrich)according to manufacturer’s instructions. RNA concentration was deter-mined by spectrometry and first strand cDNA generation and real-timePCR was performed as previously described (29, 30), using the T100and CFX 384 Real-time System (Bio-Rad), respectively. Expression ofHRPT was used as baseline and the relative expression of gene expressionwas determined as follows: DCT between gene of interest and HRPT inthe sample/DCT between gene of interest and HRPT in the in assignedunstimulated (control) sample.

Statistical analysis

Unpaired Student t test was used for comparison between two groups usingPrism (version 5.0a; GraphPad Software). Outliers were excluded fromanalysis of following Grubb’s test for one outlier, a = 0.05 (GraphPad,QuickCalcs). A p value , 0.05 was considered to indicate significantdifferences between the groups.

ResultsH. polygyrus infection impairs priming ofmycobacteria-specific T cells

To test the effect of an underlying H. polygyrus infection on in-duction of mycobacteria-specific CD4 T cells, we infected micei.v. with BCG and assessed activation of P25-TCRTg cells in ani-mals with or without worms introduced 28 d previously. P25-TCRTg cells recognize the major CD4 T cell epitope of Ag 85B,present in both BCG and M. tuberculosis (20).Ag-specific T cell priming was clearly impaired in BCG-infected

mice with chronic worm infection compared with animals free of

worms. The expansion and proliferative capacity of mycobacteria-specific P25-TCRTg cells was substantially reduced in H. poly-gyrus–coinfected animals, as measured by total number (Fig. 1A)and percentages (Fig. 1B) of P25-TCRTg cells in the spleen. Thiswas accompanied by a reduced ability of P25-TCRTg cells toproduce IFN-g upon restimulation with Ag85B (Fig. 1C, 1D).Although separated, the spleen and the gut are still in relativeproximity, and Th2 responses to H. polygyrus can be detected inthe spleen (28). To increase the distance between the site of worminfestation and the secondary infection, we delivered BCG in thefootpad. Similar to the observations described above, fewer P25-TCRTg T cells and fewer IFN-g+ P25-TCRTg T cells were foundin the skin-draining pLN 6 d after BCG footpad infection in micewith chronic H. polygyrus infection compared with worm-freemice (Fig. 1E–G). Furthermore, the cellularity of the BCG-draining pLN was notably lower in H. polygyrus–infected micecompared with worm-free mice (lymphocytes per pLN 6 d afterBCG infection; H. polygyrus/BCG: 4.56 3 106 6 8.68 3 105;BCG: 13.15 3 106 6 2.48 3 106, n = 5/group; p = 0.0115, one ofthree or more experiment with similar results). The viability ofCD4 T cells in the BCG-draining LN was high (95%) and similarbetween H. polygyrus–infected and worm-free mice. This indi-cates that the impaired T cell response observed in worm-infectedmice is not due to increased T cell death. These observationsdemonstrate that an intestinal worm infection can affect T cellpriming to infection/vaccination at sites distal to the gut.

Mice with chronic H. polygyrus infection have reduced DTHresponses to PPD

To investigate the effect of H. polygyrus on recall responses wemeasured DTH to PPD in BCG-infected/vaccinated animals withor without worm infection. In these experiments, BCG infectionwas delivered in the right footpad, and the magnitude of the DTHresponse was measured 2 wk later by recording swelling uponPPD delivery in the other footpad. This measurement can be seenas an equivalent of the PPD skin tests done in humans to assessBCG vaccination or TB infection. In addition, we also studied theeffect of depositing worm Ags proximal to the site of PPD chal-lenge. To that end, footpads were preconditioned by injectingH. polygyrus larvae 4 wk prior to PPD restimulation.Interestingly, footpad swelling was significantly reduced when

PPD was given in footpads preconditioned with L3 larvae (Fig. 2A).However, no significant effect on footpad swelling was ob-served in mice tested for PPD reactivity 4 wk after worm infectionand 2 wk after BCG infection, compared with BCG-infected,worm-free mice (Fig. 2A). Because most helminth infections arechronic in nature, we prolonged the time to BCG infection, allowingthe worm to become chronic before infection with BCG was given.Interestingly, we now found that the DTH responses were signifi-cantly diminished in worm-infected animals (Fig. 2B), implying thatit takes time before the impact of worm-mediated inhibition can beobserved on immune responses at distal sites.When the PPD injection site was preconditioned with L3 larvae,

in vitro IFN-g recall responses to PPD in the draining LN were re-duced by ∼65% compared with control LN (data not shown). How-ever, this was not seen in LN from worm-infected animals. Therewere also no significant effects of H. polygyrus infection on in vitroproduction of IL-5, IL-12, or IL-17 in LN cultures (data not shown).To determine whether fewer total T cells and Ag-specific CD4+

T cells accumulated at the site of recall response in worm-infectedmice compared with worm-free mice, we measured the infiltrationof total and P25-TCRTg CD4+ T cells in the skin where PPD wasdelivered. To facilitate the isolation of cells from skin, PPD was inthese experiments injected in the ear pinnae. We found that worm-

2264 INHIBITION OF T CELL RESPONSES TO BCG BY H. POLYGYRUS

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infected mice had fewer T cells and fewer Ag-specific P25-TCRTg cells in the skin (Fig. 2C, 2D), suggesting that the im-paired DTH response is linked to a reduction of Ag-specificT cells in the PPD-challenged tissue. There was, however, nodifference in the number or frequency of P25-TCRTg cells inPPD-draining auricular LNs (Supplemental Fig. 1A, 1B).

H. polygyrus infection increases susceptibility toTh1-controlled infections

In line with the observations that both T cell priming and recallresponses were reduced in H. polygyrus–infected mice, we foundthat these animals had a 2.3-fold higher bacterial load in the liverwhen given a systemic BCG infection (Fig. 3A). Interestingly,BCG-induced granulomas were both fewer and smaller inH. polygyrus–infected mice compared with worm-free mice (Fig. 3B,3C). This indicates that worm-infection can interfere with gran-uloma formation, leading to an increased susceptibility to myco-bacterial infection. The impaired control of mycobacteria wascoupled to reduced infiltration of both DCs and macrophages andlower expression of inducible NO synthase, IFN-g, and TNF-a inlivers of coinfected animals (Fig. 3D–H). Assessment of CFU in

spleen also indicated an increase in bacterial load in worm-infested animals, although this did not reach statistical signi-ficance (data not shown).The impaired immune response and control of BCG in worm-

infected mice was not associated with an increase in Foxp3 ex-pression or more Foxp3+ CD4 T cells (Supplemental Fig. 2A–C).The numbers of viable worms were similar in mice infected

with H. polygyrus alone and H. polygyrus–infected mice givenBCG i.v. (Fig. 3I). Thus, the established worm infection was notaffected by the superimposed BCG infection in our model.To investigate whether another Th1-controlled infection was

affected by an underlying, chronic H. polygyrus infection, animalswere coinfected with L. major in the ear. We found that H. poly-gyrus–infected mice had double the amount of parasites in theear (Fig. 4A) and a reduced cellularity in the ear-draining LN(Fig. 4B) compared with worm-free mice. In vitro recall responseto LAg by an equal number of LN cells were similar in worm-freeand worm-infected mice (data not shown), indicating that a moresensitive model (e.g., one involving TCRTg cells) is needed todetect such differences. In line with the observations shown inFig. 2, H. polygyrus– and L. major–coinfected mice had dimin-

FIGURE 1. H. polygyrus infection

interferes with T cell priming in re-

sponse to mycobacteria infection.

P25-TCRTg cells (1 3 105 to those

receiving BCG and 13 106 to control

animals/PBS group) were seeded in

Ly 5.1 mice the day before BCG

infection. The P25-TCRTg were

assessed in the spleen 6 d after i.v.

injection of 1 3 106 CFU BCG in

mice with chronic (28 d) H. polygyrus

(HP) infection or free of worms. Total

number (A) and frequency (B) of P25-

TCRTg cells and number (C) and

frequency (D) of IFN-g+ P25-TCRTg

cells following 6 h in vitro restim-

ulation with Ag85B peptide in mice

infected as described above. Total

number of T cells (E) and number (F)

and frequency (G) of IFN-g+ P25-

TCRTg cells in BCG-draining pLN

6 d after BCG footpad infection in

mice with chronic H. polygyrus in-

fection or free of worms. Data shown

are representative of two or more

experiments with three to five mice

per group. Significant differences are

indicated as follows: *p , 0.05,

**p , 0.01, ***p , 0.001.

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ished DTH responses to LAg in the footpad compared with worm-free mice (Fig. 4C). This shows that a pre-existing intestinalhelminth infection can influence host control of secondary infec-tions occurring distal to the worm.

H. polygyrus–secreted products inhibit T cell priming in vitrothrough their TGF-bR signaling capacity

To avoid host immune responses and to establish chronic infectionwithout causing detrimental pathology, helminths produce andsecrete molecules with immunomodulatory capacity (27). Suchmolecules could, directly or indirectly, be the cause of the di-minished T cell priming in response to BCG observed in worm-infected mice. To test whether worm-derived products interferewith T-cell priming, splenic DCs were treated with SWAg or theHES prior to stimulation with BCG and coculture with P25-TCRTg cells. SWAg was a relatively poor inhibitor of BCG-induced P25-TCRTg-cell IFN-g production, and significant inhi-bition was only seen when a high concentrations of SWAg con-centration was used (Fig. 5A). SWAg also inhibited BCG-inducedIL-5 production in a dose-dependent manner (Fig. 5B), reinforc-ing the general downmodulation of effector–T cell responses byworms. HES strongly inhibited IFN-g production by P25-TCRTgcells, whereas pre-exposure to BSA or LAg did not significantlyaffect IFN-g production by P25-TCRTg CD4+ T cells (Fig. 5C),indicating that the effect observed was specific to HES. IL-10 was

not detected in BCG-stimulated cultures and was not induced byHES or SWAg (data not shown).The activity of HES was sensitive to heat treatment (Fig. 5D). HES

has been shown to have a heat-sensitive TGF-b–like activity (19),suggesting that the TGF-bR activating molecule of HES-mediatedinhibition of P25-TCRTg-cell IFN-g production. In support of this,the effect of HES was reverted by inhibition of TGF-bR signaling,using the TGF-bR1/ALK5 inhibitor SB431542 (Fig. 5E). TheSWAg-mediated effect also appeared to involve TGF-bR signalingbecause the inhibition of BCG-induced responses was reversed byblockade of TGF-bR signaling (Fig. 5E). These results thus proposea role for TGF-bR signaling in worm-mediated inhibition of Tcell priming to BCG. The inhibitory effect of worm Ags in thesein vitro cultures were likely through a direct inhibition on the T cellsbecause both HES and SWAg inhibited anti-CD3/anti-CD28–induced T cell activation in absence of DCs (data not shown).Furthermore, it was not enough to simply preincubate (4 h) DCswith HES prior to coculture with T cells to observe the inhibitoryeffect on IFN-g production. That said, direct effects of worm Agon DCs have been shown by others (31) and cannot be excluded.

H. polygyrus infection reduces BCG triggered migration ofDCs from the skin to the draining LN

Given the above, we decided to investigate if there were effects ofH. polygyrus on APC in vivo. In this regard, migration of APC

FIGURE 2. DTH responses are reduced in sites preconditioned with worms and in animals with chronic H. polygyrus infection. DTH response to PPD in

C57BL/6 mice either infected orally with H. polygyrus (HP) or preconditioned with H. polygyrus L3 larvae in the “PPD” draining left footpad (L.Fp). (A)

Footpad swelling in animals infected with 1 3 106 CFU BCG in the right footpad (R.Fp) 14 d after worm infection/larval preconditioning and 14 d after

BCG (= day 28) given PPD in the contralateral footpad. (B) Footpad swelling in animals infected with BCG in the footpad 28 d after H. polygyrus infection

(chronic infection) and PPD challenged in the contralateral footpad 14 d after BCG vaccination (day 42). Total T (CD3+) cells (C) and seeded P25-TCRTg

cells (D) in ear dermis 48 h after PPD injection in Ly 5.1 mice infected with H. polygyrus and BCG as above and challenged with PPD in the ear 14 d later.

P25-TCRTg cells (1 3 105 in BCG infected and 1 3 106 in control animals) were transferred 1 d before BCG infection. Data shown are representative of

two or more experiments with three to five mice per group. Significant differences are indicated as follows: *p , 0.05, **p , 0.01, ***p , 0.001.

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from the site of infection to the draining LN is fundamental for theinitiation of a primary T cell response. The number of APCreaching the LN will accordingly influence the magnitude of thatresponse (32). Given that BCG-specific T cell expansion was re-duced in worm-infected animals, we tested the effect of chronicH. polygyrus infection on migration of APC from the site of BCGinfection to the draining LN. To track migratory cells, we injectedCFSE into the skin of the footpad and quantified the number ofCFSE+ cells detected in the draining pLN. Following BCG foot-pad infection we found that the majority of migrating (CFSEhi)cells were MHC-IIhiCD11c+/int, consistent with the phenotype ofmigratory DC (Fig. 6A). The majority of these cells were alsoCD11b+ (Fig. 6A). To address whether H. polygyrus affected such

cell migration, we measured the number of CFSEhiMHC-IIhi

CD11c+/int cells in the pLN postinfection with BCG. Mice withchronic H. polygyrus infection had fewer CFSEhiMHC-II+/hi

CD11c+/int cells in the pLN after BCG injection compared withworm-free mice (Fig. 6B, 6C). This may in part explain the re-duction in Ag-specific T cell response found in the worm-infectedmice (as shown in Fig. 1). TGF-b is a key cytokine in H. poly-gyrus–induced immune regulation (33), with the capacity to blockAg-specific CD4–T cell activation, as shown above, and with thepotential to downmodulate the migratory capacity of DCs (34). Totest whether worm-derived molecules and TGF-b could influenceBCG-induced DC migration, we conditioned the footpad withTGF-b or HES 10 d prior to BCG inoculation. As a control, we

FIGURE 3. Control of mycobacteria is impaired in mice with chronic H. polygyrus infection. Bacterial load (A), granulomas (B), and relative granuloma

area (C) in livers 21 d post i.v. infection with 1 3 106 CFU BCG in C57BL/6 mice chronically infected with H. polygyrus (HP) or free of worms. Data

shown are pooled from two to three experiments (n = 10–15/group). Frequency of DC (CD11c+MHCIIhi) (D) and monocytes (CD11b+Ly6ChiMHCII+) (E)

in livers 14 d after i.v. infection with 1 3 106 CFU in mice with chronic H. polygyrus or free of worms at the time of BCG infection. Results are pooled

from two experiments (n = 10/group). iNOS (F), IFN-g (G), and TNF-a (H) mRNA expression in livers from mice infected as in (D) and (E). The control

group received PBS injection. Data are representing two independent experiments (n = 5/group). (I) Viable adult worms in intestines of C57BL/6 mice 42 d

post-H. polygyrus infection. Mice given BCG were infected i.v. 28 d after H. polygyrus infection [as in (D)]. Results are pooled from two experiments (n =

10/group). Significant differences are indicated as follows: *p , 0.05, **p , 0.01, ***p , 0.001.

FIGURE 4. Intestinal worms can facilitate skin infection with L. major. Effect of chronic H. polygyrus (HP) infection on L. major infection (1 3 105

promastigotes) in the ear dermis: parasite load in the ear (A) and cellularity in the ear (B) dLN 5 wk after L. major infection. (C) DTH response to LAg (50

mg) delivered in the footpad 8 wk after H. polygyrus infection and 4 wk after ear infection with L. major (1 3 105 promastigotes); one of two experiments

with four to five mice per group is shown. Significant differences are indicated as follows: *p , 0.05, **p , 0.01, ***p , 0.001.

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heat-inactivated HES, thereby eliminating its TGF-b–like activity(19). In line with studies of skin cancer (34), our data suggest thatTGF-b lowers the migratory capacity of DCs. Fewer CFSEhi

MHC-IIhiCD11cint/+ cells were found in the draining pLN afterBCG infection when the site of BCG injection was preconditionedwith TGF-b (Fig. 6D). HES conditioning of the footpad also re-duced the migratory response of MHC-IIhiCD11cint/+ cells toBCG, whereas heat-inactivated HES was found to be a less potentinhibitor of the same (Fig. 6D). This finding supports worm-drivenTGF-b signaling as a potential mechanism underlying impairedresponses to BCG.

DiscussionThe efficacy of BCG vaccination is highly variable in different partsof the world. Worms, through the immune responses they evoke,have been suggested as one factor that can impair BCG vaccineefficacy and increase susceptibility to mycobacterial infection (6,35). In support of this, there is ample evidence that worms andworm products can counteract Th1 immunity and downmodulateinflammatory responses to secondary Ags (36). However, most ofthese observations have been made in systems where the worm orthe worm Ag is either in direct contact with or in close proximityto the site of inflammation/infection. Yet, most parasitic wormslive in the gut and are therefore not proximal to the site whereinjection-based vaccines are delivered. Although some findingsindicate that intestinal worms have more systemic effects on im-munity (7, 8, 37, 38), evidence that intestinal nematodes modulateimmune response in tissue distal to the worms and thereby impairimmune responses to vaccination and secondary infections isscarce and remains questioned (12, 26, 39, 40).In this study, we have experimentally addressed how a nem-

atode infection confined to the gut influences Th1 responses tosecondary infections at sites separated from the worm infection.Our results support the view that intestinal worms diminish im-mune responses to secondary vaccinations and infections. We found

that CD4+ T cell priming in response to BCG was reduced in micechronically infected with H. polygyrus, compared with worm-freeanimals. Likewise, pre-exposure to H. polygyrus Ags in vitrodramatically decreased IFN-g production by mycobacteria-specific CD4+ T cells.Similar to observations made in humans (8), we found that

DTH responses to PPD following footpad BCG infection/vaccina-tion and to L. major Ag following experimental leishmanizationwere smaller in mice with chronic H. polygyrus infection com-pared with worm-free mice. This may reflect vaccine efficacy, butmore importantly, it suggests that intestinal worms can influenceskin test–based diagnostics and possibly reduce DTH-based de-tection of TB and leishmaniasis. The notion that worms decreasethe sensitivity of recall response-based diagnosis is something thatwould need more careful investigation in clinical studies.The proximity between worm and coinfections appear to in-

fluence the suppressive effects of H. polygyrus on responses toBCG. The inhibitory effect was most evident when the distance tothe BCG effector site was small [e.g., when BCG was deliveredsystemically (i.v.) or when the effector site itself had been pre-conditioned with worm Ags (Fig. 2A)]. Time is another factor thatmay influence the inhibitory effects of H. polygyrus. The DTHresponses to PPD were attenuated in mice with chronic (.4 wk)but not acute (2 wk) worm infection, at the time when infectedwith BCG.Systemic dissemination of worm-induced Th2 or regulatory

T cells could explain how an infection confined to the gut canmodulate immunity at peripheral sites. During the first weeks ofH. polygyrus infection, worm-induced Th2 cells can be found inthe spleen (28, 37). An increase in Th2 cells could underlie thereduced priming of BCG-specific CD4+ T cells observed in thespleen. Moreover, using IL-4 reporter mice, Mohrs et al. (37)found that H. polygyrus–induced Th2 cells that spread systemi-cally have a preference for nonlymphoid organs, such as the liver.This could in turn explain why the impact of H. polygyrus on BCG

FIGURE 5. Worm-derived molecules inhibit IFN-g production by mycobacterial-specific T cells in response to BCG in through TGF-bR signaling.

Cytokine production to BCG in splenic CD11c+:P25-TCRTg cell cocultures treated with worm Ags. Splenic CD11c+ cells, from C57BL/6 mice, were

preconditioned with worm or control Ags as indicated and treated with BCG (multiplicity of infection 1). Single-cell suspensions of LN from naive P25-

TCRTg mice were then added to the CD11c+ cells, and the cells were cocultured for 5 d before supernatants were collected. Negative control cultures were

treated with medium or diluent. (A) IFN-g and (B) IL-5 in supernatant of cultures pretreated with different concentrations (5–50mg/ml) of SWAg. (C) IFN-g

in supernatant of cultures treated with 5 or 10 mg/ml H. polygyrus–secreted Ags (HES) or control Ags as indicated. (D) Effect of heat inactivation on HES

(used at 5 mg/ml) and SWAg (used at 50 mg/ml) on inhibition of BCG induced IFN-g. (E) Effect of 5 mM SB431542 on HES (5 mg/ml), SWAg (50 mg/ml),

and recombinant human (rh)TGF-b (5 ng/ml)-mediated inhibition of BCG-induced IFN-g in P25-TCRTg cultures. Data show mean 6 SEM and are

representative of two or more experiments generated from triplicate cultures. Stimulations/inhibitions were compared with cultures stimulated with BCG

alone, if not otherwise indicated. Significant differences, using Student t test, are indicated as follows: *p , 0.05, **p , 0.01, ***p , 0.001.

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load was more evident in the liver compared with the spleen. Wecould, however, not find any evidence for dissemination of Th2cells to pLN in mice with chronic H. polygyrus infection. Therewere no differences in mRNA expression of IL-4 or T cell tran-scription factors GATA-3, T-bet, and Foxp3 in pLN when com-paring H. polygyrus–infected and worm-free mice (data notshown). However, FACS analysis did reveal a modest decrease inthe percentage of T cells in skin-draining LN of mice with chronicH. polygyrus infection (percentage of lymphocytes gated asT cells in pLN; naive mice: 60.9 6 0.9%; mice with chronicH. polygyrus infection: 54.8 6 1.5%, n = 10, p = 0.0034). Thisindicates that an intestinal worm may affect lymphocyte compo-sition in skin-draining LN, which in turn could influence thesubsequent ability to respond to infection/vaccination.Reduced immunogenicity of BCG in people chronically infected

with worms has been associated with increased production ofTGF-b by PBMC (7). Indeed, TGF-b is also induced by H. poly-gyrus infection. Although primarily found at the site of infection,increased levels of serum TGF-b have also been reported inH. polygyrus–infected mice (41). We found that splenic CD4 cellsfrom mice with chronic H. polygyrus infection express more TGF-b latency-associated peptide (LAP) compared with naive mice (S2D-F). TGF-b is a pluripotent, mainly anti-inflammatory, cytokine,which can limit both Th1 and Th2 responses. H. polygyrus andother intestinal worms exploit this cytokine to facilitate thechronic establishment in the host (18, 42). Experimentally, TGF-bhas been found to be important for the control of worm-mediated

inhibition of several inflammatory diseases (43). Many nematodespecies express TGF-b homologs (44) and some, includingH. polygyrus, secrete products that can signal through the TGF-bR(19, 45). HES from H. polygyrus has previously been shown todrive regulatory (Foxp3) T cell responses and to increase pro-duction of both TGF-b and IL-10, another immune-modulatorycytokine (19). We found no evidence for an increase in Foxp3expression in H. polygyrus coinfected animals. While we cannotexclude involvement of IL-10, using a IL-10 GFP reporter mice(46) we did not find more IL-10 expressing cells in the BCG-draining pLN in worm infected compared with worm free mice(not shown).However, we found that HES can act directly on T cells and that

the TGF-bR signaling capacity of HES was needed to inhibitmycobacteria-specific T cell priming in vitro. DCs typically ac-quire regulatory properties in the presence of TGF-b (47). Al-though the effect of HES in our in vitro cocultures was mainly onthe T cells, H. polygyrus and the excretory–secretory products arealso know to facilitate regulatory and Th2-promoting DC (31, 48–50). Interestingly, we found that preconditioning the site ofBCG infection with TGF-b or HES significantly reduced BCG-triggered migration of DC (MHC-IIhiCD11c+/int cells) to thedraining LN. TGF-b can downmodulate the expression of CCR7on DCs and inhibit their migratory capacity. In mesenteric LNs,Leon et al. (51) found that H. polygyrus infection alters the ex-pression of CCR7 and CXCR5 on DCs, with implications ondevelopment of downstream Th responses. We did not find that

FIGURE 6. H. polygyrus affect DC migration in response to BCG vaccination. Tracking of cells migrating to the pLN 48–72 h after BCG infection was

done by labeling the footpad with CFSE by injection 48 h after BCG vaccination (13 106 CFU) and measuring the number of labeled (CFSEhi) cells in the

draining pLN 24 h later by FACS. (A) Gating strategy for detection of migratory (CFSE positive) cells in response to BCG infection. Numbers (B) and

frequencies (C) of CFSEhiMHC-IIhiCD11c+/int cells in pLN following BCG vaccination in mice with chronic H. polygyrus (HP) infection or free of worms.

(D) CFSEhiMHC-IIhiCD11c+/int cell numbers in pLN, where the BCG infection site (footpad) had been preconditioned of with 5 ng TGF-b, 5 mg HES, or 5

mg heat-inactivated (Hi)-HES as indicated 10 d prior to BCG injection. Results shown are representative of two or more experiments with three to five mice

per group. Control mice were preconditioned with 5 mg OVA or left untreated and only infected with BCG. Background migration was monitored by

injection of PBS in naive mice. Significant differences are indicated as follows: *p , 0.05, **p , 0.01, ***p , 0.001.

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HES affected the CCR7 or CXCR5 expression in the BCG-draining LN (data not shown). The inhibitory effect of HES onDC migration may involve more than TGF-b because HES is acomplex mixture (27). Recently, we found that the levels of CCR7do not correlate with the in vivo ability of BMDC to migrate inresponse to BCG (29). Interestingly, IL-12p40 was found to beimportant for the BCG-induced DC migration from footpad to thedraining LN (29). Others have shown that exposure of DCs toHES products downmodulates their expression of IL-12p40 (31).Similar observations have been made following exposure of DCsto TGF-b (52). In line with a role for TGF-bR signaling in HES-mediated inhibition of DC migration, heat inactivation of HES,which destroys the TGF-b–like activity of HES, significantly di-minished the inhibitory effect of HES on DC migration. Whetherthis inhibitory effect on migration involves downregulation of IL-12p40 by excretory–secretory products remains to be investigated.Taken together, our data support worm-induced TGF-bR sig-

naling as a mechanism behind helminth-mediated immune mod-ulation of effector–T cell responses.In summary, we show that a chronic worm infection confined to

the gut impacts both primary and recall immune responses tosecondary microbial challenge delivered in tissue distal to the gut.Compared with worm-free animals, mice with a chronic intestinalnematode infection had impaired T cell priming in responses toBCG, reduced DTH responses in the skin, and a higher bacterial/parasite load when infected with BCG and L. major, respectively.This implies that worms negatively can affect the diagnosis aswell as the control of intracellular infections with Mycobacteriumand Leishmania. We propose worm-evoked TGF-bR signaling asa part of the explanation as to why helminth-infected individualsare more susceptible to Th1-controlled infections and respond lesswell to immunizations dependent on such responses.

AcknowledgmentsWe thank Frank Heuts, Viviana Taylor, Zachary Darroch, Damıen

Bierschenk, and Adrian Luscombe for contributions to the study and

the Department of Microbiology, Tumor and Cell Biology animal facility

for technical support and care of animals.

DisclosuresThe authors have no financial conflicts of interest.

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