Protective effect of Schistosoma mansoni infection on allergic airway inflammation depends on the intensity and chronicity of infection

Protective effect of Schistosoma mansoni infection on allergic airway inflammation depends on the intensity and chronicity of infection Hermelijn H. Smits, PhD,a,b Hamida Hammad, PhD,b Menno van Nimwegen, BSc,b Thomas Soullie, BSc,b Monique A. Willart, BSc,b Ellen Lievers, BSc,a Jonathan Kadouch, MSc,a Mirjam Kool, BSc,b Janneke Kos-van Oosterhoud, BSc,a Andre´ M. Deelder, PhD,a Bart N. Lambrecht, PhD, MD,b* and Maria Yazdanbakhsh, PhDa* Leiden and Rotterdam, The Netherlands

Background: Population studies have suggested that chronic and intense helminth infections, in contrast to acute and mild helminth infections, might suppress allergic airway inflammation. Objective: We sought to address the question of how the chronicity and intensity of helminth infections affect allergic airway inflammation in a well-defined experimental model. Methods: C57/Bl6 mice were infected with Schistosoma mansoni, followed by sensitization and challenge with ovalbumin (OVA), and different stages and intensities of infection were studied. To this end, mice were analyzed at 8, 12, or 16 weeks, representing the acute, intermediate, or chronic phases of infection, respectively. Results: Lung lavage eosinophilia, peribronchial inflammation, and OVA-induced airway hyperresponsiveness were increased during acute infection but significantly decreased when infection progressed into chronicity. Decreases in lung lavage eosinophilia were parasite density–dependent. Similar levels of OVA-specific IgE were found during all phases of infection, whereas both OVA-specific and parasite-specific TH2 cytokine levels were significantly reduced during chronic infection. Inhibition of airway inflammation could be transferred to OVA-sensitized recipient mice by B cells and CD41 T cells from spleens of chronically, but not acutely, infected mice. This suppression was IL-10–dependent. Conclusion: During chronic, but not acute, helminth infections, suppressive mechanisms are induced that regulate immune

Basic and clinical immunology

From athe Department of Parasitology, Leiden University Medical Center, Leiden, and bthe Department of Pulmonary Medicine, Erasmus Medical Center, Rotterdam. *These authors contributed equally to the supervision of this work. Supported by VENI grants of the Dutch Foundation for Sciences (to H.H.S. and H.H.), a ZonMW program grant (to M.Y. and B.N.L.), and a grant from the Netherlands Asthma Foundation (to M.K.). Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest. Received for publication November 9, 2006; revised May 31, 2007; accepted for publication June 5, 2007. Available online August 10, 2007. Reprint requests: Hermelijn H. Smits, PhD, Center of Infectious Diseases, Department of Parasitology, P4-25, Leiden University Medical Center, Albinusdreef 2; 2333 ZA Leiden, The Netherlands. E-mail: H.H.Smits@ lumc.nl. 0091-6749/$32.00 Ó 2007 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2007.06.009

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reactions to inhaled allergens. These data confirm human epidemiologic observations in a well-controlled animal model. Clinical implications: Characterization of chronic helminth infection–induced regulatory mechanisms will help in the development of future therapeutics to treat or prevent allergic disease. (J Allergy Clin Immunol 2007;120:932-40.) Key words: helminth, allergy, immune suppression, IL-10, regulatory B cells, regulatory T cells

An inverse association between chronic helminth infections (Ascaris species, Trichuris species, hookworms, schistosomes, or filariae) and allergic disorders in tropical areas has been demonstrated.1,2 However, in areas in which helminth infections occur only sporadically3,4 or are more transient in nature,5,6 atopic disorders appear to be potentiated. Therefore it is possible that the different effect of helminth infection on allergic disease is due to differences in acute versus chronic stages of infection or due to differences in intensity of infection. The relationship between helminths and allergies is complex. Both allergy and helminth infections are characterized by high levels of circulating IgE antibodies, eosinophilia, and polarized TH2 responses.7 However, during chronic helminth infections, increased levels of the immunoregulatory cytokine IL-10 and a general Tcell hyporesponsiveness have also been reported.8,9 It has been argued that this form of immunoregulation is instrumental for long-term survival of the parasite. This suppression appears to be not strictly parasite specific but can be extended to third-party antigens, such as other pathogen-derived antigens,10,11 vaccine antigens,12,13 or ‘‘harmless’’ antigens, such as allergens. Several groups have developed combined mouse models of asthma and infection to study the causal interaction between helminth infections and the development of allergic diseases in a well-controlled manner. Both studies with rodent nematodes (eg, Heligmosomoides polygyrus) and ‘‘human’’ helminths (Ascaris species or Nippostrongylus brasiliensis) have demonstrated that infection leads to strongly reduced eosinophilic airway inflammation in the eyes or lungs.14-18 However, there are

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Intracellular IL-10 staining

also a number of animal studies showing enhanced airway inflammation and no effect or a small reduction in a few immunologic parameters associated with asthma (eg, TH2 cytokines) but without an effect on airway inflammation.19-21 Again, it is possible that different effects on allergic outcomes are due to differences in chronicity or intensity of infection. A drawback of the abovementioned studies is the lack of an experimental model with clearly defined acute and chronic stages of infection. This study aims to evaluate the susceptibility of helminth-infected mice to have allergen-induced lung inflammation and airway hyperresponsiveness (AHR) during different stages and with different intensities of infection. Infection of C57/Bl6 mice with Schistosoma mansoni is a well-defined model with distinct phases: acute infection peaks at week 8, and chronicity starts at week 12 and is fully established at about week 16.22,23 Therefore experimental schistosome infections are well suited to evaluate the role of chronicity or intensity of infection on the development of allergic airway inflammation.

METHODS Animals, parasites, and infection Female C57/Bl6 OlaHsd mice (6 weeks old; Harlan, Horst, The Netherlands) were percutaneously infected with 45 cercariae, unless stated differently, and infections lasted either until 8 (acute phase), 12 (intermediate phase), or 16 weeks (chronic phase). Livers and intestines were digested by means of overnight incubation with 5% KOH at 378C to analyze infection intensity. Egg counts were expressed as total egg number per animal.

Model of eosinophilic airway inflammation Mice were sensitized with ovalbumin (OVA; Worthington Biochemical Corp, Lakewood, NJ) or PBS by using 2 intraperitoneal injections of OVA (10 mg) or PBS emulsified in AL(OH)3 (2 mg; Imject Alum; Pierce, Rockford, Ill) at 18 and 11 days before challenge. Sensitization was started at week 5 (acute), week 9 (intermediate), or week 13 (chronic). After 1 week, all mice were challenged with 3 OVA aerosol sprays (10 mg/mL in PBS). One day after challenge, airway resistance was assessed in response to increasing concentrations of intravenously administered methacholine (SigmaAldrich, St Louis, Mo) during mechanical ventilation with a Flexivent apparatus (SCIREQ, Montreal, Quebec, Canada). Mice were killed 24 hours after the last challenge. For details on experimental procedures, see the Methods section in the Online Repository at www.jacionline.org.

Splenocytes were restimulated with phorbol 12-myristate 13acetate and ionomycin (Sigma) for 24 hours and for the last 6 hours also in the presence of Brefeldin A (Sigma). Cells were stained for surface markers (CD19 or CD4), fixed, permeabilized, and stained for IL-10 or isotype control (PharMingen, Becton Dickinson, San Diego, Calif).24

Adoptive transfer of spleen cells At day 17, OVA-sensitized animals (OVA/alum; details above) received intravenously 1 3 107 splenocytes, 5 3 106 CD191 spleen B cells (positive isolation with CD191 MACS microbeads; Miltenyi, Bergisch Gladbach, Germany), or 1 3 106 spleen CD41 T cells (negative selection with CD41 T-cell MACS isolation kit, Miltenyi) from PBS-uninfected, OVA-uninfected, PBS-infected, or OVA-infected animals at week 8, 12, or 16. In some mice IL-10 receptor antibody (250 mg; clone 1B1.3a, obtained from Dr K. Moore, DNAX, Palo Alto, Calif) or rat IgG1 (isotype control) was administered intraperitoneally at the time of splenocyte transfer. After 2 days, mice were challenged and killed as described in the Methods section in the Online Repository at www.jacionline.org.

Statistical analysis For statistical analysis, Kruskal-Wallis 1-way ANOVA and the Mann-Whitney U tests were performed. Differences were considered to be significant at a P value of less than .05.

RESULTS Chronic schistosome infection dosedependently suppressed OVA-induced lung eosinophilia Mice were infected with S mansoni 8 weeks (acute phase), 12 weeks (intermediate phase), or 16 weeks (chronic phase) before sensitization with OVA. After challenge with OVA aerosols, the cellular composition of the bronchoalveolar lavage (BAL) fluid showed airway eosinophilia, lymphocytosis, and an increase in macrophages in uninfected OVA-sensitized mice (OVA-uninfected mice) but not in uninfected PBS-immunized mice (PBS-uninfected mice; P values not shown; Figs 1, A, and E1, A in this article’s Online Repository at www.jacionline.org). The values of the OVA-uninfected group were arbitrarily set at 100% to make comparisons at different time points possible (raw data are displayed in Fig E1 in this article’s Online Repository at www.jacionline.org). At all time points, OVA-uninfected mice showed increased BAL fluid eosinophilia compared with infected PBS-immunized mice (PBS-infected mice; P values not shown; Fig 1, A, and Fig E1, A, in this article’s Online Repository at www.jacionline.org). In acutely infected OVA-sensitized mice (OVA-acute mice) significantly higher numbers of eosinophils were found in the BAL fluid compared with in OVA-uninfected mice (Fig 1, A). However, eosinophilia as a result of OVA sensitization decreased significantly in chronically infected OVA-sensitized mice (OVA-chronic mice; Fig 1, A). The amount of BAL eosinophils in PBS-infected mice was not significantly different

Basic and clinical immunology

Abbreviations used AHR: Airway hyperresponsiveness BAL: Bronchoalveolar lavage HDM: House dust mite MLN: Mediastinal lymph node OVA: Ovalbumin SEA: Soluble egg antigen Treg cell: Regulatory T cell

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FIG 1. Effect of S mansoni infection on OVA-specific eosinophilic airway inflammation. A, Mice were infected, PBS/OVA sensitized, and challenged at week 8, 12, or 16, followed by BAL fluid collection. B, Mice were infected with different numbers of cercariae (ie, 15, 30, or 45) and PBS/OVA sensitized, followed by a challenge at week 16. Each group consists of 6 to 10 mice from 3 independent experiments (mean 1 SD). *P < .05, **P < .01.

Basic and clinical immunology

from that seen in age-matched PBS-uninfected mice at all 3 time points (Fig E1 in this article’s Online Repository at www.jacionline.org). Nevertheless, a small increase in BAL fluid eosinophilia in PBS-infected mice was visible at week 16 after infection, probably reflected by the appearance of recirculating egg (Figs 1, A, and Fig E1 in this article’s Online Repository at www.jacionline.org). Interestingly, in PBS-chronic mice equal numbers of lymphocytes were found compared with in OVA-uninfected or OVA-chronic mice (Fig E1, A, in this article’s Online Repository at www.jacionline.org). Mice were infected with increasing numbers of cercariae, resulting in 3 groups of mice that were infected mildly (<15,000 eggs), moderately (15,000-30,000 eggs), or heavily (>30,000 eggs), to determine whether the intensity of infection would influence the reduction in BAL fluid eosinophilia during chronic infection. The recovery of BAL fluid eosinophils at week 16 showed a dose-dependent decrease with increasing intensity of infection (Fig 1, B), which was not seen in PBS-chronic mice (Fig E1, B, in this article’s Online Repository at www.jacionline.org). These results indicate that a natural infection with S mansoni in the mouse suppressed the development of OVA-induced airway eosinophilia at the chronic stage of infection and that this suppression is correlated with intensity of infection.

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Reduced peribronchial inflammation in chronic schistosome-infected mice Cryopreserved lung tissue sections were analyzed for cellular infiltration, eosinophilia, and goblet cell hyperplasia by means of histologic staining and morphometric analysis to investigate the degree of peribronchial and perivascular eosinophilic inflammation. OVA-uninfected mice showed a strong peribronchial and perivascular cellular infiltration (Fig E2 in this article’s Online Repository at www.jacionline.org and Fig 2, A), with high numbers of eosinophils (Fig E2 in this article’s Online Repository at www.jacionline.org and Fig 2, B) and extensive goblet cell hyperplasia (Fig E2 in this article’s Online Repository at www.jacionline.org and Fig 2, C) compared with that seen in naive mice, demonstrating a strongly manifested TH2-driven airway inflammation. OVA-acute mice and OVA-uninfected mice showed a similar degree of peribronchial and perivascular inflammation (Fig E2 in this article’s Online Repository at www.jacionline.org and Fig 2, A), with equal numbers of eosinophils (Fig E2 in this article’s Online Repository at www.jacionline.org and Fig 2, B) and a comparable extent of goblet cell hyperplasia (Fig E2 in this article’s Online Repository at www.jacionline.org and Fig 2, C). In contrast, OVA-chronic mice showed a strongly reduced cellular infiltration in the peribronchial and perivascular area (Fig E2 and 2, A), with only a few eosinophils (Fig E2 in this article’s Online Repository at www.jacionline.org and Fig 2, B) and few goblet cells in the epithelial lining of the bronchi(oles) (Fig E2 in this article’s Online Repository at www.jacionline.org and Fig 2, C) in comparison with numbers seen in OVA-uninfected or OVAacute mice. The degree of peribronchial inflammation and the number of goblet cells or eosinophils in inflammatory infiltrates in PBS-infected mice was not significantly different from that seen in age-matched PBS-uninfected mice at all time points (Fig 2, B-D). These results suggest a reduction in OVA-specific TH2 effector function in chronically infected mice. Chronic schistosome-infected animals show decreased AHR The effect of schistosome infection on increased AHR in response to direct bronchoconstrictors, such as methacholine, was analyzed by means of Flexivent measurements of airway resistance in mechanically ventilated mice at different time points, comparing acute, intermediate, and chronic phases of infection. OVA-uninfected mice showed a significantly enhanced airway resistance (at 37.5 and/or 75 and 150 mg/mL) compared with airway resistance values of PBS-uninfected or PBS-infected animals at all time points (Fig 3). In addition, airway resistance values of OVA-uninfected mice (75 and 150 mg/ mL) were also significantly increased compared with airway resistance values of OVA-intermediate and OVAchronic mice but not those of OVA-acute mice (Fig 3). At week 8 after infection, but not at weeks 12 or 16 after infection, the airway resistance values of OVA-infected

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mice were significantly enhanced (at 75 and 150 mg/mL) compared with those of PBS-uninfected or PBS-infected animals. These results indicate that during intermediate and chronic schistosome infections, there is no increased AHR, and it suggests that the development of AHR, an important clinical feature of asthma, is suppressed.

Equal levels of OVA-specific IgE in OVAinfected and OVA-uninfected animals To investigate the induction of TH2-dependent sensitization during different stages of infection, we analyzed the levels of total and OVA-specific IgE in serum. OVA-uninfected mice showed increased levels of total IgE antibodies compared with those seen in sham-immunized mice (P < .001; Fig 4, A). Infected mice showed high levels of total IgE antibodies in serum during all stages of infection (Fig 4, A). In addition, equal levels of OVA-specific IgE were measured in OVA-infected mice compared with in OVA-uninfected mice, regardless of whether the mice were at the acute or chronic stage of infection (Fig 4, B). These results show an unaffected capacity to generate allergen-specific IgE antibodies during infection, which is similar to data from human studies.8 OVA-specific cytokine responses are downregulated in chronic S mansoni–infected mice Next we addressed the question of whether cytokine responses were affected during acute, intermediate, and chronic infection by analyzing OVA-specific or helminth soluble egg antigen (SEA)–specific cytokines (IL-4,

IL-10, IL-13, and IFN-g) from lung-draining mediastinal lymph node (MLN) cells (target organ for allergic immune responses; Fig 5, A) or from splenocytes (systemic parasite-specific reactivity; Fig 5, B). OVA-specific cytokine responses of OVA-uninfected and OVA-infected mice were all significantly enhanced compared with those of PBS-uninfected or PBS-infected mice (P values not shown), except for IFN-g production, which was very low and generally around the detection limit of the assay. During acute infection (week 8), OVA-specific cytokine responses in OVA-acute mice were similar to those found in OVA-uninfected mice (Fig 5, A). In contrast, during chronic infection (week 16), all OVA-specific cytokine responses were significantly downregulated compared with those of OVA-uninfected mice and OVA-acute mice, except for IL-10 (Fig 5, A). OVA-specific IL-10 production in OVA-chronic mice was significantly decreased compared with OVA-specific IL-10 levels from OVA-uninfected mice at week 16 and OVA-intermediate mice but not OVA-acute mice (Fig 5, A). Similar data were found in OVA-stimulated spleen cultures, with the exception of a similar IFN-g production in OVA-uninfected and OVA-chronic mice (Fig E3, A, in this article’s Online Repository at www.jacionline.org). SEA-specific cytokine responses in spleens from OVAinfected and PBS-infected mice were significantly enhanced compared with those of PBS-uninfected and OVA-uninfected mice (P values not shown), with the exception of SEA-specific IFN-g production during chronic infection. SEA-specific cytokine responses from OVAinfected and PBS-infected mice were similar within each

Basic and clinical immunology

FIG 2. Effect of S mansoni infection on OVA-induced peribronchial eosinophilic inflammation. Mice were treated as in Fig 1, A. Lungs were fixed with OCT and snap-frozen. Three-micrometer sections were cut and stained with hematoxylin and eosin, periodic acid–Schiff, or major basic protein and quantified for inflammatory infiltrates (A), eosinophils (B), or goblet cells (C; mean 1 SD). Each group consists of at least 6 animals. *P < .05, **P < .01, ***P < .001.

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FIG 3. Effect of S mansoni infection on OVA-induced airway resistance. Mice were treated as in Fig 1, A. Mice were subjected to an increasing dose of methacholine during mechanical ventilation. The mean airway resistance values (1 SD) of each methacholine dose are plotted. Each group consists of 6 to 10 animals. *P < .05, **P < .01.

Basic and clinical immunology

time point of infection but significantly downregulated during chronic infection (week 16) compared with either acute (IL-13 and IFN-g) or intermediate (IL-13, IL-4, IL-10, and IFN-g) infection (Fig 5, B). SEA-specific cytokine responses in MLNs did not show the same trends as in spleens (Fig E3, B, in this article’s Online Repository at www.jacionline.org), which is probably reflected by different egg exposure dynamics because eggs only start to appear in the lungs after 12 weeks of infection and only at that moment will start to boost SEA-specific cytokines in MLN. In summary, all cytokine levels in response to both OVA and SEA (with the exception of IL-10) were decreased during chronic schistosome infection.

Adoptive transfer of splenocytes from OVAchronic mice results in an IL-10–dependent reduction of OVA-specific lung eosinophilia in sensitized recipient animals, which is attributed to B and CD41 T cells To determine whether a cellular process was responsible for the observed suppression of eosinophilic airway inflammation and AHR in OVA-chronic mice, we performed adoptive transfer experiments with MLN cells or

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splenocytes from OVA-infected mice at different stages of schistosome infection into previously OVA-sensitized recipient mice. Adoptive transfer of splenocytes from PBS-uninfected (data not shown), OVA-acute, or OVAuninfected mice in recipient mice induced equally enhanced lung eosinophilia (Fig E4 in this article’s Online Repository at www.jacionline.org) and MLN OVAspecific TH2 cytokine levels (data not shown). In contrast, adoptive transfer of splenocytes from OVA-intermediate, OVA-chronic, or PBS-chronic mice in recipient mice resulted in a significant decrease in BAL fluid eosinophils (Fig E4 in this article’s Online Repository at www.jacion line.org) with reduced MLN TH2 responses (data not shown). Interestingly, adoptive transfer of splenocytes from OVA-chronic, but not PBS-chronic, mice in naive recipient mice, followed by OVA sensitization, also resulted in reduced numbers of BAL eosinophilia (Fig E5 in this article’s Online Repository at www.jacionline.org). The capacity of PBS-chronic splenocytes to inhibit ongoing OVA-specific eosinophilic airway inflammation in OVA-sensitized mice, but not the priming of OVAspecific TH2 responses in naive mice, might reflect non– antigen-specific effector mechanisms and is the subject of future studies in our laboratory. The capacity to adoptively transfer the suppression of eosinophilic airway inflammation appeared to be restricted to the spleen because the adoptive transfer of MLN cells from OVAinfected mice at different stages of infection did not change OVA-specific lung eosinophilia in recipient mice (data not show). At present, we are comparing the 2 organs with respect to cellular composition and activation status to explain the data with respect to suppression in the adoptive transfer. Subsequent adoptive transfer experiments were performed while treating the recipient mice with blocking IL10 receptor antibodies or control rat IgG1 to study the role of IL-10. Treatment with blocking IL-10 receptor antibodies fully reversed suppression of eosinophilic airway inflammation in recipient mice after adoptive transfer of splenocytes of OVA-chronic mice (Fig 6, A). CD191 B cells and CD41 T cells were isolated from the spleens of chronically OVA-infected mice and as controls from the spleens of OVA-uninfected or PBS-uninfected mice to investigate which cell type was involved. Adoptive transfer of CD191 B cells and CD41 T cells from OVA-chronic mice resulted in significant reductions in BAL fluid eosinophilia in recipient mice for both CD191 B cells and CD41 T cells (Fig 6, B). CD191 B cells and CD41 T cells from OVA-uninfected or PBS-uninfected mice did not reduce BAL fluid eosinophilia in recipient mice. Intracellular cytokine analysis revealed that both B and CD41 T cells produced significantly increased levels of IL-10 during the chronic stage compared with the acute stage of schistosome infection (Fig 6, C). These results indicate that in chronic S mansoni–infected animals, both B cells and CD41 T cells are responsible for the adoptive transfer of suppression of allergic airway inflammation in recipient mice and that this inhibition appears to be mediated through IL-10.

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DISCUSSION The current study shows that chronic infection, but not acute infection, with S mansoni protects against the development of OVA-specific allergic airway inflammation and depends on the intensity of infection. Protection against eosinophilic airway inflammation was transferred to OVA-sensitized mice by B cells and CD41 T cells from the spleens of OVA-chronic mice and appeared to be IL-10–dependent. The observed suppression of allergic airway inflammation and lung eosinophilia in mice that were infected with S mansoni for either 12 or 16 weeks (intermediate and chronic stages) corresponds to time points in which immune modulation and T-cell hyporesponsiveness have been reported.22,23 However, OVA-specific IgE levels remained unaffected during the course of infection. In schistosome-infected human subjects increased levels of house dust mite (HDM)–specific IgE were demonstrated, whereas HDM skin prick test responses were diminished.8 In addition, long-term treatment resulted in increased skin prick test reactivity to HDM.25,26 These findings, both in human subjects and in mice, support the concept that chronic helminth infections inhibit the effector phase of allergic immune responses, whereas allergen sensitization appears to be unaffected. These findings underline the value of the present model to study mechanisms leading to protection against allergic disease and the translation to human subjects. The majority of individuals chronically infected with S mansoni are asymptomatic (90% to 95%), whereas a minority has severe hepatosplenic disease (5% to 10%). The reported lower prevalence of allergic disease in schistosomiasis has been documented in infected subjects as a whole, which largely consists of asymptomatic individuals. There are no data yet on the prevalence of allergic disorders in patients with hepatosplenomegaly. In parallel

to human subjects, mice show a similar dichotomy in symptoms,27 although the proportion of mice that have severe hepatosplenic disease varies greatly between different mouse strains compared with human subjects. BALB/c mice frequently have severe hepatosplenic syndrome, whereas this is rarely seen in C57/bl6 mice,28 making the C57/bl6 strain suitable for studying chronic asymptomatic schistosomiasis and its interaction with allergic disease in an experimental model. Recently, it was demonstrated that S mansoni infection in BALB/c mice resulted in increased AHR.19 These results can most likely be explained by the strong tendency of BALB/c mice to have uncontrolled severe hepatosplenic disease at low infection intensities. Indeed, we have observed that schistosome-infected BALB/c mice had severe hepatosplenic disease at low infection intensities, leading to a complete loss of a colony before week 16 (chronic stage). The study of different animal models could be highly valuable to delineate how genetic background influences the interaction between helminth infections and allergies. Several studies have identified a role for helminthinduced regulatory T (Treg) cells. Indeed, in patients with onchocerciasis, such antigen-specific Treg cells could be isolated and characterized by the secretion of increased levels of IL-10, TGF-b, or both and the inhibition of the proliferation of other T cells.29,30 Increased levels of IL-10 were reported in many human8,29,30 and murine studies, but the source and role of IL-10 appears to vary in different helminth infections. In some studies IL-10 was attributed to adaptive Treg cells,14,15,31 whereas in others IL-10 was linked to non-TH cell populations.18,32-34 However, a few studies have pointed out that although IL-10 levels were increased, this had no role in immune hyporesponsiveness of chronic helminth infections.14,31,35 In this study IL-10 was shown to play a central role in suppressing allergic airway inflammation in the adoptive transfer of splenocytes from chronically infected mice. In addition, spleen

Basic and clinical immunology

FIG 4. Effect of S mansoni infection on OVA-specific IgE production. Mice were treated as in Fig 1, A. After challenge, blood was collected, and total IgE (A) or OVA-specific IgE (B) levels in plasma were determined by means of ELISA. Mean and SD values are plotted for groups of 6 to 10 animals from 2 independent experiments.

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FIG 5. Effect of S mansoni infection on OVA-specific cytokine responses in MLNs and SEA-specific cytokine responses in spleens. Mice were treated as in Fig 1, A. After 8, 12, or 16 weeks, MLN and spleen cells were stimulated with OVA (10 mg/mL) or SEA (50 mg/mL). A, OVA-specific cytokines in MLN cells; B, SEA-specific cytokines in spleen cells. Mean and SD values are plotted for groups of 6 to 10 animals from 2 independent experiments. *P < .05, **P < .01.

Basic and clinical immunology

CD41 T cells from chronically infected mice were capable of (partially) downregulating lung eosinophilia in recipient mice, and this might therefore favor the concept of IL-10 producing adaptive Treg cell development during chronic schistosomiasis. This is in agreement with the capacity of S mansoni–derived molecules, such as lysophosphatidylserine, to prime human dendritic cells in vitro for the development of IL-10–producing Treg cells.36 In our experiments spleen-derived B cells from chronically infected mice also proved to be inhibitory in the adoptive transfer, and this effect was IL-10–dependent (Fig 6, B). This is in line with a previous study demonstrating a protective role for IL-10–producing B cells against the development of allergic anaphylaxis in schistosomeinfected mice.37 In addition, also in Heligmosomoides polygyrus–infected allergen-sensitized mice, B cells can transfer protection against allergic airway inflammation

(Dr M. S. Wilson, personal communication, October 2006). Furthermore, granulomatous pathology in chronic schistosome-infected mice was downregulated by a B cell–dependent mechanism requiring Fc receptor signaling.38 These studies indicate that in chronic helminth infections B cells can play a role in regulating inflammatory responses through several mechanisms. The current study demonstrates for the first time that chronic, but not acute, schistosome infection can suppress allergic airway inflammation in a dose-dependent manner. During the acute stages of infection, as the egg-driven inflammatory events are developing, the balance of effector responses to regulatory responses favors effector responses, when neutralizing the toxic egg-derived products is crucial for host survival. At this time, allergic disease is exacerbated, reflecting the underlying inflammatory events. However, as the infection progresses into the

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FIG 6. Adoptive transfer of splenocytes from chronically schistosome-infected mice in recipient mice. Mice were treated as in Fig 1, A, and spleens were collected. A, OVA-sensitized recipient mice received 10 3 106 splenocytes together with anti-IL-10 receptor or rat control IgG1. B, Recipient mice received 5 3 106 spleen B cells or 1 3 106 spleen CD41 T cells. C, Splenocytes were restimulated with phorbol 12-myristate 13-acetate/ionomycin and Brefeldin A and stained for intracellular IL-10 or isotype control. Mean and SD values are plotted for groups of 4 to 6 animals. *P < .05, **P < .01, ***P < .001.

allergy during chronic infection as a tool for therapy of allergic children. We thank Dr Franca Hartgers for help with the statistical analysis, Dr Mark Wilson for critical reading of the manuscript, and Dr Adrian Mountford for helpful discussions. REFERENCES 1. Maizels RM. Infections and allergy—helminths, hygiene and host immune regulation. Curr Opin Immunol 2005;17:656-61. 2. Yazdanbakhsh M, Wahyuni S. The role of helminth infections in protection from atopic disorders. Curr Opin Allergy Clin Immunol 2005;5: 386-91. 3. Palmer LJ, Celedon JC, Weiss ST, Wang B, Fang Z, Xu X. Ascaris lumbricoides infection is associated with increased risk of childhood asthma and atopy in rural China. Am J Respir Crit Care Med 2002; 165:1489-93. 4. Obihara CC, Beyers N, Gie RP, Hoekstra MO, Fincham JE, Marais BJ, et al. Respiratory atopic disease, Ascaris-immunoglobulin E and tuberculin testing in urban South African children. Clin Exp Allergy 2006;36:640-8. 5. Buijs J, Borsboom G, Renting M, Hilgersom WJ, van Wieringen JC, Jansen G, et al. Relationship between allergic manifestations and Toxocara

Basic and clinical immunology

chronic stages and becomes more intense, the balance tips toward a regulatory response corresponding to downmodulation of egg granuloma and reduction in TH2 cytokine responses. At this point, the regulatory environment might extend to suppress responses to third-party antigens in both an antigen-specific and antigen-nonspecific manner, as we show here with the suppression of allergic airway inflammation. Furthermore, we demonstrate that immune cells within the B- and T-cell compartments adopt regulatory properties that can transfer protection from allergic airway inflammation in an IL-10–dependent manner. These data support, in a well-controlled system, the previous observations in human subjects showing a decreased incidence of allergy among schistosome-infected children living in endemic areas. At the same time, they clarify the increased allergic symptoms in children living in areas where helminth infections occur only occasionally or with a more transient nature. Future studies should focus on targeting molecular helminth structures responsible for inducing regulatory processes and protection against

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