major secretory antigens of the helminth fasciola …differentiation of th2 cells. our data suggest...

INFECTION AND IMMUNITY, Feb. 2010, p. 793–801 Vol. 78, No. 20019-9567/10/$12.00 doi:10.1128/IAI.00573-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Major Secretory Antigens of the Helminth Fasciola hepatica Activate aSuppressive Dendritic Cell Phenotype That Attenuates Th17

Cells but Fails To Activate Th2 Immune Responses�

David J. Dowling,1† Clare M. Hamilton,1† Sheila Donnelly,2 James La Course,3 Peter M. Brophy,4John Dalton,5 and Sandra M. O’Neill1*

Parasite Immune Modulation Group, School of Nursing, Faculty of Science and Health, Dublin City University, Glasnevin, Dublin,Ireland1; Department of Biology, National University of Ireland Maynooth, Kildare, Ireland2; University of Liverpool,

Liverpool L69 7ZJ, United Kingdom3; Aberystwyth University, Aberystwyth, Ceredigion, Wales, United Kingdom4;and Institute of Parasitology, McGill University, 21,111 Lakeshore Road, Sainte Anne de

Bellevue QC H9X 3V9, Canada5

Received 21 May 2009/Returned for modification 26 June 2009/Accepted 9 November 2009

Fasciola hepatica is a helminth pathogen that drives Th2/Treg immune responses in its mammalian host. Theparasite releases a large number of molecules that are critical to inducing this type of immune response. Herewe have selected recombinant forms of two major F. hepatica secreted molecules, the protease cathepsin L(rFhCL1) and an antioxidant, sigma class glutathione transferase (rFhGST-si), to examine their interactionswith dendritic cells (DCs). Despite enzymatic and functional differences between these molecules, both inducedinterleukin-6 (IL-6), IL-12p40, and macrophage inflammatory protein 2 (MIP-2) secretion from DCs andenhanced CD40 expression. While this induction was mediated by Toll-like receptor 4 (TLR4), the subsequentintracellular signaling pathways differed; rFhCL1 signaled through p38, and rFhGST-si mediated its effect viac-Jun N-terminal kinase (JNK), p38, p-NF-�Bp65, and IRF5. Neither rFhCL1 nor rFhGST-si enhanced DCphagocytosis or induced Th2 immune responses in vivo. However, DCs matured in the presence of eitherenzyme attenuated IL-17 production from OVA peptide-specific T cells in vivo. In addition, DCs exposed toeither antigen secreted reduced levels of IL-23. Therefore, both F. hepatica FhCL1 and FhGST-si modulate hostimmunity by suppressing responses associated with chronic inflammation—an immune modulatory mecha-nism that may benefit the parasite’s survival within the host.

Dendritic cells (DCs) have a central role among innate im-mune cells in presenting antigen and priming naïve T cells todifferentiate into Th1/Th17 and Th2/Treg subsets. In responseto pathogen-associated molecular patterns (PAMPs) that bindto pattern recognition receptors (PPRs), DCs express surfacemolecules and produce cytokines that modulate the effectorfunctions of responding T cells (29). While much is known ofhow DCs respond to bacterial and viral pathogens that driveTh1/Th17 subsets (13), comparatively little is understoodabout how these cells respond to and influence the Th2/Tregadaptive immune response to helminth parasites (33).

Gene expression and proteomic analyses of DCs have re-vealed that remarkably few genes are induced following stim-ulation with helminth antigens (8, 17). DCs activated and ma-tured in the presence of helminth antigens lack the classicalmarkers, such as high levels of proinflammatory cytokines (in-terleukin-12 p70 [IL-12p70], tumor necrosis factor alpha[TNF-�], and nitric oxide) and expression of costimulatorymarkers (CD80 and CD86), observed in DCs matured withToll-like receptor (TLR) ligands such as lipopolysaccharide(LPS) (41). Additionally, TLR-mediated activation of DCs can

be inhibited significantly if they are first exposed to helminthsor helminth-derived products (24, 29).

Despite their limited maturation, helminth-primed DCs cannevertheless activate naïve T cells (35). Dendritic cells exposedto a soluble preparation of Schistosoma mansoni egg antigen(SEA) and either cocultured with naive T cells in vitro orinjected into mice can polarize T-cell responses toward a Th2phenotype (34). Similar findings have been reported for S.mansoni larval antigens (26, 27) and excretory-secretory (ES)material from Nippostrongylus brasiliensis (35), Acanthochei-lonema viteae (52), and Echinococcus granulosus (43). How-ever, Segura et al. found that adoptive transfer of DCs treatedwith ES from Heligmosomoides polygyrus resulted in suppres-sion of both Th1 and Th2 responses in recipient mice (46). Thesame DCs promoted the differentiation of T cells with a reg-ulatory phenotype and an ability to suppress effector CD4� cellproliferation and cytokine secretion (46). Therefore, a diverserange of DC phenotypes can be induced by using complexmixtures of helminth antigens. For this reason, it is importantto investigate the interactions of defined helminth-derivedmolecules with DCs and to elucidate the mechanism by whichthey alter DC function.

The liver fluke Fasciola hepatica is an important global hel-minth of humans and livestock (36). During infection, thispathogen induces potent polarized Th2/Treg immune re-sponses coincident with a suppression of Th1 cytokines (15, 16,18, 39, 40). Furthermore, infection also results in the bystandersuppression of Th1 responses to a concurrent bacterial infec-

* Corresponding author. Mailing address: Parasite Immune Modu-lation Group, School of Nursing, Faculty of Science and Health, Dub-lin City University, Glasnevin, Dublin 9, Ireland. Phone: 353-700-5455.Fax: 353-700-7919. E-mail: [email protected].

† D.J.D. and C.M.H. contributed equally to this report.� Published ahead of print on 16 November 2009.

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tion or to immunization with a Th1-inducing bacterial vaccine(6, 19). We previously demonstrated that ES molecules of F.hepatica can mimic the immunomodulatory effect observedwith active infection. One of the predominant secreted prod-ucts, a cathepsin L1 cysteine protease (FhCL1), suppressed theonset of protective Th1 immune responses to bacterial infec-tions in mice and prevented the development of a Th1 re-sponse to vaccination (11, 22). Another major antigen, com-prising 4% of F. hepatica ES material, is the antioxidantglutathione transferase (FhGST) (30), which in dimeric formsignificantly inhibited the proliferation of rat spleen cells inresponse to concanavalin A (ConA) stimulation in vitro (7).

Using recombinant forms of FhCL1 and FhGST, we showthat both molecules partially activate DCs, via the PRR TLR4.However, despite activating DCs via different intracellular sig-naling pathways, both rFhCL1- and rFhGST-treated DCs sup-pressed the development of Th17 cells and did not induce thedifferentiation of Th2 cells. Our data suggest that helminthparasites secrete multiple molecules possessing a unique mech-anism of modulation which suppresses inflammatory Th1/Th17responses with redundancy, thus permitting the uninhibiteddevelopment of Th2/Treg cells in response to other secretorymolecules.

MATERIALS AND METHODS

Animals and Ags. C57BL/6 or BALB/c mice, aged 6 to 8 weeks old, werepurchased from Harlan Ltd. (United Kingdom), and DO11.10 mice (on aBALB/c background) were purchased from Charles River Laboratories (UnitedKingdom). C3H/HeJ and C3H/HeN bone marrow cells were a gift from ChristineLoscher (Dublin City University, Ireland), and TLR4KO (on a C57BL/6 back-ground) bone marrow cells were a gift from Padraic Fallon (Trinity CollegeDublin, Ireland). All mice were maintained according to guidelines of the IrishDepartment of Children and Health.

Active Fasciola hepatica cathepsin L antigen (rFhCL1; FHU62288) and vari-ant inactive rvFhCL1 were produced in the Pichia pastoris expression system aspreviously described (11). F. hepatica GST class sigma (rFhGST-si; ABI79450)and mu (rFhGST-mu; ACF59730) were produced using Escherichia coli recom-binant expression systems as previously described (29). All enzymes exceptrvFhCL were enzymatically active, as determined using specific assays as previ-ously described (11; E. J. LaCourse, S. Perally, J. V. Moxon, G. Chemale, D. J.Dowling, S. M. O’Neill, and P. M. Brophy, unpublished data). Furthermore, allenzymes in this study showed significant activity within the culture medium.Protein concentrations were measured using a bicinchoninic acid (BCA) proteinassay kit (Pierce). Endotoxin levels were measured using the Pyrogene endotoxindetection system (Cambrex). All antigens were revealed to have endotoxin levelssimilar to those of background and complete RPMI 1640 medium (supple-mented with 5% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 �g/mlstreptomycin, 2 mM L-glutamine, and 50 �M 2-mercaptoethanol), so they weretaken to be endotoxin free (data not shown).

Isolation, maturation, and characterization of bone marrow-derived DCs.Bone marrow-derived immature DCs were prepared by culturing bone marrowcells isolated from the femurs and tibiae of mice in complete RPMI 1640 withrecombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng/ml; R&D Systems) at 37°C. On days 3 and 6 of culture, freshmedium with GM-CSF (20 ng/ml) was added to the cells (24). On day 8, cellswere harvested, counted, and stained with CD11c (Caltag Laboratories) foranalysis by flow cytometry to determine purity (�90%). For experiments, DCswere seeded into 24-well plates (Nunc) at 106/ml in complete RPMI 1640 plus 5ng/ml GM-CSF. DCs were treated with medium only, F. hepatica recombinantantigens (0 to 10 �g), LPS (100 ng/ml; Alexa), or zymosan A (5 �g/ml; Sigma-Aldrich) alone for 18 h or were stimulated for 2.5 h with recombinant antigensprior to maturation with LPS.

Supernatants from cultured DCs were tested for the production of IL-12p40,IL-12p70, IL-6, IL-10, IL-23, TNF-�, macrophage inflammatory protein 1�(MIP-1�), and MIP-2 by sandwich enzyme-linked immunosorbent assay(ELISA) (BD OptEIA ELISA sets; BD Biosciences), and nitrite was measuredusing the Griess reagent system (Promega). Expression of cell surface markers

on DCs was quantified by four-color flow cytometry, using phycoerythrin (PE)-,fluorescein isothiocyanate (FITC)-, Cy5.5-, and allophycocyanin-conjugated an-tibodies specific for CD80, CD86, CD40 (BD Biosciences), and CD11c (CaltagLaboratories). Appropriately labeled isotype-matched antibodies were used ascontrols. Acquisition was performed using a FACSCalibur flow cytometer (BDBiosciences), and analysis of results was performed using FlowJo software (TreeStar).

Protein extraction and Western blot analysis. Total protein was extractedfrom cell lysates by use of RIPA buffer containing 50 mM Tris-HCl, pH 8.0, 150mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sul-fate, and protease and phosphatase inhibitor cocktails (Sigma-Aldrich) (22).Cells were incubated in the extraction buffer on ice for 5 min before beingcentrifuged at 8,000 � g for 10 min at 4°C. Supernatants were transferred toclean tubes, and protein concentrations were determined using a BCA proteinassay kit (Pierce). Protein samples (10 to 40 �g) and prestained protein markers(Precision Plus protein standards; Bio-Rad) were separated by SDS-PAGE andblotted onto 0.45-�m Immobilon-P polyvinylidene difluoride membranes (Sigma-Aldrich). Membranes were blocked for 1 h at room temperature in 5% nonfatdried milk in phosphate-buffered saline (PBS) and incubated overnight at 4°Cwith anti-phospho-p38 (1:1,000; Cell Signaling Technology), anti-total p38 (1:1,000; Cell Signaling Technology), anti-phospho-extracellular signal-regulatedkinase (anti-phospho-ERK), anti-total ERK, anti-phospho-c-Jun N-terminal ki-nase (anti-phospho-JNK), anti-total JNK, anti-phospho-NF-�Bp65, or anti-in-terferon regulatory factor 5 (anti-IRF5) (1:1,000; Santa Cruz). Membranes werewashed in PBS with 0.05% Tween 20 (PBS-T) and incubated for 1 h at roomtemperature with peroxide-conjugated anti-rabbit IgG (1:2,000; Sigma-Aldrich).After further washing, proteins were visualized with a Super Signal kit (Pierce),exposed to film for 10 to 30 min, and processed using an FPM 100A processor(Fuji Film). Protein bands were quantified using GeneSnap acquisition andGeneTools analysis software (GeneGenius gel documentation and analysis sys-tem; Syngene). Levels of phospho-JNK, -ERK, and -p38 were normalized to totalJNK, ERK, and p38 and expressed in arbitrary units as percent increases over themedium control levels. Levels of p-NF-�Bp65 and -IRF5 were expressed inarbitrary units as percent increases over medium control levels.

HEK293 cells and NF-�B activation. rFhCL1 or rFhGST-si (10 �g/ml)-in-duced stimulation of TLR4 was tested in HEK293 cells expressing the TLR4protein linked to an NF-�B promoter (Invivogen). NF-�B activation was as-sessed by measuring the luciferase emitted from the cells.

MTS and MTT assays. Cell viability (MTS) and growth (MTT) assaysduring experimental conditions were performed by seeding DCs into 96-wellplates (5 � 104 cells/ml and 1 � 106 cells/ml, respectively) and treating themwith all antigens (0 to 10 �g/ml) for 18 h. Control cells were treated withmedium or LPS alone. Following incubation, cell viability was assessed byadding 40 �l MTS solution {3-[4,5-dimethylthiazol-2-yl]-5-(3-carboxyme-thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium plus phenazine ethosulfate(PES)} to each well. Following an incubation of 3 h at 37°C, the absorbanceof each well was read at 450 nm. Cell growth was assessed by adding a volumeof MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bro-mide) equivalent to 10% of the culture medium to each well and incubatingthe cells for 3 h at 37°C. Culture medium was then removed, the resultingMTT formazan crystals were dissolved by adding MTT solvent (equal to theoriginal culture volume), and absorbance was read at 562 nm.

Cell viability was also measured using an annexin V-FITC apoptosis detectionkit I (BD Biosciences). Briefly, 1 � 106 cells/ml were seeded into a 24-well plateand treated with recombinant antigen (10 �g/ml) and each TLR ligand, eitherseparately or together, or with medium alone. Cells were harvested 24 h later,washed twice in cold PBS, and incubated in binding buffer (0.1 M HEPES-NaOH, 1.4 M NaCl, 25 mM CaCl2) with annexin V-FITC and propidium iodide(PI) for 15 min. Following the addition of more binding buffer, cells wereimmediately analyzed by flow cytometry.

In vivo T-cell assays and cytokine measurements. For assays of in vivo T-cellpriming, DCs isolated from BALB/c mice were stimulated with rFhCL1 orrFhGST-si (10 �g/ml) prior to incubating the cells in the presence of OVApeptide (323-ISQAVHAAHAEINEAGR-329) (100 nM; GenScript Corp.) for24 h. Following a wash step with sterile endotoxin-free PBS, DCs (3 � 105) weredelivered over the sternums of naive DO11.10 mice by subcutaneous injection.After 7 days, skin draining lymph nodes (sdLN) were removed, and a single-cellsuspension of cells was plated with medium, OVA peptide (500 nM), or phorbolmyristate acetate (PMA) (25 ng/ml; Sigma-Aldrich) and anti-CD3ε MAb (1�g/ml) (clone 145-2C11; BD Biosciences). After 72 h, supernatants were re-moved for measurement of IL-4, gamma interferon (IFN-�), IL-10, and IL-17 bycommercial assay (R&D Systems).

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Phagocytosis assay. The phagocytic ability of DCs was measured using theCytoSelect 96-well phagocytosis assay (Cell Biolabs Inc.) per the manufacturer’sinstructions. Briefly, DCs from C57BL/6 mice were plated at 0.5 � 106 cells/mlin complete RPMI and incubated overnight at 37°C to allow adherence to theplate. DCs were then cultured with rFhCL1, rFhGST-si (10 �g/ml), or LPS (100ng/ml) for 2.5 h before the addition of sheep erythrocytes opsonized by IgG at aratio of 1:50. After 1 h, phagocytosis was stopped with the removal of superna-tant and adherent cells were washed with sterile PBS to remove nonphagocy-tosed erythrocytes. Adherent DCs were then lysed, substrate was added, and thenumber of engulfed erythrocytes was determined by colorimetric assay at anabsorbance of 610 nm. Negative control cells were treated with 2 �M cytocha-lasin D to block phagocytosis.

Statistics. All data were analyzed for normality prior to statistical testing.Where multiple group comparisons were made, data were analyzed using one-way analysis of variance (ANOVA). For comparisons between two groups, Stu-dent’s t test was used. In all tests, P values of �0.05 were deemed significant.

RESULTS

rFhCL1 and rFhGST-si, but not rvFhCL1 or rFhGST-mu,induce partial activation of DCs. To assess the immunomodu-latory properties of F. hepatica secreted enzymes, function-

ally active recombinant sigma class GST (rFhGST-si), muclass GST (rFhGST-mu), and cathepsin L1 (rFhCL1) weresynthesized (11; LaCourse et al., unpublished data). In ad-dition, a conformationally intact proteolytically inactivevariant of cathepsin L1 (rvFhCL1), which has the active sitecysteine replaced with a glycine, was also produced (11). Allrecombinant preparations were deemed to be endotoxinfree, as assessed using the Pyrogene endotoxin detectionsystem (Cambrex).

Initially, a series of dose-response analyses were completedto determine the optimum quantity of recombinant protein forsubsequent analyses. A concentration of 10 �g/ml for eachantigen was selected, since DC viability or growth was notaffected and this concentration stimulated optimum cellularresponses (data not shown).

Dendritic cells stimulated with rFhCL1 secreted significantlevels of IL-12p40 and IL-6, but not IL-12p70, TNF-�, nitricoxide, IL-23, or IL-10 (Fig. 1A to C and data not shown),

FIG. 1. Exposure of DCs to rFhCL1 and rFhGST-si, but not rvFhCL1 and rFhGST-mu, induces partial activation of DCs. (A to C) DCs fromC57BL/6 mice were cultured with medium (Med), rFhCL1 (10 �g/ml), rvFhCL1 (10 �g/ml), rFhGST-si (10 �g/ml), rFhGST-mu (10 �g/ml), or LPS(100 ng/ml) for 24 h, and the production of IL-12p40, IL-6, and IL-12p70 in supernatants was determined by ELISA. DCs from C57BL/6 mice werecultured with a range of concentrations of rFhCL1 (D and E) or rFhGST-si (F and G) for 24 h, and the levels of IL-12p40 and IL-6 were measuredby ELISA. Data are presented as means ( standard errors of the means [SEM]) and are representative of three experiments. *, P � 0.05; **,P � 0.01; ***, P � 0.001 (compared to unstimulated cells) (not shown). (H) Cells were harvested and analyzed by four-color flow cytometry forCD40, CD80, and CD86. Cells were gated on CD11c� cells. The histograms show data for the isotype control (filled histograms), unstimulatedcells (black lines), rFhCL1- or rFhGST-si-stimulated cells (gray lines), and LPS-stimulated cells (dotted lines). Numbers represent meanfluorescence intensities (MFI) for five different experiments.

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compared to unstimulated cells. This activation of DCs wasdependent upon the dose of rFhCL1 delivered, with increasingdoses showing correspondingly increased secretion of IL-12p40 and IL-6 (Fig. 1D and E), indicating a requirement forenzymatic activity. Consistent with this was the observationthat stimulation of DCs with the enzymatically inactive variantrvFhCL1 did not induce the secretion of any cytokines mea-sured (Fig. 1A to C and data not shown).

Treatment of DCs with rFhGST-si induced an identical cy-tokine secretion profile to that with rFhCL1 (Fig. 1A to C),although the quantity of IL-6 measured was far greater thanthat seen following exposure to rFhCL1. Similar to the casewith rFhCL1, the secretion of these cytokines was also dosedependent (Fig. 1F and G). However, rFhGST-mu did notinduce the secretion of any cytokines (Fig. 1A to C and datanot shown), suggesting that the enzyme activity required forDC activation by Fasciola GST fusion proteins may be specificto distinct subclasses.

We also determined if these enzymes could induce elevatedexpression of the costimulatory markers CD40, CD86, CD80,and major histocompatibility complex class II (MHC II) onDCs. Both rFhCL1 and rFhGST-si stimulated a significantincrease in CD40 expression compared to the isotype control(Fig. 1H). The expression of CD80, CD86 (Fig. 1H), and MHCII (data not shown) was not altered following exposure to theserecombinants. Both rvFhCL1 and rFhGST-mu failed to alterthe expression levels of all cell surface markers measured (datanot shown).

We have given due consideration to the possibility that re-sidual endotoxin (whose major component is LPS) in the an-tigen preparations contributed to the activation of DCs de-scribed here. However, all of the data indicate that the effectsseen were due to the activity of the recombinant proteins, notthe presence of endotoxin. For example, the phenotype of DCactivation described for rFhCL1 and rFh-GST-si was differentfrom that induced by LPS (Fig. 1A to C and H). These enzymeswere produced from the same expression systems as rvFhCL1and rFhGST-mu, respectively, both of which had no measur-able effect on the activation status of DCs, suggesting that anyeffect seen was unlikely to be due to endotoxin contamination.Finally, the effects of rFhCL1 and rFhGST-si on DC activationwere shown to be heat labile, since they were no longer ob-served following boiling (data not shown), which contraindi-cates the involvement of endotoxin.

The partial activation of DCs by rFhCL1 and rFhGST-si isTLR4 dependent. Since previous reports have shown that hel-minth antigens can induce partial maturation of DCs throughTLR4 activation (22, 26), we went on to determine if theactivation of DCs by rFhCL1 and rFhGST-si was dependentupon the TLR4 pathway. The secretion of IL-12p40 and IL-6by rFhCL1 and rFhGST-si was significantly reduced in theabsence of the TLR4 receptor (TLR4KO) (Fig. 2A and B).While a low level of IL-6 secretion was measured in responseto rFhCL1 in the TLR4-deficient DCs, this was not significantcompared to cells stimulated with culture medium. Similar tothe cytokine results, rFhCL1, rFhGST-si, and LPS failed toinduce any alteration in CD40 expression in the absence ofTLR4 (Fig. 2C). As expected, TLR4-deficient DCs failed torespond to LPS. In addition, zymosan A, a TLR4-independent,TLR2-dependent agonist, induced the secretion of comparable

quantities of IL-12p40 and IL-6 in both wild-type and TLR4-deficient strains of mice, confirming the integrity of TLR sig-naling in the knockout DCs. These results confirm that activa-tion of DCs by rFhCL1 and rFhGST-si is dependent upon thepresence of TLR4.

C3H/HeJ mice exhibit a functional point mutation within the

FIG. 2. The partial activation of DCs by rFhCL1 and rFhGST-si isTLR4 dependent. DCs from C57BL/6 (background strain; WT),TLR4-deficient (TLR4KO), C3H/HeN, and C3H/HeJ mice were cul-tured with medium (Med), rFhCL1 (10 �g/ml), rFhGST-si (10 �g/ml),zymosan (5 �g/ml), or LPS (100 ng/ml) for 24 h, and the quantities ofIL-12p40 (A and D) and IL-6 (B and E) in supernatants were deter-mined by ELISA. Data are presented as means ( SEM) and arerepresentative of three experiments. ***, P � 0.001 compared tocontrol mice. (C and F) Dendritic cells were harvested and analyzed byfour-color flow cytometry, gated on CD11c� cells, for the expression ofCD40. In each histogram, the stimulated cells are represented by theblack line and the isotype control by the filled histogram. Numbersrepresent mean fluorescence intensities (MFI) for three separate ex-periments.



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Pro712 residue of the TIR domain of TLR4, preventing theproduction of cytokines from DCs in response to bacterial LPS(42). However, the TLR4-dependent response of these cells tohelminth-secreted ES-62 remained intact. In contrast, stimu-lation of DCs isolated from C3H/HeJ mice with either rFhCL1or rFhGST-si did not increase the expression of CD40 or thesecretion of IL-6 or IL-12p40 (Fig. 2D to F). The backgroundwild-type mice, C3H/HeN, which possess a fully functionalTLR4 protein, had increased CD40 expression and secretedIL-6 or IL-12p40 (Fig. 2D to F), as expected, in response toboth antigens.

rFhCL1 and rFhGST-si induce the secretion of chemokinesfrom DCs. Recently, it was shown that Th1-promoting DCsselectively express elevated MIP-1� and promote recruitmentof Th1 cells (20, 32), while MIP-2, a chemokine associated withhelminth infection (38), promotes the recruitment of Th2 cells.Given the role played by these chemokines in the selectivehoming of immune cells, we sought to characterize the profilesof MIP-1� (CCL3) and MIP-2 (CXCL2) chemokines producedby DCs following treatment with rFhCL1 and rFhGST-si.rFhCL1 and rFhGST-si induced both MIP-1� and MIP-2 fromDCs (Fig. 3A and B). However, these enzymes again showed adifferent profile from that for LPS with both enzymes, inducingsignificantly less MIP-1� than LPS but comparable levels ofMIP-2. The polarized secretion of MIP-2 by these enzymescould influence a Th2 microenvironment by directly recruit-ing Th2 cells to the site of infection. As shown in Fig. 3A andB, medium controls produced endogenous levels of bothchemokines.

The secretion of MIP-1� was TLR4 dependent, since theproduction of this chemokine was absent in DCs isolated fromTLR4-deficient mice. In contrast, the secretion of MIP-2 ap-peared to be partially dependent upon TLR4 (Fig. 3A and B),suggesting the involvement of a coreceptor. Further studies arerequired to identify this receptor and also to characterize a rolefor MIP-2 in Fasciola infection.

rFhCL1 and rFhGST-si differentially activate MAPKs (p38,ERK, and JNK), NF-�B, and IRF5 in DCs compared to LPS.Since the partial activation of DCs by rFhCL1 and rFhGST-siis mediated through TLR4, we investigated whether these an-tigens activated intracellular mitogen-activated protein kinases(MAPKs) associated with the TLR4 signaling pathway. Toaddress this question, the levels of phosphorylation of ERK,

p38, and JNK in DCs following treatment with rFhCL1 andrFhGST-si were determined by Western blotting, using LPS asa positive control.

As expected, LPS caused the activation of all three signaltransducers in comparison to medium, with distinct kineticssimilar to previously published data (Fig. 4A and C) (3, 4). Incontrast, only an increase in the activation of p38 was detectedfollowing treatment with rFhCL1. In addition, this was ob-served at a much later time point (60 min) than that seen forLPS activation. Stimulation of DCs with rFhGST-si inducedthe phosphorylation of JNK and p38, but not ERK, at mosttime points tested (Fig. 4A and C). While the kinetic profile ofJNK and p38 activation was similar to that for LPS, peaking at30 min, the level of phosphorylation activated by rFhGST-siwas significantly lower than that with LPS.

The transcription factor NF-�B is primarily associated withTLR innate immune cell activation, typically DC maturation,and is involved in regulating the expression of a large numberof inflammatory mediators (25). The phosphorylation of theDNA binding subunit p65 is an indicator of NF-�B activation(25). As expected, LPS stimulation of DCs led to an increase inthe presence of phosphorylated NF-�Bp65 (p-NF-�Bp65) at15, 30, and 60 min (Fig. 4A and C). Similarly, rFhGST-siinduced a significant increase in the level of p-NF-�Bp65 at 15,30, and 60 min. However, the extents of phosphorylation mea-sured at 15 and 60 min were less than those seen for LPS (Fig.4C). Activation of NF-�Bp65 was not detected in DCs stimu-lated by rFhCL1 at any time point examined. To confirm thesefindings, human recombinant HEK293 cells, functionally ex-pressing TLR4 linked to an NF-�B reporter gene, werestimulated with rFhGST-si, rFhCL1, and LPS (Fig. 4B).Consistent with the Western blot analyses, the data indi-cated that rFhGST-si but not rFhCL1 activated NF-�B anddemonstrated that this was dependent upon TLR4.

Since no phosphorylation of NF-�Bp65 was detected follow-ing treatment of DCs with rFhCL1, we investigated whetherthis enzyme activated the IRF5 transcription factor. Followingactivation of TLR4, IRF5 directly interacts with the TLR4adaptor protein MyD88 and functions as a key transcriptionfactor for TLR4-dependent induction of IL-6 and IL-12p40,independent of NF-�B and the MAPKs (50). Since we havedemonstrated that rFhCL1-induced secretion of IL-6 and IL-12p40 is dependent upon the presence of TLR4 (Fig. 2B), wemeasured the levels of IRF5 in DCs activated with rFhCL1 andrFhGST-si by Western blotting (Fig. 4A and C). However, onlyrFhGST-si, not rFhCL1, significantly enhanced the activationof IRF5 compared to that in unstimulated cells at 15 and30 min.

rFhCL1- and rFhGST-si-treated DCs do not phagocytoseantigen. The functional hallmark of DC maturation is theability to phagocytose antigens in combination with MHC II.Considering that both enzymes failed to induce the expressionof MHC II or to fully activate DCs, we investigated the abilityof Fasciola enzyme-activated DCs to phagocytose opsonizedsheep erythrocytes. DCs fully matured by LPS stimulationdemonstrated an ability to efficiently phagocytose antigen, witha significant increase (P � 0.01) in the number of engulfederythrocytes compared with medium controls (Fig. 5). In con-trast, rFhCL1- and rFhGST-si-treated DCs engulfed the same

FIG. 3. Chemokine secretion is observed in DCs matured withrFhCL1 and rFhGST-si. DCs from C57BL/6 (wild-type backgroundstrain) and TLR4-deficient mice were cultured with medium, rFhCL1(10 �g/ml), rFhGST-si (10 �g/ml), or LPS (100 ng/ml) for 24 h, and theproduction of MIP-1� (A) and MIP-2 (B) in supernatants was deter-mined by ELISA. Data are presented as means SEM and arerepresentative of three experiments. ��, P � 0.01; ***, P � 0.001(compared to wild-type mice).



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number of erythrocytes as immature unstimulated DCs, indi-cating an inability to phagocytose antigen (Fig. 5).

rFhCL1 and rFhGST-si bias the specific immune response byattenuating Th17 immune responses by OVA-specific T cells.Following phagocytosis, the role of fully matured DCs is topresent antigen to T cells, thus inducing antigen-specific im-

mune responses. We examined the ability of subcutaneouslyinjected differentially activated DCs pulsed with OVA to primeT-helper-cell responses in the skin draining lymph nodes ofDO11.10 transgenic mice, a strain expressing a T-cell receptorspecific for OVA peptide (26).

Skin draining lymph node cells isolated from mice which re-ceived DCs secreted antigen-specific IL-4, IL-10, IFN-�, andIL-17 (Fig. 6A to D) in response to OVA stimulation ex vivo.However, lymph node cells from mice that received DCs culturedwith either rFhCL1 or rFhGST-si and subsequently matured inthe presence of OVA had significantly reduced antigen-specificIL-17 production in response to stimulation with OVA (Fig. 6B).No significant differences were observed in the antigen-specificproduction of IL-4, IL-10, and IFN-� (Fig. 6A, C, and D).

Since IL-23 from DCs is important in driving the secretion ofIL-17 from CD4� T cells (1), we determined whether DCsexposed to rFhCL1 or rFhGST-si were less capable of produc-ing IL-23. DCs were exposed to rFhCL1 and rFhGST-si 2.5 hbefore activation with LPS. After 18 h of incubation in thepresence of LPS, the quantity of IL-23 in the supernatant wasmeasured. Concurrent with the ability to suppress IL-17-spe-cific responses, treatment of DCs with rFhCL1 and rFhGST-siprior to LPS stimulation resulted in significant suppression ofIL-23 production (Fig. 7).

FIG. 4. Differential activation of MAPKs, NF-�B, and IRF5 in DCs treated with rFhCL1 and rFhGST-si compared to that in DCs treated withLPS. (A) DCs from C57BL/6 mice were cultured with culture medium, rFhGST-si (10 �g/ml), rFhCL1 (10 �g/ml), or LPS (100 ng/ml). At 15, 30,and 60 min, cells were lysed and total cellular protein was extracted. Protein (10 �g/sample) was separated in a 10% SDS-PAGE gel, transferredto a polyvinylidene difluoride (PVDF) membrane, and sequentially probed for phosphorylated JNK (p-JNK), p-p38, p-ERK, total JNK (t-JNK),total p38, total ERK, p-NF-�Bp65, and IRF5. Representative blots from three experiments are shown. (B) Recombinant HEK-293 cellsfunctionally expressing TLR4 protein linked to an NF-�B luciferase reporter gene were stimulated with LPS, rFhGST-si (10 �g/ml), or rFheCL1(10 �g/ml) for 24 h, and representative data for two experiments are shown. (C) Densitometric analysis was performed on all immunoblots. p-JNK,p-p38, and p-ERK values were normalized to total JNK, ERK, or p38, and all values are expressed in arbitrary units as percent increases over themedium control group value. *, P � 0.05; ***, P � 0.001 (compared with medium control group).

FIG. 5. rFhCL1- and rFhGST-si-treated DCs do not phagocytoseantigen. DCs from C57BL/6 mice were cultured with medium, LPS(100 ng/ml), rFhCL1, or rFhGST-si (10 �g/ml) for 2.5 h before theaddition of opsonized sheep erythrocytes at a ratio of 1:50. After 1 h,the extent of phagocytosis was assessed by measuring the number ofengulfed erythrocytes, using a colorimetric assay. Data are presentedas means ( SEM) and are representative of three experiments. ��,P � 0.01 compared to untreated medium control group.



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DISCUSSION

We have previously demonstrated that F. hepatica ES mod-ulates T-cell responses in mice (15). Here we demonstrate thattwo major components of ES, cathepsin L and GST-si, alterthe function of DCs and, consequently, the ensuing T-cellresponse. Both antigens partially activate DCs to secrete IL-12p40, IL-6, and MIP-2 and to express CD40 in a TLR4-dependent manner. While the partial activation of DCs andthe development of Th2 responses are typical of helminthinfections (41), we found that rFhCL1- or rFhGST-si-stimu-lated DCs did not induce the differentiation of Th2 cells. Incontrast, these antigens attenuated the development of a Th17immune response, a function never previously described forhelminth-activated DCs. Therefore, despite sharing some phe-notypic characteristics of other helminth antigen-activatedDCs, rFhCL1- and rFhGST-si-activated DCs exhibited a dif-ferent activation state and effector function.

A strong correlation exists between Th1/Th17 immune re-sponses and the development of severe inflammation-medi-ated pathology in helminth-infected mice (9, 44). Therefore,regulation of IL-17 is critical to the control of inflammatorypathology associated with helminth infection. It has been sug-gested previously that the strong polarized Th2 environmentpromoted by helminths prevents the development of Th1/Th17immune responses (5). However, our data indicate that Fasci-ola secreted molecules actually suppress the differentiation ofTh17 cells, independently of Th2 cells, by altering the functionof DCs. Consistent with this hypothesis was the observationthat Fasciola antigen-activated DCs secreted reduced levelsof IL-23, a cytokine important in the expansion and survivalof Th17 cells (31).

The attenuation of Th17 development could also be ex-plained by the presence of the IL-12p40 homodimer, IL-12p80.Under a number of inflammatory conditions, the secretion of

IL-12p40 was closely associated with increased levels of IL-12p80 (12, 21, 53). Recent studies have reported a bioactiverole for IL-12p80 in preventing the biological activity of IL-23by blocking its binding to IL-12r1, one of the two chains in theIL-23 receptor complex (12, 21, 53). We know that the immu-nological assay employed in this study to detect IL-12p40 levelscross-reacts with IL-12p80, making it possible that this antag-onistic cytokine is secreted by DCs in response to these mol-ecules. rFhCL1 or rFhGST-si also stimulated DCs to secreteIL-6, whose role in IL-17 attenuation is less clear given itsinvolvement in the generation of Th17 cells. However, IL-6may be important in the differentiation and growth of otherT-cell subsets during infection (37).

The demonstration that two biochemically distinct enzymesinhibited Th17 immune responses would suggest that this im-munological outcome is critical for the success of F. hepaticainfection. This is supported by the fact that these moleculesutilize alternative pathways ensuring redundancy in the processof DC-mediated suppression of T-cell responses. Indeed, otherFasciola antigens induce a DC phenotype, independent of p38,ERK, JNK, or NF-�B, that suppresses Th1 immune responses(24). Helminth-activated DCs that have a capacity to promoteTh2 responses appear to be dependent upon the phosphory-lation of ERK (1, 2, 51) and display a rapid but transientincrease in the nuclear translocation of NF-�B (51). In con-trast, neither rFhCL1 nor rFhGST-si activated ERK andrFhGST-si induced a more persistent activation of NF-�B in DCsthan that observed for other helminths. Such distinct activationpatterns may explain the functional capacity of Fasciola-treated DCs to suppress Th17 cells rather than to promote Th2cells. In addition, IRF5 deregulation can impact the develop-ment of Th17-mediated autoimmune diseases (23). This studyis the first to report IRF5 activation by helminth antigens, andIRF5 activation by rFhGST-si may be required for the induc-tion of IL-6 and IL-12p40 or may be involved in the subsequentpolarization of T-cell responses by rFhGST-si-activated DCs.

Both rFhCL1 and rFhGST-si induced a suppressor DC phe-notype that was dependent upon the presence of the TLR4receptor, thus identifying these two enzymes as possible novelhelminth PAMPs. TLRs, while implicated in the recognition ofhelminth parasite products by DCs, may not be critical in thedevelopment of Th2 immune responses, since helminth-asso-ciated Th2 responses are not abrogated in the absence of TLRs

FIG. 7. rFhGST-si and rFhCL1 suppress the ability of DCs to se-crete IL-23 in response to LPS. DCs from C57BL/6 mice were culturedwith medium, rFhCL1, or rFhGST-si (10 �g/ml) 2.5 h prior to LPSstimulation (100 ng/ml). After 18 h, IL-23 in the supernatants wasmeasured by sandwich ELISA. Data are presented as means ( SEM)and are representative of three experiments. ***, P � 0.001 comparedwith LPS group.FIG. 6. DCs activated with rFhCL1 and rFhGST-si attenuate Th17

immune responses. DCs were cultured with OVA (100 nM) followingexposure to medium, rFhGST-si (10 �g/ml), or rFheCL1 (10 �g/ml)overnight at 37°C. (A to D) Stimulated DCs were subcutaneouslyinjected over the sternums of naïve DO11.10 mice. After 7 days, localskin draining lymph nodes were removed for restimulation in vitro withOVA (500 ng/ml) or PMA/anti-CD3 (25 ng/ml and 1 �g/ml). After72 h, supernatants were analyzed by ELISA for the presence of IFN-�,IL-17, IL-10, and IL-4. Data are means ( SEM) for three individualwells for four individual mice and are representative of three experi-ments. *, P � 0.05; ***, P � 0.001 (compared with OVA group).



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(28). Instead, TLR ligation by helminth-derived antigens isrecognized as a mechanism to limit the development of Th1cytokine-mediated inflammation. For example, the filarialnematode ES product ES-62 inhibits IL-12 secretion from DCsin response to classic stimulators, such as LPS/IFN-� (22), ina TLR4-dependent manner. Similarly, both rFhCL1 andrFhGST-si inhibit IL-23 secretion from LPS-matured DCs. F.hepatica secreted products therefore induce a phenotype ofDCs, via interaction with TLR4, that is hyporesponsive toclassical stimulation. However, unlike the case in other hel-minth studies, the consequence of this for Fasciola is a reduc-tion in the development of inflammatory Th17 responses.

Considering that a diverse range of helminth products arerecognized by TLR4, it is not surprising that these moleculesutilize the same receptor. However, the variation in transcrip-tional activation of DCs by rFhCL1 and rFhGST may reflectdifferences in their enzyme activity. At least seven species-independent classes of cytosolic GST are known and may bedistinguished from each other on the basis of substrate speci-ficity and inhibitor sensitivity (45, 47). Three GST classes,representing at least six distinct enzymes, have been identifiedin F. hepatica (10, 45). We have not determined if enzymeactivity is important for the activation of DCs described here.However, the sigma but not mu class induced the suppressorDC phenotype, which indicates that enzyme activity may beimportant. Given the dearth of knowledge on pathogen-de-rived GST interactions with DCs, it is not possible to speculateon a mechanism at this stage.

We have shown that the enzyme activity of rFhCL1, a mem-ber of the papain-like family of cysteine proteases (48, 49), isimportant for its immunomodulatory function. Similar pro-teases from this family, such as gingipain, a protease fromPrevotella intermedia, and bromelain, a protease extracted frompineapple, both cleave CD14 from the surfaces of monocytes,rendering them hyporesponsive to LPS (14). However, we haveshown that CD14 surface expression in response to rFhCL1 isunaltered (data not shown). While we do not know howrFhCL1 activates different TLR4 signaling, it is feasible that itmay cleave peptides from the TLR4 receptor which, uponrelease, could disrupt subsequent binding of coreceptors, ulti-mately leading to the inactivation of signaling pathways.

The demonstration that two biologically distinct molecules,using different pathways, activate DCs with the same capacityto modulate developing adaptive responses supports the viewthat helminth parasites induce a heterogenous population ofinnate immune cells to control the outcome of infection. Themajority of research investigating this phenomenon has usedpreparations of parasite secretions, which represent a compos-ite of many different proteins, lipids, and sugars. A number ofthese molecules, including CL1 and GST, are universally ex-pressed by all helminth parasites and suggest the existence ofa common mechanism(s) of modulation. Understandinghow each individual antigen from helminth antigen prepa-rations affects DC maturation and function may shed lighton the importance of discrete immunomodulatory mecha-nisms that collectively lead to the immune responses asso-ciated with helminth infection.

ACKNOWLEDGMENTS

This work was supported by the Dublin City University Faculty ofScience and Health Targeted Research Development Fund, the Eu-ropean Union (DELIVER; grant FOOD-CT-2005-023025), and theBBSRC (grant BB/C503638/2).

We thank Carolyn Wilson (DCU) for technical support.

REFERENCES

1. Aggarwal, S., N. Ghilardi, M. H. Xie, F. J. de Sauvage, and A. L. Gurney.2003. Interleukin-23 promotes a distinct CD4 T cell activation state charac-terized by the production of interleukin-17. J. Biol. Chem. 278:1910–1914.

2. Agrawal, A., S. Dillon, T. L. Denning, and B. Pulendran. 2006. ERK1�/�mice exhibit Th1 cell polarization and increased susceptibility to experimen-tal autoimmune encephalomyelitis. J. Immunol. 176:5788–5796.

3. Agrawal, S., A. Agrawal, B. Doughty, A. Gerwitz, J. Blenis, T. Van Dyke, andB. Pulendran. 2003. Cutting edge: different Toll-like receptor agonists in-struct dendritic cells to induce distinct Th responses via differential modu-lation of extracellular signal-regulated kinase-mitogen-activated protein ki-nase and c-Fos. J. Immunol. 171:4984–4989.

4. Arrighi, J. F., M. Rebsamen, F. Rousset, V. Kindler, and C. Hauser. 2001. Acritical role for p38 mitogen-activated protein kinase in the maturation ofhuman blood-derived dendritic cells induced by lipopolysaccharide, TNF-alpha, and contact sensitizers. J. Immunol. 166:3837–3845.

5. Bazzone, L. E., P. M. Smith, L. I. Rutitzky, M. G. Shainheit, J. F. Urban, T.Setiawan, A. M. Blum, J. V. Weinstock, and M. J. Stadecker. 2008. Coin-fection with the intestinal nematode Heligmosomoides polygyrus markedlyreduces hepatic egg-induced immunopathology and proinflammatory cyto-kines in mouse models of severe schistosomiasis. Infect. Immun. 76:5164–5172.

6. Brady, M. T., S. M. O’Neill, J. P. Dalton, and K. H. Mills. 1999. Fasciolahepatica suppresses a protective Th1 response against Bordetella pertussis.Infect. Immun. 67:5372–5378.

7. Cervi, L., G. Rossi, and D. T. Masih. 1999. Potential role for excretory-secretory forms of glutathione-S-transferase (GST) in Fasciola hepatica. Par-asitology 119:627–633.

8. Chaussabel, D., R. T. Semnani, M. A. McDowell, D. Sacks, A. Sher, and T. B.Nutman. 2003. Unique gene expression profiles of human macrophages anddendritic cells to phylogenetically distinct parasites. Blood 102:672–681.

9. Cheever, A. W., R. H. Duvall, T. A. Hallack, Jr., R. G. Minker, J. D. Malley,and K. G. Malley. 1987. Variation of hepatic fibrosis and granuloma sizeamong mouse strains infected with Schistosoma mansoni. Am. J. Trop. Med.Hyg. 37:85–97.

10. Chemale, G., R. Morphew, J. V. Moxon, A. L. Morassuti, E. J. LaCourse, J.Barrett, D. Johnston, and P. M. Brophy. 2006. Proteomic analysis of gluta-thione transferases from the liver fluke parasite Fasciola hepatica. Proteo-mics 23:6263–6273.

11. Collins, P. R., C. M. Stack, S. M. O’Neill, S. Doyle, T. Ryan, G. P. Brennan,A. Mousley, M. Stewart, A. G. Maule, J. P. Dalton, and S. Donnelly. 2004.Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica)virulence: propeptide cleavage sites and autoactivation of the zymogen se-creted from gastrodermal cells. J. Biol. Chem. 279:17038–17046.

12. Cooper, A. M., and S. A. Khader. 2006. IL-12p40: an inherently agonisticcytokine. Trends Immunol. 1:33–38.

13. Curtis, M. M., and S. S. Way. 2009. Interleukin-17 in host defence againstbacterial, mycobacterial and fungal pathogens. Immunology 126:177–185.

14. Deschner, J., A. Singhal, P. Long, C. C. Liu, N. Piesco, and S. Agarwal. 2003.Cleavage of CD14 and LBP by a protease from Prevotella intermedia. Arch.Microbiol. 179:430–436.

15. Donnelly, S., S. M. O’Neill, M. Sekiya, G. Mulcahy, and J. P. Dalton. 2005.Thioredoxin peroxidase secreted by Fasciola hepatica induces the alternativeactivation of macrophages. Infect. Immun. 73:166–173.

16. Donnelly, S., C. M. Stack, S. M. O’Neill, A. A. Sayed, D. L. Williams, andJ. P. Dalton. 2008. Helminth 2-Cys peroxiredoxin drives Th2 responsesthrough a mechanism involving alternatively activated macrophages. FASEBJ. 22:4022–4032.

17. Ferret-Bernard, S., R. S. Curwen, and A. P. Mountford. 2008. Proteomicprofiling reveals that Th2-inducing dendritic cells stimulated with helminthantigens have a ‘limited maturation’ phenotype. Proteomics 5:980–993.

18. Flynn, R. J., C. Mannion, O. Golden, O. Hacariz, and G. Mulcahy. 2007.Experimental Fasciola hepatica infection alters responses to tests used fordiagnosis of bovine tuberculosis. Infect. Immun. 75:1373–1381.

19. Flynn, R. J., and G. Mulcahy. 2008. The roles of IL-10 and TGF-beta incontrolling IL-4 and IFN-gamma production during experimental Fasciolahepatica infection. Int. J. Parasitol. 38:1673–1680.

20. Gafa, V., M. E. Remoli, E. Giacomini, M. C. Gagliardi, R. Lande, M. Severa,R. Grillot, and E. M. Coccia. 2007. In vitro infection of human dendritic cellsby Aspergillus fumigatus conidia triggers the secretion of chemokines forneutrophil and Th1 lymphocyte recruitment. Microbes Infect. 8:971–980.

21. Gillessen, S., D. Carvajal, P. Ling, F. J. Podlaski, D. L. Stremlo, P. C.Familletti, U. Gubler, D. H. Presky, A. S. Stern, and M. K. Gately. 1995.



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist.Eur. J. Immunol. 1:200–206.

22. Goodridge, H. S., F. A. Marshall, K. J. Else, K. M. Houston, C. Egan, L.Al-Riyami, F. Y. Liew, W. Harnett, and M. M. Harnett. 2005. Immunomodu-lation via novel use of TLR4 by the filarial nematode phosphorylcholine-containing secreted product, ES-62. J. Immunol. 174:284–293.

23. Gutierrez-Roelens, I., and B. R. Lauwerys. 2008. Genetic susceptibility toautoimmune disorders: clues from gene association and gene expressionstudies. Curr. Mol. Med. 6:551–561.

24. Hamilton, C. M., D. J. Dowling, C. E. Loscher, R. M. Morphew, P. M.Brophy, and S. M. O’Neill. 2009. Fasciola hepatica tegumental antigen sup-presses dendritic cell maturation and function. Infect. Immun. 6:2488–2498.

25. Hayden, M. S., and S. Ghosh. 2008. Shared principles in NF-kappaB signal-ing. Cell 132:344–362.

26. Jenkins, S. J., J. P. Hewitson, S. Ferret-Bernard, and A. P. Mountford. 2005.Schistosome larvae stimulate macrophage cytokine production throughTLR4 dependent and -independent pathways. Int. Immunol. 17:1409–1418.

27. Jenkins, S. J., and A. P. Mountford. 2005. Dendritic cells activated withproduct released by schistosome larvae drive Th2-type immune responses,which can be inhibited by manipulation of CD40 costimulation. Infect. Im-mun. 73:395–402.

28. Kane, C. M., E. Jung, and E. J. Pearce. 2008. Schistosoma mansoni eggantigen-mediated modulation of Toll-like receptor (TLR)-induced activa-tion occurs independently of TLR2, TLR4, and MyD88. Infect. Immun.12:5754–5759.

29. Kapsenberg, M. L. 2003. Dendritic-cell control of pathogen-driven T-cellpolarization. Nat. Rev. Immunol. 3:984–993.

30. LaCourse, E. J., M. Hernandez-Viadel, J. R. Jefferies, C. Svendsen, D. J.Spurgeon, J. Barrett, P. Kille, and P. M. Brophy. 2009. Glutathione trans-ferase (GST) as a candidate molecular based biomarker for soil toxin expo-sure in the earthworm Lumbricus rubellus. Environ. Pollut. 157:2459–2469.

31. Reference deleted.32. Lebre, M. C., T. Burwell, P. L. Vieira, J. Lora, A. J. Coyle, M. L. Kapsenberg,

B. E. Clausen, and E. C. De Jong. 2005. Differential expression of inflam-matory chemokines by Th1- and Th2-cell promoting dendritic cells: a role fordifferent mature dendritic cell populations in attracting appropriate effectorcells to peripheral sites of inflammation. Immunol. Cell Biol. 5:525–535.

33. MacDonald, A. S., and R. M. Maizels. 2008. Alarming dendritic cells for Th2induction. J. Exp. Med. 205:13–17.

34. MacDonald, A. S., A. D. Straw, B. Bauman, and E. J. Pearce. 2001. CD8�dendritic cell activation status plays an integral role in influencing Th2response development. J. Immunol. 167:1982–1988.

35. Maizels, R. M., A. Balic, N. Gomez-Escobar, M. Nair, M. D. Taylor, and J. E.Allen. 2004. Helminth parasites—masters of regulation. Immunol. Rev. 201:89–116.

36. McManus, D. P., and J. P. Dalton. 2006. Vaccines against the zoonotictrematodes Schistosoma japonicum, Fasciola hepatica and Fasciola gigantica.Parasitology 133(Suppl.):S43–S61.

37. Mudter, J., and M. F. Neurath. 2007. IL-6 signaling in inflammatory boweldisease: pathophysiological role and clinical relevance. Inflamm. Bowel Dis.8:1016–1023.

38. Muller, K., S. Bischof, F. Sommer, M. Lohoff, W. Solbach, and T. Laskay.2003. Differential production of macrophage inflammatory protein 1gamma(MIP-1gamma), lymphotactin, and MIP-2 by CD4(�) Th subsets polarizedin vitro and in vivo. Infect. Immun. 11:6178–6183.

39. O’Neill, S. M., M. T. Brady, J. J. Callanan, G. Mulcahy, P. Joyce, K. H.Mills, and J. P. Dalton. 2000. Fasciola hepatica infection downregulates Th1responses in mice. Parasite Immunol. 22:147–155.

40. O’Neill, S. M., K. H. Mills, and J. P. Dalton. 2001. Fasciola hepatica cathep-sin L cysteine proteinase suppresses Bordetella pertussis-specific interferon-gamma production in vivo. Parasite Immunol. 23:541–547.

41. Perrigoue, J. G., F. A. Marshall, and D. Artis. 2008. On the hunt forhelminths: innate immune cells in the recognition and response to helminthparasites. Cell. Microbiol. 9:1757–1764.

42. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. Van Huffel, X. Du, D.Birdwell, E. Alejos, M. Silva, C. Galanos, M. Freudenberg, P. Ricciardi-Castagnoli, B. Layton, and B. Beutler. 1998. Defective LPS signaling inC3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085–2088.

43. Rigano, R., B. Buttari, E. Profumo, E. Ortona, F. Delunardo, P. Margutti, V.Mattei, A. Teggi, M. Sorice, and A. Siracusano. 2007. Echinococcus granu-losus antigen B impairs human dendritic cell differentiation and polarizesimmature dendritic cell maturation towards a Th2 cell response. Infect.Immun. 4:1667–1678.

44. Rutitzky, L. I., L. Bazzone, M. G. Shainheit, B. Joyce-Shaikh, D. J. Cua, andM. J. Stadecker. 2008. IL-23 is required for the development of severeegg-induced immunopathology in schistosomiasis and for lesional expressionof IL-17. J. Immunol. 180:2486–2495.

45. Salvatore, L., G. Wijffels, J. L. Sexton, M. Panaccio, S. Mailer, I. McCauley,and T. W. Spithill. 1995. Biochemical analysis of recombinant glutathioneS-transferase of Fasciola hepatica. Mol. Biochem. Parasitol. 69:281–288.

46. Segura, M., Z. Su, C. Piccirillo, and M. M. Stevenson. 2007. Impairment ofdendritic cell function by excretory-secretory products: a potential mecha-nism for nematode-induced immunosuppression. Eur. J. Immunol. 37:1887–1904.

47. Sheehan, D., G. Meade, V. M. Foley, and C. A. Dowd. 2001. Structure,function and evolution of glutathione transferases: implications for classifi-cation of non-mammalian members of an ancient enzyme superfamily. Bio-chem. J. 360:1–16.

48. Stack, C. M., S. Donnelly, J. Lowther, W. Xu, P. R. Collins, L. S. Brinen, andJ. P. Dalton. 2007. The major secreted cathepsin L1 protease of the liverfluke, Fasciola hepatica: a Leu-12 to Pro-12 replacement in the noncon-served C-terminal region of the prosegment prevents complete enzyme au-toactivation and allows definition of the molecular events in prosegmentremoval. J. Biol. Chem. 22:16532–16543.

49. Stack, C. M., C. R. Caffrey, S. M. Donnelly, A. Seshaadri, J. Lowther, T. F.Tort, P. R. Collins, M. W. Robinson, W. Xu, J. H. McKerrow, C. S. Craik,S. R. Geiger, R. Marion, L. S. Brinen, and J. P. Dalton. 2008. Structural andfunctional relationships in the virulence-associated cathepsin L proteases ofthe parasitic liver fluke, Fasciola hepatica. J. Biol. Chem. 283:9896–9908.

50. Takaoka, A., H. Yanai, S. Kondo, G. Duncan, H. Negishi, T. Mizutani, S.Kano, K. Honda, Y. Ohba, T. W. Mak, and T. Taniguchi. 2005. Integral roleof IRF-5 in the gene induction programme activated by Toll-like receptors.Nature 434:243–249.

51. Thomas, P. G., M. R. Carter, A. A. Da’dara, T. M. DeSimone, D. A. Harn.2005. A helminth glycan induces APC maturation via alternative NF-kappaB activation independent of I kappa B alpha degradation. J. Immunol.175:2082–2090.

52. Whelan, M., M. M. Harnett, K. M. Houston, V. Patel, W. Harnett, and K. P.Rigley. 2000. A filarial nematode-secreted product signals dendritic cells toacquire a phenotype that drives development of Th2 cells. J. Immunol.12:6453–6460.

53. Wysocka, M., M. Kubin, L. Q. Vieira, L. Ozmen, G. Garotta, P. Scott, and G.Trinchieri. 1995. Interleukin-12 is required for interferon-gamma produc-tion and lethality in lipopolysaccharide-induced shock in mice. Eur. J. Im-munol. 3:672–676.

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