hepatica Tegumental Coat Impairs Mast Cells' Ability To Drive Th1 Immune Responses

This information is current as Krisztina V. Vukman, Paul N. Adams, Martin Metz, Marcus of September 28, 2021. Maurer and Sandra M. O'Neill J Immunol 2013; 190:2873-2879; Prepublished online 15 February 2013; doi: 10.4049/jimmunol.1203011

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Fasciola hepatica Tegumental Coat Impairs Mast Cells’ Ability To Drive Th1 Immune Responses

Krisztina V. Vukman,* Paul N. Adams,* Martin Metz,† Marcus Maurer,† and Sandra M. O’Neill*

The parasitic induces strong Th2 and T-regulatory immune responses while simultaneously suppressing Th1-driven immune responses to bystander microbial . It also prevents the initiation of Th1-mediated autoimmune disorders in mice through the suppression of Th17 and Th1 immune responses, and this can be mimicked by parasite-derived molecules. We have isolated F. hepatica tegumental coat Ag (FhTeg) and demonstrated its suppressive effect in vivo by directly targeting dendritic cells, impairing their ability to drive Th1 responses. Mast cells are critical in promoting Th1 protective immunity during bacterial and in driving Th1-mediated pathological conditions in autoimmune . In this article, we show that FhTeg inhibits the ability of mast cells to drive the Th1 by suppressing cytokine secretion (TNF-a, Downloaded from IL-6, IFN-g, and IL-10) and ICAM1 expression in mast cells stimulated with LPS or heat-inactivated Bordetella pertussis Ag. These heat-inactivated B. pertussis Ag/LPS–stimulated mast cells fail to promote Th1 immune responses in CD4+ T cells when pretreated with FhTeg, and a role for ICAM1 in this process was demonstrated. FhTeg suppresses the activation of transcription factors in the TLR signaling pathway, which explains the decrease in cytokine production and cell surface marker expression. We demonstrated that FhTeg suppresses MAPK and NF-kB activation and enhances SOCS3 expression, which could explain its negative effect on the TLR pathways. We conclude that FhTeg targets innate immune cells, inhibiting their ability to drive Th1 http://www.jimmunol.org/ immune responses. The Journal of Immunology, 2013, 190: 2873–2879.

ast cells are associated with helminth infection but significantly impacts populations in the developing world (12). are mainly studied in the context of IgE-mediated re- However, they can prevent the initiation and perpetuation of many M sponses and Th2 immunity. Mast cells express path- Th1 immune disorders, such as multiple sclerosis and Crohn’s ogen recognition receptors such as TLRs and, when activated with (13, 14), in these populations. Helminth-driven regulatory TLR ligands, are a potent source of Th1 proinflammatory medi- networks, through the induction of regulatory cytokines TGF-b ators, such as TNF-a and IFN-g. Mast cells promote Th1-driven and IL-10, can suppress these proinflammatory Th1 immune by guest on September 28, 2021 defense against bacterial and protozoan infections (1–4), and responses (14, 15). Furthermore, helminths release molecules that in the absence of mast cells these infections are significantly ex- directly impair the ability of innate immune cells to drive Th1/Th17 acerbated (5, 6), demonstrating a critical role for mast cells. Mast immune responses (10, 16). Understanding these mechanisms could cells are also associated with many Th1-mediated autoimmune lead to the development of novel anti-inflammatory therapeutic disorders, such as multiple sclerosis and Crohn’s disease, in which targets and expand our understanding of helminth–microbial TNF-a–secreting mast cells are important contributors to immune interactions in populations. disorders (7–9). Fasciola hepatica, a parasitic flatworm also known as the Helminths and the products they release exert effects that impair common liver fluke, can suppress protective Th1-driven immunity Th1 immunity to bystander microbial infections, such as against bystander infections such as and Bordetella and tuberculosis (10, 11), and this impairment of Th1 immunity pertussis infection (17–19). It can also attenuate experimental au- toimmune encephalomyelitis (EAE) through the suppression of Th17 and Th1 immune responses (20). *Parasite Immune Modulation Group, School of Biotechnology, Faculty of Science Fasciola tegumental coat is shed from the parasite every 2–3 h and Health, Dublin City University, Glasnevin, Dublin 9, Ireland; and †Department of Dermatology and , Charite´-Universita¨tsmedizin Berlin, 10117 Berlin, Germany and is in constant contact with host immune cells. Previously, we Received for publication October 31, 2012. Accepted for publication January 7, have isolated F. hepatica tegumental coat Ag (FhTeg) and ex- 2013. amined its interactions with innate immune cells. Most of the This work was supported by the Programme for Research in Third Level Institutions isolated from FhTeg (21–23) and the contents of the (PRTLI) Cycle 4—the PRTLI is cofunded through the European Regional Develop- glycocalyx (24) are identified. More than 50 different proteins are ment Fund, part of the European Union Structural Funds Programme 2007–2013; and by European Cooperation in Science and Technology Action BM1007, “Mast Cells present in the preparation, and the biological active component and —Targets for Innovative Therapies.” or components of FhTeg have yet to be identified. Currently, the Address correspondence and reprint requests to Dr. Sandra O’Neill, Parasite Immune focus is on elucidating FhTeg’s function and role in immunomo- Modulation Group, Biotechnology, Faculty of Science and Health, Dublin City Uni- dulation. We have demonstrated the Th1-suppressive effect of versity, Glasnevin, Dublin 9, Ireland. E-mail address: [email protected] FhTeg in vivo by inhibiting the release of proinflammatory cyto- Abbreviations used in this article: BMMC, bone marrow–derived ; BP, heat-inactivated Bordetella pertussis Ag; EAE, experimental autoimmune enceph- kines in the mouse model of septic shock (25). We also demon- alomyelitis; FhTeg, Fasciola hepatica tegumental coat Ag; PCMC, peritoneum- strated that FhTeg alters the ability of dendritic cells to prime Th1 derived cultured mast cell; qPCR, quantitative PCR; SOCS3, suppressor of cytokine immune responses by rendering them hyporesponsive to TLR signaling 3. activation, suppressing the production of a select panel of in- Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 flammatory cytokines and costimulatory molecules. The sup- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203011 2874 HELMINTH SUPPRESSES MAST CELL PROINFLAMMATORY RESPONSES pressive effect is not mediated through the common MAPKs Coculture studies and cytokine measurements found in the TLR pathway and is independent of MyD88 and CD4+ T cells were isolated using the MACS CD4+ T Cell Isolation Kit TLR4. We have shown that FhTeg suppresses the transcription (Miltenyi Biotec, Surrey, U.K.) and were used if the purity was $ 95%, factor NF-kB p65, which is involved in the transcription of many as determined by flow cytometry (FITC-conjugated CD4, clone: L3T4; Th1-promoting factors (25). This study examines FhTeg’s inter- BD Biosciences). BMMCs and peritoneum-derived cultured mast cells action with mast cells to determine if its modulatory effect can (PCMCs) were cultured in the presence of FhTeg (10 mg/ml) 2.5 h before stimulation with LPS (100 ng/ml) or BP (100 per cell) for 24 h. impair mast cells’ ability to promote Th1 immunity, and the Washed BMMCs and PCMCs or supernatant was added to CD4+ cells (1:1 possible mechanism involved in this process will be discussed. cell/cell or supernatant/ex vivo media ratio) in plates coated with anti-CD3 (1 mg/ml; BD Biosciences) for 72 h, and supernatants were tested for IFN- g, IL-4, IL-5, and IL-10 by commercial ELISA (BD Biosciences). In some Materials and Methods experiments, anti-ICAM1 blocking Ab (clone: YN1/1.4; Abcam, Cam- and Ags bridge, U.K.) was added to mast cells for 30 min, and then mast cells were washed prior to coculturing with CD4+ cells. C57BL/6 mice, 6–8 wk old, were purchased from Charles River (Carrentrilla, Ireland). Mice were kept under specific –free con- RNA extraction and RT-PCR ditions at the Bioresource Unit, Faculty of Health and Science, Dublin City University, Dublin, Ireland. All mice were maintained according to the BMMCs were stimulated with or without FhTeg (10 mg/ml) 2.5 h prior to guidelines of the Irish Department of Children and Health. Ethical ap- stimulation with BP, LPS, or PBS. Cells were harvested and washed after proval for mice experiments was obtained from the Dublin City University 5, 15, 30, and 60 min before resuspending in TRISure (Bioline, London, ethics committee and the Irish Department of Children and Health. U.K.), and RNA was extracted according to the manufacturer’s instruc- FhTeg was prepared as previously published (25). Endotoxin levels were tions. To eliminate genomic DNA contamination, RNA samples were Downloaded from determined using the PyroGene Endotoxin Detection System (Cambrex/ treated with RNase-free DNase (Invitrogen, Paisley, U.K.); cDNA was Lonza, Walkersville, MD). FhTeg yielded levels similar to background synthesized using a reverse transcriptase kit (Promega, Madison, WI) levels, and they were less than the lower limit of detection in this assay according to the manufacturer’s protocol. (,0.01 EU/ml). Heat-inactivated Bordetella pertussis (BP) was a kind gift The quantitative PCR (qPCR) transcription analysis was carried out on a from Prof. Bernie Mahon, National University of Ireland Maynooth, real-time thermal cycler ABI7900FH (Applied Biosystems, Paisley, U.K.), Maynooth, Ireland (26). All concentrations were determined using using Maxima SYBR Green Master Mix according to the manufacturer’s a bicinchoninic acid protein assay kit (Pierce/Fischer, Dublin, Ireland). protocol (Fermentas/Fischer, Dublin, Ireland) in triplicate on 96-well

MicroAmp Optical Plates (Applied Biosystems). Three internal reference http://www.jimmunol.org/ experiments genes were used: b-actin (NM_007393.3), GAPDH (NM_008084.2), and Gusb (b-glucuronidase, NM_010368.1). Samples were maintained for C57BL/6 mice were i.p. injected with PBS or FhTeg (10 mg) 1 h prior to the 10 min at 95˚C as the initial step, then 15 s at 95˚C and 60 s at 60˚C, injection with BP (5 mg) every third day for 10 d (total of four injections). through 40 cycles. After amplification, melting curves were recorded (from Peritoneal cells were obtained from the mice 24 h after the last injection by 50 to 95˚C) to check product size and homogeneity. Pfaffl’s method was injecting 10 ml PBS into the peritoneal cavity. Cell numbers and viability used to calculate relative changes in gene transcription (30). The PCR were monitored using trypan blue staining. Percentages of mast cells were efficiency was determined using eight-step dilution series (0.3–20 ng determined by measuring cell surface expression of c-kit and FcεRI and by cDNA). Primers are listed in Table I. Kimura staining (27). Cells were stained for intracellular TNF-a expres- sion immediately after peritoneal lavage. Protein extraction and Western blot analysis BMMCs and PCMCs were stimulated with FhTeg (10 mg/ml) 2.5 h prior by guest on September 28, 2021 Isolation, maturation, and characterization of bone to stimulation with LPS (100 ng/ml) or BP (100 bacteria per cell) for 5, marrow– and peritoneum-derived cultured mast cells 15, 30, or 60 min. Total protein was extracted from cell lysates, using ra- dioimmunoprecipitation assay buffer containing protease and phosphatase Bone marrow–derived mast cells (BMMCs) were generated from the inhibitor cocktails. Protein samples (10–40 mg) and prestained protein femoral and tibia bone marrow cells of C57BL⁄6 mice and maintained in complete IMDM in the presence of 10% heat-inactivated FCS, 100 U/ml markers (See Blue Plus 2; Invitrogen) were separated by SDS-PAGE and m penicillin/streptomycin, and 30% WEHI-3–conditioned IMDM medium blotted onto 0.45- m Immobilon-P polyvinylidene fluoride membranes (ATTC TIB-68) as a source of the murine growth factor IL-3 for 4 wk (28). (Millipore, Carrigtwohill, Ireland). Membranes were stained according to Peritoneal cells were obtained from C57BL/6 mice after i.p. injection of standard Western blot protocol using anti–phospho-ERK (phospho-ERK; Cell Signaling Technology, Beverly. MA), anti–total-ERK, anti–phospho- 10 ml sterile PBS and then cultured in RPMI 1640 medium supplemented p38, anti–total-p38, anti–phospho-JNK, anti–total-JNK, anti–NF-kB p65, with 10% FCS and 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, b 10 ng/ml recombinant mouse IL-3 (Calbiochem/Merck, Darmstadt, Ger- anti-SOCS3, and anti– -actin primary Abs and peroxide-conjugated anti- many), and 30 ng/ml recombinant mouse stem cell factor at 37˚C, as rabbit IgG (Sigma-Aldrich, Dublin, Ireland) secondary Ab. Proteins were previously described (29). At 48 h later, nonadherent cells were removed visualized with a chemiluminescent HRP substrate (Millipore), exposed to and replaced by fresh culture medium for an additional 7 d. Of the total film, and processed using an FPM 100A processor (Fuji Film). Protein cells, .95% were identified as mast cells on the basis of c-kit and FcεRI bands were quantified using ImageJ analysis software (imagej.nih.gov). Levels of phospho-ERK, phospho-p38, phospho-JNK, and phospho–NF- cell surface expression or Kimura staining. Cell number and viability were kB p65 were normalized to total-ERK, total-p38, total-JNK, and b-actin monitored using trypan blue staining. To test functionality, b-hexosamin- idase release from mast cells was measured as previously described (29). and expressed in arbitrary units as percentage increases over the medium Mast cells were cultured with LPS (100 ng/ml; Alexis, San Diego, CA), control levels. BP (100 bacteria per cell), PMA (25 ng/ml), or A23187 (1 mM), or with FhTeg (10 mg/ml) 2.5 h prior to stimulation with BP or LPS. After 6 or 24 h, levels of TNF-a, IL-6, IL-4, IL-5, IL-10, and IFN-g were measured Table I. List of primers for qPCR transcription analysis in supernatant by commercial ELISA (BD Biosciences, Oxford, U.K.). Cells were incubated for 15 min with CD16/CD32 (FcgIII/II; BD Biosciences) to block FcR prior to flow cytometric analysis. Cells were Product stained with PE/FITC/APC-conjugated anti-FcεRI (clone: MAR1; eBio- Name Sequence (59→39) Size (bp) Tm (˚C) science, Hatfield, U.K.), anti-CD117 (c-kit; clone: 2B8), CD54 (ICAM1; b-actin AGTCCCTCACCCTCCCAAA 266 61.83 clone: YN1/1.7.4), anti-CD252 (OX40L; clone: RM134L), CD40L (clone: CTGCCTCAACACCTCAACC 59.25 MRI), CD28 (BD 37.51), or the appropriate IgG isotype control, for GAPDH CAAGAAGGTGGTGAAGCAG 112 56.92 30 min at 4˚C in the dark. In multicolor experiments, unstained, single- GAAGGTGGAAGAGTGGGAGTT 59.59 stained, and fluorescence minus one controls were also used. mAb binding Gusb ACCAGCCACTATCCCTACTCA 200 58.68 was analyzed by flow cytometry (FACSAria; BD Biosciences) in the GCCACAGACCACATCACAAC 60.01 presence of DNase to avoid aggregation, using FACSDiva and FlowJo SOCS3 GGTTCTGCTTTGTCTCTCCTATGT 95 60.19 Software. Dead cells and cell debris were gated out on the basis of their TGTGTTTGGCTCCTTGTGTG 60.79 forward and side scatter profile. C-kit and FcεRI double-positive cells were designated as mast cells. Tm, Temperature. The Journal of Immunology 2875

Statistics moved and cells were washed. Supernatant was added to CD4+ + All data were analyzed for normality prior to statistical testing by Origin 6.1 T cells, or CD4 T cells were cocultured with washed mast cells. (OriginLab) software. When multiple group comparisons were made, data When cocultured with BP- and LPS-stimulated BMMCs or were analyzed using one-way ANOVA. For comparisons between two PCMCs, or when cultured with supernatant from these cells, CD4+ groups, the Student t test was used. In all tests, p , 0.05 was deemed T cells secreted significant levels of IFN-g, IL-10, and IL-5 (Fig. significant (*p # 0.05, **p # 0.01). 3), but no IL-4 (data not shown). Significantly lower levels of IFN- g, IL-10, and IL-5 cytokines were obtained when T cells were Results cocultured with BP- and LPS-stimulated BMMCs or PCMCs in FhTeg inhibits TNF-a expression in mast cells in vivo the presence of FhTeg, or when the T cells were cultured with F. hepatica suppresses protective Th1-driven immunity against supernatant from these cells (Fig. 3). BMMCs, PCMCs, and T B. pertussis (18), and because mast cells have an important role cells alone were used as negative controls; and levels of IFN-g, in protection against bacterial infections, we wanted to determine IL-10, IL-4, or IL-5 were not significantly higher than background if FhTeg can inhibit the production of proinflammatory cytokines levels and were significantly less than levels in cocultured samples by mast cells in vivo. In this study, we first demonstrated that BP, when measured after 72 h. when injected into the peritoneal cavity of C57BL/6 mice, induced ICAM1 expression on mast cells is important in mast high numbers of TNF-a–expressing mast cells (Fig. 1). When cell–T cell communication, and its expression is also inhibited these mice were coinjected with FhTeg, mast cells expressed sig- by FhTeg nificantly less TNF-a than did those in BP-injected mice, confirming that FhTeg can inhibit the production of proinflammatory cytokines OX40L, CD40L, CD28, and ICAM1 are thought to have a critical Downloaded from by mast cells in vivo (Fig. 1). role in mast cell–T cell communication (33–35). BMMCs and PCMCs express low levels of OX40L, CD40L, and CD28 (data FhTeg suppresses cytokine secretion from LPS- or not shown), and these were not significantly enhanced in the BP-stimulated mast cells presence of LPS and BP. In contrast, ICAM1 was highly expressed To confirm our in vivo findings, we stimulated BMMCs and on these cells, and this was enhanced following stimulation with

PCMCs in vitro with BP or LPS as a positive control in the presence LPS and BP (Fig. 4). FhTeg did not enhance OX40L, CD40L, http://www.jimmunol.org/ or absence of FhTeg, and cytokine secretion was measured in CD28, and ICAM1 on mast cells (data not shown). Because supernatant after 24 h. Both BMMCs (IL-6 and TNF-a) and PCMCs ICAM1 was highly expressed, we stimulated mast cells with BP (IL-6, TNF-a, IL-10, and IFN-g) secreted significant levels of and LPS in the presence of FhTeg and demonstrated that FhTeg cytokines; however, in the presence of FhTeg, cytokine secretion significantly inhibited ICAM1 expression on BMMCs (data not was significantly suppressed (Fig. 2; p # 0.05 to p # 0.001). We shown) and PCMCs (Fig. 4; p # 0.05). We confirmed that ICAM1 2 2 repeated these experiments with BMMCs from STAT6 / mice, was important in mast cell–CD4+ T cell communication by adding and we demonstrated that FhTeg had the same suppressive effect anti-ICAM1 blocking Ab to activated mast cells a half hour prior 2 2 2 2 in STAT6 / mast cells (data not shown). However, STAT6 / to washing and coculturing with CD4+ T cells. Like FhTeg, it mast cells secreted more Th1 cytokines after BP stimulation. caused a significant decrease in IFN-g, IL-10, and IL-5 secretion by guest on September 28, 2021 CD4+ T cells cocultured with FhTeg-treated mast cells secrete (Fig. 5A–C). less IL-10, IFN-g, and IL-5 The FhTeg suppressive effect is not specific to NF-kB p65 Mast cells, when cocultured with naive CD4+ cells in the presence expression, as it inhibits MAPK activation in mast cells of anti-CD3, can promote Th1 immune responses, and this can be We previously showed that FhTeg suppresses the activation of enhanced when mast cells are pretreated with bacterial Ags or NF-kB in LPS-stimulated dendritic cells (25), and this was exam- TLR ligands (31–33). BMMCs and PCMCs were stimulated with ined in mast cells. It was first confirmed that BP can induce NF-kB BPandLPSinthepresenceorabsenceofFhTegfor24hto p65 (at 15 and 30 min) expression in BMMCs (Fig. 6A–C). FhTeg determine if mast cell ability to promote cytokine production from suppressed the activation of NF-kB in LPS- and BP-stimulated naive T cells can be inhibited. After 24 h, supernatants were re- mast cells (Fig. 6A–C), and this was confirmed in PCMCs at 15 and 30 min (Fig. 6D–F). To test whether this effect was specific to NF-kB, the phos- phorylation of ERK, JNK, and p38 was measured. First it was confirmed that BP can induce phospho-ERK, -JNK, and -p38 expression in mast cells, and in this study it was demonstrated that BP can significantly enhance phospho-ERK, but not phospho-JNK and -p38. We also showed that FhTeg suppressed the activation of phospho-ERK in LPS- and BP-stimulated mast cells (at 5, 30, and 60 min), and these results were confirmed in PCMCs at 15 and 30 min (Fig. 6D–F). SOCS3 transcription and expression is up-regulated by FhTeg in mast cells FIGURE 1. FhTeg inhibits TNF-a expression in mast cells in vivo. As both the NF-kB and MAPK pathways are suppressed by C57BL/6 mice were injected with FhTeg (10 mg) or PBS prior to being FhTeg, we hypothesize that this Ag targets signaling molecules immunized by BP (10 mg) four times for 10 d. Peritoneal mast cells were intracellularly stained for TNF-a. Mast cells were identified on the basis of further up the TLR4 signaling pathway or FhTeg may induce c-kit and FcεRI expression by flow cytometry. Data are presented as the negative regulators of the TLR pathway. First, the expression of mean 6 SEM of two independent experiments, with three to five mice in suppressor of cytokine signaling 3 (SOCS3), a known inhibitor of each group. *p # 0.05 compared with control group. Fluorescence minus the TLR4 signaling pathway, was measured by qPCR (36) (Table one control is indicated with dashed line. MFI, Mean fluorescence intensity. I). The SOCS3 RNA level was significantly higher at 5 and 15 2876 HELMINTH SUPPRESSES MAST CELL PROINFLAMMATORY RESPONSES

FIGURE 2. FhTeg suppresses cytokine expression in LPS- or BP-stimulated BMMCs and PCMCs. Mast cells were stimulated with and without FhTeg

(10 mg/ml) for 2.5 h prior to stimulation with vehicle (PBS), LPS (100 ng/ml), BP (100 bacteria per cell), or PMA (20 ng/ml) for 24 h. With a commercial Downloaded from ELISA, TNF-a (A) and IL-6 (B) levels were measured in the supernatants of BMMCs, whereas TNF-a (C), IL-6 (D), IFN-g (E), and IL-10 (F) cytokine expression was measured in supernatants of PCMCs. Data are presented as the mean 6 SEM of three independent experiments. +p # 0.05, ++p # 0.01 compared with vehicle (PBS) group, *p # 0.05, **p # 0.01 compared with BP and LPS group. min after LPS stimulation and at 5 min after BP stimulation in

Discussion http://www.jimmunol.org/ FhTeg-treated BMMCs than in control cells (Fig. 7A, 7B). As This study sheds light on the anti-inflammatory effects of helminth SOCS3 was upregulated by FhTeg in early time points and it is Ags on the Th1 proinflammatory properties of mast cells. FhTeg known to be one of the most important inhibitors of the TLR4 impairs mast cells’ ability to drive Th1 immune responses by in- signaling pathway, these results were confirmed by Western blot hibiting the release of key mediators, such as TNF-a, IL-6, IFN-g, analysis. The FhTeg/LPS treatment experiment was repeated, and and IL-10. It also suppresses the expression of ICAM1, a cell proteins at 5, 15, 30, and 60 min were extracted. As with the surface molecule that has an important role in mast cell–T cell qPCR results, in the early time points SOCS3 was upregulated in crosstalk (35). Although studies have shown the importance of FhTeg-treated samples, followed by LPS- and BP-induced SOCS3 ICAM1 expression on T cells in mast cell–T cell communication, expression in latter time points (Fig. 7 C). here we demonstrate that ICAM1 expression on mast cells is also by guest on September 28, 2021

FIGURE 3. FhTeg suppresses the activation of T cells by LPS- or BP-stimulated mast cells. BMMCs and PCMCs were incubated with and without FhTeg (10 mg/ml) for 2.5 h following stimulation with vehicle (PBS), LPS (100 ng/ml), or BP (100 bacteria per cell) for 24 h. Anti-CD3–stimulated naive CD4+ cells were cocultured with the washed cells (A–F) (1:1) or with the supernatants (G–L) for 72 h. Supernatants were collected, and IFN-g (A, B, G, H), IL-10 (C, D, I, J), and IL-5 (E, F, K, L) expression was measured using a commercial ELISA. Data are presented as the mean 6 SEM of three independent experiments. p # 0.05, **p # 0.01 compared with BP and LPS group. The Journal of Immunology 2877

demonstrating that FhTeg can suppress Th1 immunity in vivo by directly rendering innate immune cells, such as dendritic cells, hyporesponsive to TLR activation (25), a characteristic of helminth-activated dendritic cells (37). The suppression of Th1 immune response is critical for F. he- patica to survive within its host, as immunity to Fasciola infection requires strong Th1 inflammatory responses (38). We have shown previously that IL-42/2 mice, which are predisposed toward a Th1 immune response, are less susceptible to F. hepatica infection, with reduced liver damage, compared with IFN-gR2/2 mice, which elicited strong Th2 responses and are highly susceptible FIGURE 4. FhTeg suppresses the upregulation of ICAM1 expression to infection (39). Vaccine efficacy using F. hepatica excretory– on LPS- or BP- stimulated mast cells. PCMCs were stimulated with or secretory molecules or using recombinant F. hepatica secretory without FhTeg (10 mg/ml) for 2.5 h prior to stimulation with vehicle enzymes is associated with enhanced lymphocyte proliferation (PBS), LPS (100 ng/ml), or BP (100 bacteria per cell) for 24 h. ICAM1 and IFN-g expression (10, 40). expression was measured by flow cytometry. Data are presented as the We have previously shown that mice coinfected with Fasciola 6 p # mean SEM of three independent experiments. * 0.05 compared exhibit delayed clearance of B. pertussis from their lungs, which is with BP and LPS group. associated with a decrease in Ag-specific Th1 immune responses

(17). This finding was also observed in mice coinjected with Downloaded from critical in this process. Our results suggest that in T cell–mast cell B. pertussis and Fasciola Ags (41). In this article, we demon- communication both the mast cell–derived cytokines and cell-to- strated that FhTeg injected into mice suppresses B. pertussis– cell contact are crucial to induce sufficient immune responses, as induced TNF-a expression in mast cells, suggesting that mast we showed that the inhibition of cytokine secretion and cell sur- cells may be targeted by helminth Ags during bystander infec- face molecule upregulation by FhTeg both impaired the mast tions. This idea could explain the failure of F. hepatica coinfected cells’ ability to prime T cells. Our study supports previous findings mice to clear B. pertussis infection because mast cells secrete http://www.jimmunol.org/ proinflammatory mediators like TNF-a and IFN-g that mediate host defense against B. pertussis (3, 4). Similarly, mast cells have a critical role during EAE in mice, as mast cell knockout mice are protected from developing this disease (42); this could also ex- plain how Fasciola could attenuate EAE in mice (20). Further studies are required to determine the role of mast cells in this process, which would shed further light on the impact of hel- minths on communicable and noncommunicable diseases. FhTeg suppressed LPS-induced NF-kB and MAPK pathway by guest on September 28, 2021 (ERK) activation in mast cells. NF-kB and MAPKs are important signaling molecules that lead to the expression of ICAM1 (43) and the secretion of proinflammatory cytokines. Our findings correlate with other studies on dendritic cells, as mansoni Ags reduce LPS-stimulated phosphorylation of p38, JNK, and ERK in murine dendritic cells (44). S. haematobium and lumbricoides infections were shown to suppress LPS-induced p38 phosphorylation, but not ERK activation, in dendritic cells (45, 46). Studies demonstrate that NF-kB2/2 mice infected with muris show increased susceptibility to infection, with impaired Th2/Treg immune responses and the phosphorylation of NF-kB and ERK supposed to be crucial in helminth-induced Th2 immune responses (47, 48). The fact that FhTeg does not induce, but rather suppresses, the activation of these proteins supports our unpublished finding that FhTeg does not drive Th2 immunity; rather, it has a role in the suppression of Th1-type responses. FhTeg induces negative regulators of the TLR pathway because FhTeg enhanced expression of SOCS3 in mast cells. SOCS3 is a member of a family of molecules that are one of the main inhibitory groups playing an important role in autoregulation of pathogen- induced inflammatory responses (49). Previous studies showed FIGURE 5. Effect of FhTeg on LPS- or BP-stimulated mast cells can increased SOCS3 RNA levels in blood cells from children with be mimicked by anti-ICAM1 Ab when mast cells are cocultured with Ascaris Trichuris S. haematobium + helminth ( spp., spp., or ) infec- anti-CD3–stimulated naive CD4 cells. PCMCs were stimulated with tion. They suggested that the higher level of SOCS3 might be and without FhTeg (10 mg/ml) for 2.5 h prior to stimulation with PBS, responsible for the increased severity of bystander Plasmodium LPS (100 ng/ml), or BP (100 bacteria per cell) for 24 h. Anti-ICAM1 Ab (1 mg/ml) was added to mast cells 30 min before cells were washed infection, which requires Th1 immune responses (50). It is likely and cocultured with anti-CD3–stimulated naive CD4+ Tcellsfor72h. that F. hepatica has the same mechanism to suppress Th1 immune Supernatants were tested for IFN-g (A), IL-10 (B),andIL-5(C), using responses during bystander infection, but further studies are required. commercial ELISA. Data are presented as the mean 6 SEM of three Mast cells were shown to contribute to the immune response independent experiments. against Strongyloides ratti, ,andNippostrongylus 2878 HELMINTH SUPPRESSES MAST CELL PROINFLAMMATORY RESPONSES

FIGURE 6. FhTeg suppresses phospho-ERK and NF-kB p65 expression in BP- and LPS-stimulated mast cells. BMMCs and PCMCs were stimulated with and without FhTeg (10 mg/ml) for 2.5 h prior to stimulation with PBS, LPS (100 ng/ml), or BP (100 bacteria per cell) for 5, 15, 30, and 60 min. Protein samples were analyzed by Western blot for phosphorylated-ERK (p-ERK), total-ERK (t-ERK), NF-kB p65, and b-actin expression (A, D). Values of p-ERK and NF-kB p65 were normalized to t-ERK and b-actin and are expressed in arbitrary units as percentage increases over PBS control value (B,C, E, F). Downloaded from Densitometry data are presented as the mean 6 SEM of three independent experiments. *p # 0.05, **p # 0.01 compared with control group. brasiliensis in rat and mouse models (51–53). The fecundity of multaneously protecting them against Th1-mediated autoimmune Heligneosomoides polygyrus was higher in mast cell–deficient disorders. Further studies will help us to determine how FhTeg mice than in wild-type controls (54). However, these studies enhances SOCS3 expression, which may lead to potential identi- indicate that the mast cell phenotype is important in driving fication of novel means by which helminths modulate innate im- http://www.jimmunol.org/ Th2 immunity that is essential for clearing gut parasites (55). mune cells. Because these cells are involved in Th1 immune We previously showed that mast cell number increases mark- disorders like autoimmune diseases, inflammatory bowel disease, edly in the peritoneal cavity and liver of F. hepatica–infected or Crohn’s disease, in which TNF-a secretion from these cells and FhTeg-injected mice and that the interaction of FhTeg with is critical to pathological characteristics (8, 9), in the future this mast cells does not promote Th2 immune response or suppress finding might lead to the development of a therapeutic inhibitor IgE-mediated mast cell activation (56). Given that Th1 immune for pathogenic cell phenotypes. responses are important in immune protection against Fasciola infection, it is possible that in the early stages of infection mast Acknowledgments by guest on September 28, 2021 cells are recruited to contribute to the overall Th1 milieu and We thank Dr. Tama´s Visnovitz (National University of Ireland Maynooth, this is inhibited by the release of FhTeg by Fasciola into the Maynooth, Ireland) and Keith Rochfort (Dublin City University, Dublin, microenvironment. This idea is supported by FhTeg’s interac- Ireland) for helpful advice; Dr. Phil Cummins (Dublin City University) for tion with mast cells and dendritic cells, as it directly inhibits providing facilities during qPCR experiments; and the staff of Bioresource their ability to drive Th1 immune responses. Unit, at Dublin City University, for excellent technical assistance. In summary, FhTeg impairs mast cells’ ability to drive Th1 immune responses, which sheds further light on how helminths Disclosures leave populations susceptible to bystander infections while si- The authors have no financial conflicts of interest.

FIGURE 7. SOCS3 transcription and expression are upregulated by FhTeg in mast cells. BMMCs were stimulated with and without FhTeg (10 mg/ml) for 2.5 h prior to stimulation with PBS (P), LPS (L; 100 ng/ml), or BP (B; 100 bacteria per cell) for 5, 15, 30, and 60 min. Total RNA was extracted, and after reverse transcription cDNA was analyzed with qPCR for SOCS3 (A, B). RNA expression was normalized to GAPDH, b-actin, and Gusb control genes and was shown relative to PBS control, using Pfaffl’s method. Protein samples were analyzed by Western blot for SOCS3 and b-actin expression (C). Values of proteins were normalized to b-actin and are expressed in arbitrary units as percentage increases over PBS control value. Data are presented as the mean 6 SEM of three independent experiments. *p # 0.05, **p # 0.01 compared with control group. The Journal of Immunology 2879

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