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Keywords:

  • Allergy;
  • Eosinophils;
  • IgE;
  • Infection;
  • Macrophages

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGEMENTS
  8. Conflict of interest
  9. References

Allergic asthma is less prevalent in countries with parasitic helminth infections, and mice infected with parasites such as Heligmosomoides polygyrus are protected from allergic airway inflammation. To establish whether suppression of allergy could be mediated by soluble products of this helminth, we tested H. polygyrus excretory-secretory (HES) material for its ability to impair allergic inflammation. When HES was added to sensitising doses of ovalbumin, the subsequent allergic airway response was suppressed, with ablated cell infiltration, a lower ratio of effector (CD4+CD25+Foxp3) to regulatory (CD4+Foxp3+) T (Treg) cells, and reduced Th1, Th2 and Th17 cytokine production. HES exposure reduced IL-5 responses and eosinophilia, abolished IgE production and inhibited the type 2 innate molecules arginase-1 and RELM-α (resistin-like molecule-α). Although HES contains a TGF-β-like activity, similar effects in modulating allergy were not observed when administering mammalian TGF-β alone. HES also protected previously sensitised mice, suppressing recruitment of eosinophils to the airways when given at challenge, but no change in Th or Treg cell populations was apparent. Because heat-treatment of HES did not impair suppression at sensitisation, but compromised its ability to suppress at challenge, we propose that HES contains distinct heat-stable and heat-labile immunomodulatory molecules, which modulate pro-allergic adaptive and innate cell populations.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGEMENTS
  8. Conflict of interest
  9. References

Allergic asthma has dramatically increased in prevalence in developing countries, exceeding 5% of the Western European and North American populations [1]. This contrasts with much lower asthma incidence in tropical countries that have significant levels of parasite infections, and these observations have stimulated research into the effects of parasitic organisms on human allergy [2-4]. From these studies, it has emerged that allergic diseases are least common in parts of the world with high helminth endemicity, and that within endemic populations the prevalence of atopy is significantly lower in individuals with chronic worm infections [5-10].

While a causal link between human helminth infections and reduced allergy has yet to be proven [2], chemotherapeutic clearance of intestinal helminths can result in accentuated atopic responsiveness [11-13] and helminth infection blocks overt allergy in a number of animal models [14-17]. The degree to which helminths may influence allergy, however, is clearly variable between different parasite species [18], and is probably dependent upon the duration and intensity of infection [2, 19], each factors that need to be taken into account in assessing this interaction.

The concept that helminths may act in a beneficial way for therapy of immunological disorders is one that has been gaining interest in recent years, especially since reports that the majority of inflammatory bowel disease (IBD) patients infected with Trichuris suis, the pig whipworm, went into remission [20, 21]. Subsequent trials in allergic rhinitis have tested both T. suis therapy [22] and the effects of the human hookworm Necator americanus [23], and the latter parasite has also been trialled for coeliac disease [24, 25]. While these later studies did not find any clinical improvement, significant suppression of inflammatory gut immune responses occurred with N. americanus [25, 26]. Beyond clinical trials, there have also been remarkable reports of remission of multiple sclerosis in patients serendipitously acquiring intestinal helminth infections [27-29] and a case history of an IBD patient self-medicated with the human species Trichuris trichiura resulting in both remission of symptoms and a switch in mucosal immune reactivity toward IL-22 [30].

Prompted by reports from human helminth-infected populations, we previously studied the effects of a model intestinal nematode parasite, Heligmosomoides polygyrus, on the expression of allergic airway inflammation in an experimental system using ovalbumin (OVA) and house dust mite allergy [31]. We found that mice carrying a chronic infection with this helminth showed profound suppression of lung eosinophilia, airway tissue inflammation and type 2 cytokine production in response to airway challenge. Moreover, mice that had been sensitised to allergen were protected from airway allergy if they subsequently became infected; hence, this parasite may confer a therapeutic effect in reversing the allergic state in vivo. This protective effect could also be adoptively transferred with either regulatory T (Treg) cell [31], or B-cell transfer [32], in both cases in an IL-10 independent manner. Together with a number of other studies on H. polygyrus infection in allergy [33, 34], colitis [35-37] and diabetes [38, 39], it is clear that this helminth downregulates multiple effector pathways of the adaptive immune response [40].

Therapy of human immune pathologies with live helminth parasites continues to be evaluated, but will remain an empirical process. Moreover, permitted doses of live parasites are minimal due to ethical and logistical constraints [41], such that exposure levels and duration do not approach those seen in endemic populations. Future therapeutic applications of live helminths will inevitably be limited by the same factors, arguing the case for developing non-living molecular derivatives based on helminth immunomodulatory products; such molecules should also permit a more mechanistic understanding of interactions with the host immune system [42]. Indeed, a number of parasite-derived products have shown some protective ability in immunopathology models [16, 17]. Products of Ancylostoma caninum [43, 44], Hymenolepis diminuta [45], Schistosoma mansoni [44] and Trichinella spiralis [46] all contain immunomodulators which can suppress pathology in mouse models of colitis, while somatic constituents of Ascaris suum can inhibit allergic inflammation [47, 48], as can secretions of Nippostrongylus brasiliensis [49]. Physiologically, active immunomodulators are most likely to be secreted from live parasites into their host environment [50], and in this report we focus on the H. polygyrus excretory-secretory (HES) products as potential modulators of airway allergy in a mouse model.

HES is a complex mixture of proteins, carbohydrates and lipids, many of which have been defined by proteomics [51] and monoclonal antibody analysis [52]. HES is known to downmodulate dendritic cell responses [53] and includes a molecule that binds to and signals through the mammalian TGF-β receptor [54]. Similarly to mammalian TGF-β, HES induces Foxp3 expression in stimulated CD4+ T cells in vitro, which are functionally suppressive and able to suppress airway allergic pathology. Like other nematode organisms, the H. polygyrus genome includes TGF-β family members [55], however it is as yet unclear if these are responsible for the TGF-β signal.

We have now tested HES for its effects in a mouse allergy model and show that it replicates the suppression of pathology seen in the same model with live H. polygyrus infection [31]. We report below that co-administration of HES with sensitising allergen inhibits both innate and adaptive arms of the immune response, including eosinophilia, type 2 innate response markers, antibody generation and effector T-cell reactivity. Moreover, airway eosinophilia could be suppressed to a significant extent even when HES was administered at airway challenge to mice previously sensitised to allergen in the absence of the helminth products, opening the door to future research into therapeutic application to alleviate immunopathological conditions.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGEMENTS
  8. Conflict of interest
  9. References

Inhibition of airway allergy in mice co-sensitised with HES

We first tested the ability of HES products to alter the outcome of an airway allergy model that has previously been shown to be suppressed during live infection with H. polygyrus [31]. Mice were sensitised with two injections of alum-precipitated OVA in PBS on days 0 and 14; we added soluble HES to the suspension to avoid any functional denaturation that might occur through alum precipitation. All animals received an airway challenge of OVA on days 28, 29 and 30. As shown in Fig. 1, addition of HES at sensitisation resulted in profound suppression of the airway allergic inflammation measured by total cell numbers as well as inflitrates of eosinophils and CD4+ T-helper cells in the bronchoalveolar lavage (Fig. 1A) and lung tissue (Fig. 1B). As well as absolute cell numbers, proportions of bronchoalveolar lavage (BAL) SiglecF+CD11c eosinophils were also sharply reduced with HES administration (Fig. 1C). Haematoxylin and Eosin (H&E) and periodic acid-Schiff (PAS) staining of formalin-fixed lung sections also showed reduced inflammatory infiltrate and mucus production with HES administration (Fig. 1D).

image

Figure 1. HES suppresses airway allergy. Twenty micrograms of HES was co-administered with each sensitisation injection of OVA-Alum on day 0 and 14, and challenges of 30 min of 1% OVA aerosol were given at days 28–30. Bronchioalveolar lavage (BAL) fluid and lung tissue for cell preparation were collected at day 31. Shown are (A) the numbers of total BAL cells and cell types, (B) the numbers of total lung preparation cells and cell types, (C) a representative flow cytometry plot of SiglecF versus CD11c expression on BAL cells, and (D) representative haematoxylin and eosin (H&E, 20× magnification) and periodic acid-Schiff (PAS, 200× magnification) staining of formalin-fixed lung sections. (A–C) Results are representative of at least two repeat experiments each with — four to six mice per group. (D) Arrows indicate mucus-producing cells. Significant differences shown compare OVA/Alum to OVA/Alum + HES. Error bars are SEM. *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA, with Tukey's post test.

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Inhibition is not due to antigenic competition

To address whether the suppression of OVA-specific responsiveness by HES was due to a simple antigen competition effect by the additional proteins, we repeated these experiments using bovine serum albumin (BSA) admixed with OVA-alum (Fig. 2). BSA co-administration could not replicate the potent suppression of BAL total and eosinophil cell numbers seen with HES administration (Fig. 2 A–C). We also found that when OVA-alum was co-administered with HES, but not with BSA, anti-OVA IgG1 titres were diminished more than tenfold (Fig. 2D) and IgE almost completely ablated (Fig. 2E). Anti-OVA IgG2a titres were also reduced (data not shown) indicating that a shift to Th1 responsiveness had not occurred.

image

Figure 2. The suppressive effects of HES are not due to antigen competition. Twenty micrograms of HES or bovine serum albumin (BSA) were co-administered with each sensitisation injection of OVA-Alum on day 0 and 14, and following challenge at days 28–30, bronchoalveolar lavage (BAL) fluid and serum were collected at day 31. Shown are (A) the total BAL cell numbers, (B) the SiglecF+CD11c (eosinophil) proportion of total BAL cells, (C) the SiglecF+CD11c (eosinophil) BAL cell numbers, (D) OVA-specific IgG1 measured by ELISA, and (E) OVA-specific IgE measured by ELISA. Results are representative of two repeat experiments each with four to six mice per group. Error bars are SEM. ***p < 0.001, one-way ANOVA, with Tukey's post test.

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Inhibition by HES is mediated by heat-stable components

Because HES is a complex mixture of multiple proteins and glycoconjugates [51], we next determined whether the functional effect of HES was mediated by a heat-stable component. HES was pre-incubated at 100°C and compared with untreated HES for its ability to suppress airway inflammation. As shown in Figure 3, heat-treated HES (HT-HES) is as potent as HES at suppressing total numbers (Fig. 3A) and eosinophilic infiltrate into the BAL (Fig. 3B). HT-HES was similar to untreated HES in reduction of OVA-specific IgG1 (Fig. 3C) and IgE (Fig. 3D).

image

Figure 3. The suppressive effects of HES are heat stable. Twenty micrograms of HES or heat-treated HES (HT-HES) was co-administered with each sensitisation injection of OVA-Alum on day 0 and 14, and following challenge at days 28–30, BAL fluid and serum were collected at day 31. Shown are (A) the total BAL cell numbers, (B) SiglecF+CD11c eosinophil numbers, (C) OVA-specific IgG1 measured by ELISA, and (D) OVA-specific IgE measured by ELISA. Results are representative of four repeat experiments each with four to six mice per group. Error bars are SEM. *p < 0.05; **p < 0.01, one-way ANOVA, with Tukey's post test.

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Inhibition by HES is not accounted for by the activity of TGF-β

Because HES contains a functional mimic of TGF-β, a known inhibitor of airway allergy [56], we compared the effects of 20 μg of HES administration with that of the equivalent activity of TGF-β (4 ng) [54]. As shown in Figure 4, TGF-β co-administration showed no indication of any suppression of eosinophilic BAL infiltrates, in contrast to almost total ablation by HES (Fig. 4A and B). HES also suppressed IL-4, IL-5, IL-13 and IFN-γ expression in the lung homogenate, while TGF-β did not affect these cytokines (Fig. 4C–F); the abolition of the OVA-induced IFN-γ response gives further evidence that Th1 responsiveness does not replace the Th2 response following HES exposure. Levels of IL-10 in the lung were found not to differ between mice that had received HES and control mice (data not shown).

image

Figure 4. HES protects against airway inflammation more potently than TGF-β. Twenty micrograms of HES or 4 ng recombinant TGF-β was co-administered with each sensitisation injection of OVA-Alum on day 0 and 14, and following challenge at days 28–30, lung tissue was collected and homogenised at day 31. (A) The total BAL cell numbers and (B) the SiglecF+CD11c eosinophil numbers are shown. Levels of the cytokines (C) IL-4, (D) IL-5, (E) IL-13, (F) IFN-γ and IL-10 were measured using BD Flex sets, and normalised by dividing the cytokine concentration (in pg/mL) by the total protein concentration (in mg/mL) of the lung homogenate. Levels of IL-10 were below detection limits in this experiment, but were either unchanged or reduced with HES compared with that of OVA alone in other experiments where IL-10 was detectable. Results are representative of at least two experiments, each with four to six mice per group. Error bars are SEM. *p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA, with Tukey's post test.

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Type 2 innate response markers are suppressed by HES

Key innate cell populations such as alternatively activated macrophages (AAMs) are involved in both anti-parasite [57, 58] and asthmatic [59, 60] immune responses. Together with epithelial cells [61], AAMs express high levels of type 2 innate response markers, such as RELMα and Ym1 (where RELM is resistin-like molecule). HES administration was found to potently suppress expression of both of these products (Fig. 5A and B). Real-time RT-PCR confirmed this finding (data not shown), with expression levels of additional type 2 innate response markers Arginase-1 and acidic mammalian chitinase (AMCase) suppressed by HES (although AMCase did not reach statistical significance) (Fig. 5C and D). Transcription of the type 2 response epithelial cell marker RELM-β was also reduced with HES administration (Fig. 5E), as was the chemokine eotaxin/CCL11 (Fig. 5F). Recombinant mammalian TGF-β could not replicate any of these effects of HES administration. Immunohistochemical staining for RELM-α (Fig. 5G) shows production both in epithelial and interstitial cells, many of which appear to be macrophages by morphology. Although overall RELMα staining was reduced with HES administration, production by both epithelial and interstitial cells could still be detected (Fig. 5G, right panel).

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Figure 5. HES suppresses type 2 innate response markers, while recombinant mammalian TGF-β cannot. Cell-free BAL supernatants from the mice shown in Fig. 4 were tested by ELISA for levels of (A) RELM-α and (B) Ym1 protein by ELISA. RNA was prepared from lung tissue and qRT-PCR used to assess levels of (C) Arg-1, (D) AMCase, (E) RELM-β, and (F) eotaxin transcription relative to GAPDH. Data are representative of two repeat experiments, each with four to six mice per group. (G) Formalin-fixed lung tissue sections from five individual mice in each group of the second experiment were stained for RELM-α by immunohistochemistry (five mice per group). Arrows indicate interstitial cell staining. Error bars are SEM. *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA, with a Tukey's post test.

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T-helper cytokine expression is suppressed by HES administration

The innate and antibody responses associated with asthma were suppressed by HES, so we assessed whether this was accompanied by a reduced T-cell cytokine response in the lung. We found that lung CD4+ T-cell production of IL-4, IL-10, IL-13, IL-17A and IFN-γ were all suppressed by co-administration of HES (Fig. 6A). To distinguish whether suppression was due to reduced Th2 differentiation or inhibition of cytokine production by Th2-committed T cells, we administered OVA with and without HES to 4-get mice; in these mice, commitment to the Th2 pathway initiates GFP production prior to the expression of IL-4 protein [62]. Co-staining of lung lymphocytes for GFP and intracellular IL-4 protein showed that HES co-administration reduced both GFP+IL-4 (committed to Th2 but not currently producing IL-4) and GFP+IL-4+ (Th2 cells currently producing IL-4) populations (Fig. 6B and C), indicating that HES interferes with the initial differentiation of Th2 cells.

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Figure 6. HES treatment reduces T-cell cytokine production. HES was co-administered with OVA-Alum sensitisations in 4-get mice. Following challenge at days 28–30, cells were prepared from liberase/DNAse-digested lung tissue, stimulated with PMA and Ionomycin in the presence of Brefeldin A, and stained for CD4 and intracellular IL-4, IL-10, IL-13, IL-17A, and IFN-γ for flow cytometry. Shown are (A) the IL-4+, IL-10+, IL-13+, IL-17A+, and IFN-γ+ proportions of the CD4+ population, (B) representative flow cytometry plots of GFP versus IL-4 in CD4+ lymphocytes, and (C) the proportions of CD4+ cells which were GFP+IL-4 and GFP+IL-4+. Data are representative of two repeat experiments, each with four to six mice per group. Error bars are SEM. *p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA, with Tukey's post test.

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Treg cell numbers do not expand with HES treatment

HES has been previously shown to induce expression of the Treg cell canonical transcription factor Foxp3 in stimulated CD4+ cells in vitro [54]. We therefore assessed levels of CD4+Foxp3+ cells in the lung after HES administration. Figure 7A shows that Treg cell frequencies within the CD4+ population in the lung tissue are higher following OVA sensitisation alone than with HES co-administration. Activation of Treg cells, as assessed by CD103 expression by CD4+Foxp3+ cells, was also unaffected by HES administration (Fig. 7B). However, proportions of activated effector T-helper cells (CD4+CD25+Foxp3) are very significantly decreased with HES administration (Fig. 7C). Hence, the ratio of activated effector T-helper to Treg cells is significantly reduced in mice receiving HES co-administration, potentially shifting the balance in favour of Treg cells (Fig. 7D). Representative FACS plots are shown in Figure 7E.

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Figure 7. HES treatment reduces Treg- and Teffector-cell accumulation. Twenty micrograms of HES was co-administered with each sensitisation injection of OVA-Alum on day 0 and 14, and following challenge at days 28–30, cells were prepared from liberase/DNAse-digested lung tissue and stained for CD4, CD25, CD103, and Foxp3 for flow cytometry. Shown are (A) the Foxp3+ proportion of CD4+ cells, (B) the CD103+ proportion of CD4+Foxp3+ cells, (C) the percentage of CD25+ cells among CD4+Foxp3 cells, (D) the ratio of activated effector T cells (CD4+Foxp3CD25+) to Treg cells (CD4+Foxp3+), and (E) representative flow cytometry plots of CD4+ cells showing Foxp3 versus CD25. Data are shown as mean + SEM are representative of two repeat experiments, each with four to six mice per group. *p < 0.05; ***p < 0.001, one-way ANOVA, with Tukey's post test.

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Inhibition of airway eosinophilia by HES administered with allergen challenge

To test the potential of HES components to suppress established responses, we next administered HES with the OVA intratracheal challenges to mice that had first been sensitised with OVA-alum alone. In this challenge model, we again found significantly lower numbers of BAL total cells, eosinophils and CD4+ T-helper cells (Fig. 8A). However, in the lung tissue only eosinophils showed any marked negative trend, and none of the changes reached statistical significance (Fig. 8B). In this challenge setting, only intact and not heat-inactivated HES was able to suppress total (Fig. 8C) and eosinophil (Fig. 8D) cell numbers, and the effect was primarily on eosinophil numbers rather than the proportions of eosinophils within the total inflammatory infiltrate (Fig. 8E). As in HES at sensitisation, replacement of HES with an equivalent amount of recombinant mammalian TGF-β did not protect against eosinophil accumulation (Fig. 8F).

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Figure 8. HES treatment at challenge suppresses eosinophil responses, while heat-treated HES (HT-HES) or mammalian TGF-β does not. All mice were sensitised with OVA-Alum on day 0 and 14, then 20 μg HES (or controls of 20 μg HT HES or 4 ng recombinant mammalian TGF-β) were co-administered with challenge of 20 μg OVA protein administered intratracheally on days 28–30. Bronchoalveolar lavage (BAL) cells and lung tissue for cell preparation were collected at day 31. Shown are (A) the total number of BAL cells and cell types, (B) the numbers of total lung preparation cells and cell types, (C) the total BAL cell numbers with HT HES control, (D) SiglecF+CD11c BAL eosinophil numbers with HT HES control, (E) SiglecF+CD11c proportions of total BAL cells with HT HES control, and (F) SiglecF+CD11c BAL eosinophil numbers with TGF-β control. Results are representative of three repeat experiments, each with four to six mice per group. Error bars are SEM. *p < 0.05; ***p < 0.001, one-way ANOVA, with Tukey's post test.

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In recipients of intratracheal HES with OVA, BAL fluid also contained lower levels of IL-5, while IL-4 appeared unchanged (Fig. 9A and B). Levels of RELM-α and Ym1 in the BAL fluid were also lower with HES administration and unaffected by HT-HES, although only attaining significance with Ym1 (Fig. 9C and D). Transcription of the type 2 innate response markers, Arginase-1 and AMCase, again tended to be suppressed (Fig. 9E and F), whereas RELM-β was unaltered (Fig. 9G). Likewise, levels of eotaxin/CCL11 showed a downward trend with HES administration which was absent with heat-inactivated HES (Fig. 9H).

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Figure 9. HES treatment at challenge suppresses type 2 response markers. Cell-free BAL supernatants from the experiment shown in Figure 8 B–D were tested (A and B) by CBA for levels of (A) IL-4 and (B) IL-5, and (C and D) by ELISA for levels of (C) RELM-α and (D) Ym1 protein. (E–H) RNA was prepared from lung tissue and qRT-PCR used to assess levels of (E) Arginase-1, (F) AMCase, (G) RELM-β, and (H) eotaxin transcription relative to GAPDH. Results are representative of two repeat experiments, each with four to six mice per group. Error bars are SEM. *p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA, with Tukey's post test.

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We again determined frequencies of Foxp3+ and CD103+Foxp3+ Treg cells in allergic mice. Levels of Foxp3+ and CD103+Foxp3+ Treg cells increased in OVA-sensitised and challenged animals, and in contrast to HES given at sensitisation when HES was given at challenge this was unchanged (Fig. 10A and B). CD4+Foxp3CD25+-activated effector cells were again increased with asthma induction and were also unchanged by HES administration (Fig. 10C), as was the ratio of activated effector T-helper cells (Fig. 10D). Representative FACS plots are shown in Figure 10E. To further examine the Th2 response with HES at challenge, lung tissue CD4+ cells were stained for intracellular IL-4 and IL-13 (Fig. 10F and G), but consistent with undiminished activation of Foxp3 T cells, no difference in expression of these Th2 cytokines was seen with HES at challenge.

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Figure 10. HES at challenge does not affect T-cell accumulation or cytokine production in the lung. (A–E) Lung tissue cells were prepared from the experiment shown in Figure 8A and stained for CD4, CD25, CD103, and Foxp3 for flow cytometry. Shown are (A) the Foxp3+ proportion of CD4+ cells, (B) the CD103+ proportion of CD4+Foxp3+ cells, (C) the percentage of CD25+ cells among CD4+Foxp3 cells, (D) the ratio of activated effector T cells (CD4+Foxp3CD25+) to Treg cells (CD4+Foxp3+), and (E) representative flow cytometry plots of CD4+ cells showing Foxp3 verses CD25. Results are shown as mean + SEM are representative of two repeat experiments with — four to six mice per group. (F and G) In the second experiment, lung tissue cells from five individual mice were stimulated with PMA, Ionomycin, and Brefeldin A for 4 h, and stained for CD4 and intracellular (F) IL-4 and (G) IL-13 by flow cytometry. *p < 0.05; **p < 0.01, one-way ANOVA, with Tukey's post test.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGEMENTS
  8. Conflict of interest
  9. References

The influence of infections on the induction and progression of allergic disease is under increasing scrutiny in both epidemiological and experimental studies [63-65]. Helminth parasites in particular are associated with a degree of protection from allergic reactivity [3, 4]. However, due to the heterogeneity of human exposure and the diversity of helminth species, current evidence is not always consonant with a causal link between infection and reduction of allergy [2]. Hence, investigations with defined laboratory models are essential to dissect the interactions that are likely to occur in humans, and to exploit any molecular principle from parasites that may be beneficial in ameliorating immunopathology.

In this report, we show that the murine intestinal helminth H. polygyrus releases soluble factors that potently suppress immune responses and pathology in an experimental model of allergic inflammation. These molecules suppress a wide span of innate and adaptive immune responses if co-administered with the initial sensitising injections and key markers of innate inflammation (in particular, eosinophilia) when given to mice only at the challenge phase. Because of the breadth of effects, and the fact that initial suppression is mediated by heat-stable components and the challenge inhibition by heat-labile molecules, we hypothesise that HES contains multiple suppressive factors, those that can suppress initiation of adaptive immune responses, and factors which inhibit recruitment or expansion of allergic innate effector cells to the lung.

The suppression of allergy when helminth products are given at sensitisation may be common to other parasites, as N. brasiliensis ES adsorbed with OVA on Alum reduces the subsequent allergic response [49]. However, suppression of the BAL eosinophil response when ES is given at challenge has not, to our knowledge, been previously shown. A possible mechanism by which HES could inhibit sensitisation is by enzymatic modification of OVA; however, the fact that HT-HES is effective at sensitisation makes it unlikely that any enzymatic effect is responsible. While the protective component in the challenge setting is heat-labile, T-cell responses to OVA are unchanged in this model, again arguing that there is no defect in presentation of OVA to T cells due to modification of the allergen. Hence, in both cases we favour the hypothesis that HES acts directly on inducer and effector components of the immune response.

In blocking adaptive responses, we found not only that lung Th2 cytokine levels were profoundly inhibited by HES co-administration at sensitisation, but also the Th1 cytokine IFN-γ, arguing that allergy inhibition was not achieved simply by immune deviation toward the Th1 mode. Intracellular cytokine staining showed suppression of Th2, Th1 and Th17 cells with HES co-administration and the suppression of OVA-specific antibody was also observed across all isotypes, consistent with this conclusion. Among the innate populations, the abrogation of eosinophil infiltration (along with eosinophil-stimulating factors IL-5 and eotaxin/CCL11) was highly significant, as this subset is most closely associated with airway pathology [66, 67]. Downregulation of eotaxin in particular could lead to the suppression of eosinophil recruitment to the airways seen with HES administration at challenge. Due to the brief time frame of the challenge protocol, this observation implies that HES may directly impact on the eosinophil population, and does not act indirectly through an adaptive (eg Th2) compartment which is unaffected by the challenge-only administration of HES.

We also observed striking changes in type 2 innate response markers. These markers, such as Ym1 and RELM-α, were initially discovered in AAM, and more recently shown to be produced at high levels by epithelial cells in allergic inflammation models [61]. We had hypothesised that HES similar to secretions from other helminths [68] can drive AAM, which would in turn act in an immunosuppressive mode to block the Th2 allergic immune response. In contrast, we saw instead a highly significant reduction in type 2 innate response markers, indicating an unexpected inhibition of AAM. Because these type 2 innate response markers are exquisitely Th2-dependent (through IL-4Rα), these changes might simply be downstream consequences of a suppressed Th2 response in the lung. However, the clear suppression of Ym1 by HES at challenge argues, as with eosinophils, that inhibition may act directly on innate cells and not through an intermediary Th2 cell. It is possible, however, that allergic eosinophil expansion is dependent upon AAMs in the lung, in a manner parallel to that recently reported for adipose tissue [69]. Because the apparent roles of AAMs in helminth immunity and asthma exacerbation vary between experimental models, it will be fascinating to dissect their activity further in this system, especially with the new range of specific antibodies and inhibitors of AMM products now becoming available [70].

When HES was administered with OVA at the sensitisation phase of the airway allergy model, we also observed downregulation of the type 2 immune response epithelial cytokine RELM-β. Interestingly, this molecule has been more closely associated with the intestinal epithelium, and in that setting acts as an innate defensin-like molecule to inhibit survival of H. polygyrus adult worms [71]. Inhibition of RELM-β in the airways may nevertheless contribute to amelioration of allergy, as RELM-β-deficient mice show reduced goblet cell hyperplasia and other inflammatory reactions following allergen challenge [72].

Mice treated with HES at sensitisation also showed very potent suppression of anti-OVA IgG1 and especially IgE responses. It is interesting to speculate how HES may interrupt humoral immunity so profoundly. H. polygyrus infection is known to induce a regulatory population of B cells, which can suppress airway allergy on transfer [31]. Thus, in the context of HES administration, suppression of OVA-specific IgE responses may reflect skewing of the B-cell population to a regulatory phenotype. However, as shown by Wilson et al., neither infection with H. polygyrus [31] nor transfer of the regulatory B-cell population [32] resulted in suppression of the allergen-specific IgG1 or IgE response. Therefore, HES may invoke a mechanism to inhibit anti-parasite antibody responses, which when co-administered with a bystander allergen (in this case, OVA), extends to that antigen also. In H. polygyrus infections, neither the T-cell- nor B-cell- mediated suppression of asthma is dependent on IL-10, and as no increase in total, or CD4+ T cell-derived, IL-10 was seen with HES administration, other host regulatory cytokines (eg TGF-β) may be involved.

Our results show that HES at sensitisation can potently inhibit Th2 responses and pathology in a widely studied OVA-induced asthma model. This model depends on the systemic sensitisation of mice with OVA protein in an aluminium hydroxide adjuvant, inducing a Th2 response that can be recalled in the airways. However, the induction of allergy in humans is more likely to be induced directly in the airways by exposure to environmental allergens. Thus, we are presently extending our results with other regimens giving allergen repeatedly without adjuvant directly into the airways. Furthermore, suppression of Th2 responses, however they are initiated, is of relevance not only to allergy and the control of Th2-mediated parasite expulsion from the host. For example, it has been noted that immunity induced by vaccines, most of which employ alum adjuvant, is suppressed in helminth endemic areas [17, 73], possibly through pathways replicated by HES.

Our laboratory has recently completed a proteomic analysis of HES [51], which will facilitate identification of the molecular product(s) in HES that mediate protection. In addition to ascertaining the suppressive molecular principle(s) from H. polygyrus, future research needs also to characterise the immunological mechanism and pathway of suppression. In the first instance, the question should be addressed of which host cells are targeted by HES. Previous work on H. polygyrus infection has highlighted the parasite's suppressive effects through dendritic cells [74-76], Treg [31, 77, 78] and regulatory B cells [31]. Other work on products derived from H. polygyrus has shown roles for induction of suppressive dendritic cells [53] and Treg cells [54]. HES contains a TGF-β mimic which is capable of inducing Treg cells in vitro [54], and we hypothesised that HES could suppress allergic immune responses through this pathway. Our data, however, argue against suppression dependent on the TGF-β pathway or Treg induction: an equivalent amount of recombinant mammalian TGF-β could not suppress allergic immune responses at sensitisation or at challenge; HES suppression at sensitisation was heat-stable, while the HES TGF-β signal is heat labile [54]; and Treg proportions, numbers and activation levels were lower with HES administration at sensitisation than in positive controls. Therefore, we hypothesise that HES contains other immunomodulatory factors than the TGF-β mimic and these are largely responsible for the suppressive effect seen with HES administration at sensitisation. When HES is administered challenge, the suppression by HES is heat-labile and therefore the suppression could be due to the TGF-β mimic. However, the Treg population was unchanged by HES administration at challenge and mammalian TGF-β could not replicate the suppressive effect, therefore we believe it is unlikely that the TGF-β mimic is entirely responsible. We are presently carrying out experiments using TGF-βR signalling inhibitors to formally exclude this possibility.

In summary, here we present data showing that HES administration can replicate the suppressive effects of H. polygyrus infection on airway allergy. These effects are likely to be mediated by multiple factors, and are unlikely to be solely attributed to the TGF-β-like activity contained in HES. As HES could also suppress airway eosinophilia once an immune response had been established, we propose that it may contain possible therapeutics for human disease. We are now using fractionation, proteomic and genomic techniques to attempt to identify the suppressive factor(s) in HES.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGEMENTS
  8. Conflict of interest
  9. References

Parasites and HES

The life cycle of Heligmosomoides polygyrus bakeri was maintained as described elsewhere [79]. Adult excretory-secretory products were prepared as previously described [80], and when tested in the Limulus Amoebocyte Lysate assay (Lonza, Slough, UK), was found to contain less than 0.1 IU LPS per μg protein. Heat inactivation was performed by incubation at 100°C for 20 min. Class IV OVA and BSA were purchased from Sigma.

Mice

BALB/c or 4-get [62] (heterozygous with BALB/c) mice were bred in-house at the University of Edinburgh and accommodated according to Home Office regulations.

Airway allergy model

Induction of airway allergic inflammation was performed as previously described [31]. Briefly, mice were sensitised with two injections (day 0 and 14) of 20 μg OVA precipitated with alum, i.p. At days 28, 29 and 30, mice were challenged with an aerosol of 1% OVA protein in PBS or by intratracheal instillation of 20 μg OVA in PBS, as indicated. At day 31, the bronchoalveolar space was lavaged and lungs taken for analysis. Serum was collected at this time point.

Lung histology and immunohistochemistry

Lungs were lavaged with, and collected into, 10% buffered formalin, incubated overnight at 4°C and then transferred to 70% ethanol. Lungs were then paraffin embedded and sectioned, before being stained with standard protocols for H&E and PAS. For immunohistochemical staining, sections were stained with rabbit anti-RELM-α (Peprotech, London, UK) and a goat anti-rabbit secondary antibody (Vector labs, Peterborough, UK). Staining was detected with horseradish peroxidase and 3,3-diaminobenzidine.

Flow cytometry

Single cell suspensions of lung tissue were prepared by digesting the right lobes of the lung in 2 U/mL liberase TL (Roche, Welwyn Garden City, UK) and 80 U/mL DNase (Invitrogen, Paisley, UK) at 37°C with agitation for 25 min, followed by maceration through a 70 μm cell strainer. Cells were phenotyped by labelling with fluorescently labelled antibodies to CD4, CD11b, CD11c, CD25, CD103, Foxp3, GR1 and SiglecF, or the relevant isotype controls before analysing by FACS (all antibodies from BD Biosciences, Oxford, UK).

Where intracellular cytokine staining was carried out, cells were stimulated for 4 h at 37°C with 500 ng/mL Phorbol Myristate Acetate, 1 μg/mL Ionomycin and 10 μg/mL Brefeldin A (Sigma, Dorset, UK). Cells were stained for CD4 and then permeabilised using the manufacturers instructions with the BD Biosciences Fixation/Permeabilisation kit. Cells were then stained for IFN-γ, IL-4, IL-10, IL-13 and IL-17A. After permeabilisation, GFP in 4-get mice was detected using polyclonal rabbit anti-GFP (Ebioscience, Hatfield, UK), followed by anti-rabbit IgG-Alexa Fluor 488 (Invitrogen). Live/dead Aqua (Invitrogen) was used to exclude dead cells. Samples were analysed by flow cytometry using Becton-Dickinson FACSCanto or LSR-II flow cytometers. BAL and lung tissue cell types were characterised as: CD11c+SiglecF+ (alveolar macrophages), SiglecF+CD11c (eosinophils), CD11bhiCD11cGR1hiSiglecF (neutrophils) and CD4+CD11b (T-helper cells).

ELISA

Antibody levels in serum were measured on OVA-coated ELISA plates and serial dilutions of serum. To measure IgE levels, serum was first depleted of IgG using 4 Fast Flow Protein G Sepharose beads (GE Healthcare, Little Chalfont, UK), according to the manufacturer's instructions. IgG1 was detected with goat anti-mouse IgG1-HRP (Southern Biotech, Birmingham, AL, USA) and IgE was detected using rat anti-mouse IgE-biotin followed by streptavidin-alkaline phosphatase (Sigma).

The type 2 innate response markers RELM-α and Ym-1 were measured by ELISA, using the rabbit anti-mouse RELM-α, then biotinylated rabbit anti-mouse RELM-α, compared to a recombinant RELM-α standard (Peprotech), or the Chitinase 3-like/ECF-L (Ym1) Duoset kit (R&D Systems, Abingdon, UK) according to manufacturer's instructions.

Cytokine CBA

Cytokine levels were detected in cell-free BAL supernatant or lung homogenate. A section of the left lobe of the lung was homogenised using a Tissuelyser (QIAGEN, Crawley, UK) in 0.5 mL of 1× cell lysis buffer (Cell Signaling, Danvers, MA, USA), containing 1 μM PMSF (Sigma). IL-4, IL-5, IL-10, IL-13 and IFN-γ levels were detected using BD CBA Flex-set kits and were acquired on a BD FACSArray. Levels of cytokines in lung homogenates were normalised by dividing by total protein concentration, as assessed by Bradford assay compared to a BSA standard (Pierce, Cramlington, UK).

RT-PCR

A section of the left lobe of the lung was homogenised on a Tissuelyser (QIAGEN) in Trizol reagent (Invitrogen), and RNA was prepared according to manufacturers instructions. Reverse transcription was performed using 1–2 μg of RNA, 2 μL ten reverse transcriptase buffer, 2 μL 25 mM dNTP mix, 1 μL of 50 U/μL Moloney murine leukaemia virus reverse transcriptase, 0.5 μL of 40 U/μL RNAsin, 1 μL of 0.4 μg/mL Oligo dT primer and made up to 20 μL with diethylpyrocarbonate (DEPC)-treated water. A PCR block (Peltier Thermal Cycler, Bio-Rad, Hemel Hempstead, UK) was used for the transcription reaction, 20°C for 10 min, 37°C for 60 min and 99°C for 5 min.

Transcript levels of the genes of interest were measured by real-time PCR using the Lightcycler 480 II (Roche). PCR amplifications were carried out in 10 μL total volume made up of 1 μL cDNA, 5 μL SYBR Green (Roche), 0.3 μL of each primer (10 μM) (Table 1) and 3.4 μL DEPC-treated water (Invitrogen). The amplifications were performed using the following protocol: 30-s denaturation at 95°C, 5 s for primers to anneal at 65°C, 12-s elongation at 72°C for 40 cycles. Primers used are shown in Table 1.

Table 1. Primers used for real-time PCR
GeneForward primerReverse primerAmplicon length
Arg-1CAGAAGAATGGAAGAGTCAGCAGATAT-GCAGGGAGT-CACC249
AMCaseCCCTTG-GCA-TATC-CACTGAACAGAA-TCCACTGCC-TCCAG125
GAPDHATGACA-TCAAGAA-GGTGGTGCATACCA-GGAAATG-AGCTTG177
RELM-βCGTCTC-CCTTTTC-CCACTGCAGGAG-ATCGTCTTAG-GCTCTT114
EotaxinCACGGTCA-CTTCCTTC-ACCTTGG-GGATCTT-CTTACT-GGTCA92

Statistics

All data were analysed using Prism 5 (Graphpad, La Jolla, CA, USA). Groups were analysed by one-way ANOVA, with a Tukey's post test comparing all groups to the OVA only positive control. Unless otherwise indicated differences are not significant. *** p < 0.001; **p < 0.01; *p < 0.05.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGEMENTS
  8. Conflict of interest
  9. References

We gratefully acknowledge grant funding support from the Medical Research Council (to M.T.O'G and K.J.F), the American Asthma Foundation (to N.B. and H.J.McS), Asthma UK (to T.E.S) and the Wellcome Trust. We also thank Yvonne Harcus and Elaine Robertson for assistance with the H. polygyrus life cycle.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGEMENTS
  8. Conflict of interest
  9. References

The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGEMENTS
  8. Conflict of interest
  9. References
  • 1
    Eder, W., Ege, M. J. and von Mutius, E., The asthma epidemic. N. Engl. J. Med. 2006. 355: 22262235.
  • 2
    Cooper, P. J., Interactions between helminth parasites and allergy. Curr. Opin. Allergy. Clin. Immunol. 2009. 9: 2937.
  • 3
    Flohr, C., Quinnell, R. J. and Britton, J., Do helminth parasites protect against atopy and allergic disease? Clin. Exp. Allergy 2009. 39: 2032.
  • 4
    Smits, H. H., Everts, B., Hartgers, F. C. and Yazdanbakhsh, M., Chronic helminth infections protect against allergic diseases by active regulatory processes. Curr. Allergy Asthma Rep. 2010. 10: 312.
  • 5
    Araujo, M. I., Lopes, A. A., Medeiros, M., Cruz, A. A., Sousa-Atta, L., Solé, D. and Carvalho, E. M., Inverse association between skin response to aeroallergen and Schistosoma mansoni infection. Int. Arch. Allergy Immunol. 2000. 123: 145148.
  • 6
    van den Biggelaar, A., van Ree, R., Roderigues, L. C., Lell, B., Deelder, A. M., Kremsner, P. G. and Yazdanbakhsh, M., Decreased atopy in children infected with Schistosoma haematobium : a role for parasite-induced interleukin-10. Lancet 2000. 356: 17231727.
  • 7
    Nyan, O. A., Walraven, G. E. L., Banya, W. A. S., Milligan, P., Van Der Sande, M., Ceesay, S. M., Del Prete, G. et al., Atopy, intestinal helminth infection and total serum IgE in rural and urban adult Gambian communities. Clin. Exp. Allergy 2001. 31: 16721678.
  • 8
    Scrivener, S., Yemaneberhan, H., Zebenigus, M., Tilahun, D., Girma, S., Ali, S., McElroy, P. et al., Independent effects of intestinal parasite infection and domestic allergen exposure on risk of wheeze in Ethiopia: a nested case-control study. Lancet 2001. 358: 14931499.
  • 9
    Dagoye, D., Bekele, Z., Woldemichael, K., Nida, H., Yimam, M., Hall, A., Venn, A. J. et al., Wheezing, allergy, and parasite infection in children in urban and rural Ethiopia. Am. J. Respir. Crit. Care Med. 2003. 167: 13691373.
  • 10
    Cooper, P. J., Intestinal worms and human allergy. Parasite Immunol. 2004. 26: 455467.
  • 11
    Lynch, N. R., Hagel, I., Perez, M., Di Prisco, M. C., Lopez, R. and Alvarez, N., Effect of anthelmintic treatment on the allergic reactivity of children in a tropical slum. J. Allergy Clin. Immunol. 1993. 92: 404411.
  • 12
    van den Biggelaar, A. H., Rodrigues, L. C., van Ree, R., van der Zee, J. S., Hoeksma-Kruize, Y. C., Souverijn, J. H., Missinou, M. A. et al., Long-term treatment of intestinal helminths increases mite skin-test reactivity in Gabonese schoolchildren. J. Infect. Dis. 2004. 189: 892900.
  • 13
    Flohr, C., Tuyen, L. N., Quinnell, R. J., Lewis, S., Minh, T. T., Campbell, J., Simmons, C. et al., Reduced helminth burden increases allergen skin sensitization but not clinical allergy: a randomized, double-blind, placebo-controlled trial in Vietnam. Clin. Exp. Allergy. 2009. 40: 131142.
  • 14
    Wilson, M. S. and Maizels, R. M., Regulation of allergy and autoimmunity in helminth infection. Clin. Rev. Allerg. Immunol. 2004. 26: 3549.
  • 15
    Fallon, P. G. and Mangan, N. E., Suppression of Th2-type allergic reactions by helminth infection. Nat. Rev. Immunol. 2007. 7: 220230.
  • 16
    Danilowicz-Luebert, E., O'Regan, N. L., Steinfelder, S. and Hartmann, S., Modulation of specific and allergy-related immune responses by helminths. J. Biomed. Biotechnol. 2011. 2011: 821578.
  • 17
    McSorley, H. J. and Maizels, R. M., Helminth infections and host immune regulation. Clin. Rev. Microbiol. 2012. In press.
  • 18
    Leonardi-Bee, J., Pritchard, D. and Britton, J., Asthma and current intestinal parasite infection: systematic review and meta-analysis. Am. J. Respir. Crit. Care Med. 2006. 174: 514523.
  • 19
    Smits, H. H., Hammad, H., van Nimwegen, M., Soullie, T., Willart, M. A., Lievers, E., Kadouch, J. et al., Protective effect of Schistosoma mansoni infection on allergic asthma depends on intensity and chronicity of infection. J. Allergy Clin. Immunol. 2007. 120: 932940.
  • 20
    Summers, R. W., Elliott, D. E., Qadir, K., Urban, J. F., Jr., Thompson, R. and Weinstock, J. V., Trichuris suis seems to be safe and possibly effective in the treatment of inflammatory bowel disease. Am. J. Gastroenterol. 2003. 98: 20342041.
    Direct Link:
  • 21
    Summers, R. W., Elliott, D. E., Urban, J. F., Jr., Thompson, R. A. and Weinstock, J. V., Trichuris suis therapy for active ulcerative colitis: a randomized controlled trial. Gastroenterology 2005. 128: 825832.
  • 22
    Bager, P., Arnved, J., Rønborg, S., Wohlfahrt, J., Poulsen, L. K., Westergaard, T., Petersen, H. W. et al., Trichuris suis ova therapy for allergic rhinitis: a randomized, double-blind, placebo-controlled clinical trial. J. Allergy Clin. Immunol. 2010. 125: 123130.
  • 23
    Blount, D., Hooi, D., Feary, J., Venn, A., Telford, G., Brown, A., Britton, J. et al., Immunologic profiles of persons recruited for a randomized, placebo-controlled clinical trial of hookworm infection. Am. J. Trop. Med. Hyg. 2009. 81: 911916.
  • 24
    Daveson, A. J., Jones, D. M., Gaze, S., McSorley, H., Clouston, A., Pascoe, A., Cooke, S. et al., Effect of hookworm infection on wheat challenge in celiac disease–a randomised double-blinded placebo controlled trial. PLoS One 2011. 6:e17366.
  • 25
    McSorley, H. J., Gaze, S., Daveson, J., Jones, D., Anderson, R. P., Clouston, A., Ruyssers, N. E. et al., Suppression of inflammatory immune responses in celiac disease by experimental hookworm infection. PLoS One 2011. 6:e24092.
  • 26
    Gaze, S., McSorley, H. J., Daveson, J., Jones, D., Bethony, J. M., Oliveira, L. M., Speare, R. et al., Characterising the mucosal and systemic immune responses to experimental human hookworm infection. PLoS Pathog. 2012. 8:e1002520.
  • 27
    Correale, J. and Farez, M., Association between parasite infection and immune responses in multiple sclerosis. Ann. Neurol. 2007. 61: 97108.
  • 28
    Correale, J. and Farez, M., Helminth antigens modulate immune responses in cells from multiple sclerosis patients through TLR2-dependent mechanisms. J. Immunol. 2009. 183: 59996012.
  • 29
    Correale, J. and Farez, M. F., The impact of parasite infections on the course of multiple sclerosis. J. Neuroimmunol. 2011. 233: 611.
  • 30
    Broadhurst, M. J., Leung, J. M., Kashyap, V., McCune, J. M., Mahadevan, U., McKerrow, J. H. and Loke, P., IL-22+ CD4+ T cells are associated with therapeutic Trichuris trichiura infection in an ulcerative colitis patient. Sci. Transl. Med. 2010. 2: 60ra88.
  • 31
    Wilson, M. S., Taylor, M., Balic, A., Finney, C. A. M., Lamb, J. R. and Maizels, R. M., Suppression of allergic airway inflammation by helminth-induced regulatory T cells. J. Exp. Med. 2005. 202: 11991212.
  • 32
    Wilson, M. S., Taylor, M. D., O'Gorman, M. T., Balic, A., Barr, T. A., Filbey, K., Anderton, S. M. et al., Helminth-induced CD19+CD23hi B cells modulate experimental allergic and autoimmune inflammation. Eur. J. Immunol. 2010. 40: 16821696.
  • 33
    Bashir, M. E., Andersen, P., Fuss, I. J., Shi, H. N. and Nagler-Anderson, C., An enteric helminth infection protects against an allergic response to dietary antigen. J. Immunol. 2002. 169: 32843292.
  • 34
    Kitagaki, K., Businga, T. R., Racila, D., Elliott, D. E., Weinstock, J. V. and Kline, J. N., Intestinal helminths protect in a murine model of asthma. J. Immunol. 2006. 177: 16281635.
  • 35
    Elliott, D. E., Setiawan, T., Metwali, A., Blum, A., Urban, J. F., Jr. and Weinstock, J. V., Heligmosomoides polygyrus inhibits established colitis in IL-10-deficient mice. Eur. J. Immunol. 2004. 34: 26902698.
  • 36
    Sutton, T. L., Zhao, A., Madden, K. B., Elfrey, J. E., Tuft, B. A., Sullivan, C. A., Urban, J. F., Jr. et al., Anti-inflammatory mechanisms of enteric Heligmosomoides polygyrus infection against trinitrobenzene sulfonic acid-induced colitis in a murine model. Infect. Immun. 2008. 76: 47724782.
  • 37
    Hang, L., Setiawan, T., Blum, A. M., Urban, J., Stoyanoff, K., Arihiro, S., Reinecker, H. C. et al., Heligmosomoides polygyrus infection can inhibit colitis through direct interaction with innate immunity. J. Immunol. 2010. 185: 31843189.
  • 38
    Saunders, K. A., Raine, T., Cooke, A. and Lawrence, C. E., Inhibition of autoimmune type 1 diabetes by gastrointestinal helminth infection. Infect. Immun. 2006. 75: 397407.
  • 39
    Liu, Q., Sundar, K., Mishra, P. K., Mousavi, G., Liu, Z., Gaydo, A., Alem, F. et al., Helminth infection can reduce insulitis and type 1 diabetes through CD25- and IL-10-independent mechanisms. Infect. Immun. 2009. 77: 53475358.
  • 40
    Maizels, R. M., Hewitson, J. P., Murray, J., Harcus, Y., Dayer, B., Filbey, K. J., Grainger, J. R. et al., Immune modulation and modulators in Heligmosomoides polygyrus infection. Exp. Parasitol. 2012. 132: 7689.
  • 41
    Mortimer, K., Brown, A., Feary, J., Jagger, C., Lewis, S., Antoniak, M., Pritchard, D. et al., Dose-ranging study for trials of therapeutic infection with Necator americanus in humans. Am. J. Trop. Med. Hyg. 2006. 75: 914920.
  • 42
    Harnett, W. and Harnett, M. M., Therapeutic immunomodulators from nematode parasites. Exp. Rev. Mol. Med. 2008. 10:e18.
  • 43
    Cançado, G. G., Fiuza, J. A., de Paiva, N. C., Lemos, L. D., Ricci, N. D., Gazzinelli-Guimarães, P. H., Martins, V. G. et al., Hookworm products ameliorate dextran sodium sulfate-induced colitis in BALB/c mice. Inflamm. Bowel. Dis. 2011. 17: 22752286.
  • 44
    Ruyssers, N. E., De Winter, B. Y., De Man, J. G., Loukas, A., Pearson, M. S., Weinstock, J. V., Van den Bossche, R. M. et al., Therapeutic potential of helminth soluble proteins in TNBS-induced colitis in mice. Inflamm. Bowel. Dis. 2009. 15: 491500.
  • 45
    Johnston, M. J. G., Wang, A., Catarino, M. E. D., Ball, L., Phan, V. C., MacDonald, J. A. and McKay, D. M., Extracts of the rat tapeworm, Hymenolepis diminuta, suppress macrophage activation in vitro and alleviate chemically induced colitis in mice. Infect. Immun. 2010. 78: 13641375.
  • 46
    Motomura, Y., Wang, H., Deng, Y., El-Sharkawy, R. T., Verdu, E. F. and Khan, W. I., Helminth antigen-based strategy to ameliorate inflammation in an experimental model of colitis. Clin. Exp. Immunol. 2009. 155: 8895.
  • 47
    Itami, D. M., Oshiro, T. M., Araujo, C. A., Perini, A., Martins, M. A., Macedo, M. S. and Macedo-Soares, M. F., Modulation of murine experimental asthma by Ascaris suum components. Clin. Exp. Allergy 2005. 35: 873879.
  • 48
    McConchie, B. W., Norris, H. H., Bundoc, V. G., Trivedi, S., Boesen, A., Urban, J. F., Jr. and Keane-Myers, A. M., Ascaris suum-derived products suppress mucosal allergic inflammation in an IL-10 independent manner via interference with dendritic cell function. Infect. Immun. 2006. 74: 66326641.
  • 49
    Trujillo-Vargas, C. M., Werner-Klein, M., Wohlleben, G., Polte, T., Hansen, G., Ehlers, S. and Erb, K. J., Helminth derived products inhibit the development of allergic responses in mice. Am. J. Respir. Cell Mol. Biol. 2007. 175: 336344.
  • 50
    Hewitson, J. P., Grainger, J. R. and Maizels, R. M., Helminth immunoregulation: the role of parasite secreted proteins in modulating host immunity. Mol. Biochem. Parasitol. 2009. 167: 111.
  • 51
    Hewitson, J. P., Harcus, Y., Murray, J., van Agtmaal, M., Filbey, K. J., Grainger, J. R., Bridgett, S. et al., Proteomic analysis of secretory products from the model gastrointestinal nematode Heligmosomoides polygyrus reveals dominance of venom allergen-like (VAL) proteins. J. Proteomics 2011. 74: 15731594.
  • 52
    Hewitson, J. P., Filbey, K. J., Grainger, J. R., Dowle, A. A., Pearson, M., Murray, J., Harcus, Y. et al., Heligmosomoides polygyrus elicits a dominant nonprotective antibody response directed at restricted glycan and peptide epitopes. J. Immunol. 2011. 187: 47644777.
  • 53
    Segura, M., Su, Z., Piccirillo, C. and Stevenson, M. M., Impairment of dendritic cell function by excretory-secretory products: a potential mechanism for nematode-induced immunosuppression. Eur. J. Immunol. 2007. 37: 18871904.
  • 54
    Grainger, J. R., Smith, K. A., Hewitson, J. P., McSorley, H. J., Harcus, Y., Filbey, K. J., Finney, C. A. M. et al., Helminth secretions induce de novo T-cell Foxp3 expression and regulatory function through the TGF-β pathway. J. Exp. Med. 2010. 207: 23312341.
  • 55
    McSorley, H. J., Grainger, J. R., Harcus, Y. M., Murray, J., Nisbet, A., Knox, D. P. and Maizels, R. M., daf-7-related TGF-β homologues from trichostrongyloid nematodes show contrasting life cycle expression patterns. Parasitology 2010. 137: 159171.
  • 56
    Hansen, G., McIntire, J. J., Yeung, V. P., Berry, G., Thorbecke, G. J., Chen, L., DeKruyff, R. H. et al., CD4+ T helper cells engineered to produce latent TGF-β1 reverse allergen-induced airway hyperreactivity and inflammation. J. Clin. Invest. 2000. 105: 6170.
  • 57
    Kreider, T., Anthony, R. M., Urban, J. F., Jr. and Gause, W. C., Alternatively activated macrophages in helminth infections. Curr. Opin. Immunol. 2007. 19: 448453.
  • 58
    Loke, P., Gallagher, I., Nair, M. G., Zang, X., Brombacher, F., Mohrs, M., Allison, J. P. et al., Alternative activation is an innate response to injury that requires CD4+ T cells to be sustained during chronic infection. J. Immunol. 2007. 179: 39263936.
  • 59
    Byers, D. E. and Holtzman, M. J., Alternatively activated macrophages as cause or effect in airway disease. Am. J. Respir. Cell Mol. Biol. 2010. 43: 14.
  • 60
    Melgert, B. N., Oriss, T. B., Qi, Z., Dixon-McCarthy, B., Geerlings, M., Hylkema, M. N. and Ray, A., Macrophages: regulators of sex differences in asthma? Am. J. Respir. Cell Mol. Biol. 2010. 42: 595603.
  • 61
    Munitz, A., Cole, E. T., Atar-Karo, D., Finkelman, F. D. and Rothenberg, M. E., Resistin-like molecule alpha regulates IL-13-induced chemokine production but not allergen-induced airway responses. Am. J. Respir. Cell Mol. Biol. 2012. 46: 703713.
  • 62
    Voehringer, D., Shinkai, K. and Locksley, R. M., Type 2 immunity reflects orchestrated recruitment of cells committed to IL-4 production. Immunity 2004. 20: 267277.
  • 63
    Bach, J. F., The effect of infections on susceptibility to autoimmune and allergic diseases. N. Engl. J. Med. 2002. 347: 911920.
  • 64
    Schaub, B., Lauener, R. and von Mutius, E., The many faces of the hygiene hypothesis. J. Allergy Clin. Immunol. 2006. 117: 969977.
  • 65
    Maizels, R. M., Pearce, E. J., Artis, D., Yazdanbakhsh, M. and Wynn, T. A., Regulation of pathogenesis and immunity in helminth infections. J. Exp. Med. 2009. 206: 20592066.
  • 66
    Hamelmann, E. and Gelfand, E. W., IL-5-induced airway eosinophilia–the key to asthma? Immunol. Rev. 2001. 179: 182191.
  • 67
    Lee, J. J., Dimina, D., Macias, M. P., Ochkur, S. I., McGarry, M. P., O'Neill, K. R., Protheroe, C. et al., Defining a link with asthma in mice congenitally deficient in eosinophils. Science 2004. 305: 17731776.
  • 68
    Allen, J. E. and MacDonald, A. S., Profound suppression of cellular proliferation mediated by the secretions of nematodes. Parasite Immunol. 1998. 20: 241247.
  • 69
    Wu, D., Molofsky, A. B., Liang, H. E., Ricardo-Gonzalez, R. R., Jouihan, H. A., Bando, J. K., Chawla, A. et al., Eosinophils sustain alternatively activated macrophages in adipose tissue. Science 2011. 332: 243247.
  • 70
    Sutherland, T. E., Andersen, O. A., Betou, M., Eggleston, I. M., Maizels, R. M., van Aalten, D. and Allen, J. E., Analyzing airway inflammation with chemical biology: dissection of acidic mammalian chitinase function with a selective drug-like inhibitor. Chem. Biol. 2011. 18: 569579.
  • 71
    Herbert, D. R., Yang, J.-Q., Hogan, S. P., Groschwitz, K., Khodoun, M. V., Munitz, A., Orekov, T. et al., Intestinal epithelial cell secretion of RELM-β protects against gastrointestinal worm infection. J. Exp. Med. 2009. 206: 29472957.
  • 72
    Mishra, A., Wang, M., Schlotman, J., Nikolaidis, N. M., DeBrosse, C. W., Karow, M. L. and Rothenberg, M. E., Resistin-like molecule-β is an allergen-induced cytokine with inflammatory and remodeling activity in the murine lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 2007. 293:L305L313.
  • 73
    Borkow, G. and Bentwich, Z., Chronic parasite infections cause immune changes that could affect successful vaccination. Trends Parasitol. 2008. 24: 243245.
  • 74
    Balic, A., Smith, K. A., Harcus, Y. and Maizels, R. M., Dynamics of CD11c+ dendritic cell subsets in lymph nodes draining the site of intestinal nematode infection. Immunol. Lett. 2009. 127: 6875.
  • 75
    Li, Z., Liu, G., Chen, Y., Liu, Y., Liu, B. and Su, Z., The phenotype and function of naturally existing regulatory dendritic cells in nematode-infected mice. Int. J. Parasitol. 2011. 41: 11291137.
  • 76
    Smith, K. A., Hochweller, K., Hämmerling, G. J., Boon, L., Macdonald, A. S. and Maizels, R. M., Chronic helminth infection mediates tolerance in vivo through dominance of CD11clo CD103 DC population. J. Immunol. 2011. 186: 70987109.
  • 77
    Finney, C. A.M., Taylor, M. D., Wilson, M. S. and Maizels, R. M., Expansion and activation of CD4+CD25+ regulatory T cells in Heligmosomoides polygyrus infection. Eur. J. Immunol. 2007. 37: 18741886.
  • 78
    Rausch, S., Huehn, J., Kirchhoff, D., Rzepecka, J., Schnoeller, C., Pillai, S., Loddenkemper, C. et al., Functional analysis of effector and regulatory T cells in a parasitic nematode infection. Infect. Immun. 2008. 76: 19081919.
  • 79
    Camberis, M., Le Gros, G. and Urban, J., Jr., Animal model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus. In Coico, R. (Ed.), Current protocols in immunology, John Wiley and Sons, Inc., 2003, 55: 19.12.119.12.17.
  • 80
    Harcus, Y., Nicoll, G., Murray, J., Filbey, K., Gomez-Escobar, N. and Maizels, R. M., C-type lectins from the nematode parasites Heligmosomoides polygyrus and Nippostrongylus brasiliensis. Parasitol. Int. 2009. 58: 461470.
Abbreviations
AAM

alternatively activated macrophage

AMCase

acidic mammalian chitinase

BAL

bronchoalveolar lavage

HES

Heligmosomoides polygyrus excretory-secretory products

IBD

inflammatory bowel disease

PAS

periodic acid-Schiff

RELM

resistin-like molecule