Helminth-Modified Pulmonary Immune Response Protects
Mice from Allergen-Induced Airway Hyperresponsiveness1
Niamh E. Mangan,* Nico van Rooijen,†Andrew N. J. McKenzie,‡and Padraic G. Fallon2*
It has been shown that the presence of certain helminth infections in humans, including schistosomes, may reduce the propensity
to develop allergies in infected populations. Using a mouse model of schistosome worm vs worm ? egg infection, our objective was
to dissect the mechanisms underlying the inverse relationship between helminth infections and allergies. We have demonstrated
that conventional Schistosoma mansoni egg-laying male and female worm infection of mice exacerbates airway hyperresponsive-
ness. In contrast, mice infected with only schistosome male worms, precluding egg production, were protected from OVA-induced
airway hyperresponsiveness. Worm-infected mice developed a novel modified type 2 cytokine response in the lungs, with elevated
allergen-specific IL-4 and IL-13 but reduced IL-5, and increased IL-10. Although schistosome worm-only infection is a laboratory
model, these data illustrate the complexity of schistosome modulation of host immunity by the worm vs egg stages of this helminth,
with the potential of infections to aggravate or suppress allergic pulmonary inflammation. Thus, infection of mice with a human
parasitic worm can result in reduced airway inflammation in response to a model allergen. The Journal of Immunology, 2006,
and mucus hypersecretion that results in intermittent airway ob-
struction (1). The immune etiology of asthma is complex, but ge-
netic and immunological analyses of atopic individuals have re-
vealed that Th2-type cytokines are causally associated with
allergies (2, 3) with a type 2 cytokine response being characterized
by increased (Th2) cell development and production of IL-4, -5, -9,
and -13 resulting in IgE production, mucus hyperplasia, and
The prevalence of a range of atopic diseases is rising and with
respect to allergic asthma there has been an almost 2-fold increase
in incidence in the past two decades (5). The rate at which the
incidence of allergic disease is rising implicates a recent change in
environmental influences on this process, i.e., the hygiene hypoth-
esis (5). This hypothesis proposes that a shift in the immune sys-
tem toward type 1 immunity upon early exposure to infections
such as bacterial (6, 7) and viral (8) infections protects against
allergic diseases by reducing the expression of Th2 cytokines gen-
erally evoked by allergens. An alternative explanation holds that
certain parasitic helminth infections may protect against allergic
disorders because human populations with high rates of parasitic
helminth infections, which induce an immunological shift toward
sthma is an atopic inflammatory disorder of the airways
that is characterized by increased airway hyperrespon-
siveness (AHR),3eosinophil infiltration of the airways,
the “allergic” Th2 responses, have a reduced prevalence of allergic
disorders (9). Schistosoma spp. are tropical helminth parasites,
characteristically associated with being potent inducers of Th2 cy-
tokine responses including eosinophilia and IgE responses (10),
that have been postulated to ameliorate atopic disorders in
Recent experimental studies have shown that mice or rats in-
fected with rodent nematode parasites have reduced allergic re-
sponses (11–14). We have previously demonstrated that Schisto-
soma mansoni infection protects mice from anaphylaxis through a
regulatory mechanism induced by the worm (15). In this study, we
have evaluated whether S. mansoni infection of mice, the mouse
being the preferred animal model for studies on the immunobiol-
ogy of schistosomiasis (10), altered susceptibility of the animals to
OVA-induced AHR, which is also widely used as a model of hu-
man pulmonary inflammation (16). We have identified that the
worm stage of S. mansoni infection modulates mice so they are
refractory to AHR. This is the first formal demonstration of a
mechanism that human parasitic worms use to suppress allergen-
induced airway inflammation.
Materials and Methods
Female BALB/c mice were purchased from Harlan at 6–8 wk of age.
Outbred male or female Tyler’s Original (TO) mice, also from Harlan,
were obtained for egg and worm production. IL-13-deficient (IL-13?/?)
mice were provided by Dr. A. McKenzie (Medical Research Council Lab-
oratory of Molecular Biology, Cambridge, U.K.) and were bred in-house
(17). Mice were housed in individually ventilated and filtered cages under
positive pressure (Techniplast). Food and water were supplied ad libitum.
Sentinel mice were screened to ensure specific pathogen-free status. All
animal experiments were performed in compliance with Irish Department
of Health and Children regulations.
A Puerto Rican strain of S. mansoni was maintained by passage in male or
female outbred TO strain mice and albino Biomphalaria glabrata snails
served as intermediate hosts. Female BALB/c, 6–8 wk of age (Harlan),
were infected percutaneously with 30 mixed male and female cercariae for
a conventional infection where eggs are laid (worm ? egg infections), or
mice were infected with 30 male cercariae for a worm infection where no
eggs are present. The sex of cercariae shed from individual snails was
*School of Biochemistry and Immunology, Trinity College, Dublin, Ireland;†Vrije
Univeriteit, Vrije, The Netherlands; and‡Medical Research Council Laboratory of
Molecular Biology, Cambridge, United Kingdom
Received for publication July 19, 2005. Accepted for publication October 24, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the Wellcome Trust and Science Foundation Ireland.
2Address correspondence and reprint requests to Dr. Padraic G. Fallon, School of
Biochemistry and Immunology, Trinity College, Dublin, Ireland. E-mail address:
3Abbreviations used in this paper: AHR, airway hyperresponsiveness; DPBS, Dul-
becco’s PBS; Penh, enhanced pause; Mch, methacholine; GL, pulmonary resistance;
Cdyn, pulmonary compliance; BAL, bronchoalveolar lavage; EPO, eosinophil per-
oxidase; PAS, periodic acid-Schiff; FSC, forward side scatter; SSC, side scatter.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
determined by PCR as described (18). To remove worms from worm-
infected mice, animals were orally treated with the schistosomicidal drug
Praziquantel (Sigma-Aldrich; 100 mg/kg orally for 5 consecutive days).
Cell surface phenotyping was analyzed using Tricolor-conjugated anti-
CD19 (6D5; Caltag Laboratories), anti-CD4 (CT-CD4; Caltag Laborato-
ries), anti-CD8 (5H10; Caltag Laboratories), anti-F4/80 (F4/80; Caltag
Laboratories), and PE-conjugated anti-CD25 (PC61 5.3; BD Pharmingen),
anti-Syndecan-1 (CD138; 281-2 BD Pharmingen), anti-CD5 (Ly-1; BD
Pharmingen), anti-IgM (?-chain specific; The Jackson Laboratory), anti-
CD11b (mac-1, M1/70; BD Pharmingen), anti-CCR3 (83101; R&D Sys-
tems), and FITC-conjugated anti-GL7 (GL7; BD Pharmingen). Intracellu-
lar cytokine staining was with PE-conjugated anti-IL-4 (BVD6-24G2) and
FITC-conjugated anti-IL-10 (JES5-2A5) was obtained from Caltag
Hybridoma culturing and Ab production
Anti-IL-10R (1B1.3a), anti-CD25 (PC61 5.3) were purchased from Amer-
ican Type Culture Collection. The anti-CD4 (YTS 191) hybridoma was
kindly provided by Prof. A. Cooke (University of Cambridge, Cambridge,
U.K.) and Prof. H. Waldman (University of Oxford, Oxford, U.K.). The
above hybridoma cell lines were cultured in RPMI 1640 and supernatants
were precipitated in 50% ammonium sulfate followed by dialysis against
Dulbecco’s PBS (DPBS; pH 7.2) (Sigma-Aldrich). Ab was purified on
Protein G (Sigma-Aldrich) separation columns and protein was quantified
before use. All Abs were tested for endotoxin contamination and were
confirmed to have ?0.5 endotoxin units/mg (Chromogenic LAL). Macro-
phages were depleted by the treatment of mice with liposomes containing
dichloromethylene bisphosphonate (clodronate liposomes), prepared as de-
scribed (19). Clodronate was a gift of Roche Diagnostics.
Ag sensitization and challenge
Mice were sensitized with OVA (fraction V: Sigma-Aldrich) and airways
challenged with OVA to induce pulmonary allergic inflammation and AHR
(see Fig. 1). On days 1 and 14, mice received an i.p. injection of 100 ?l of
a sensitization solution containing 20 ?g of OVA and 2 mg of Imject Alum
(Pierce). Mice received a 20-min aerosol challenge with 1% OVA in DPBS
from days 28–30. Control mice received an aerosol of PBS.
OVA has high levels of endotoxin contamination (?1.5–3 endotoxin
units/mg) which previously has been shown to alter the development of
AHR (20). It is impossible to completely remove endotoxin from OVA
preparations (20), while the addition of polymyxin B, to inactivate LPS, is
also inappropriate due to direct affects of the antibiotic on cell activation
(21). Therefore, every effort was made to maintain sterile procedures to
minimize the endotoxin contamination in all OVA preparations. OVA was
prepared using endotoxin-free reagents and all glassware and equipment
were endotoxin decontaminated (0.5 mM NaOH). Endotoxin was partially
removed (Detoxi-gel endotoxin removing gel columns; Pierce) and OVA
preparations were extensively dialyzed against sterile DPBS (Sigma-Al-
drich) using dialysis cassettes (Pierce). After these measures, the OVA
used in this study had ?0.5 (range 0.186–0.447) endotoxin units/mg
Pulmonary assessment of enhanced pause (Penh)
Lung reactivity of OVA-sensitized mice was assessed using a protocol by
Hamelmann et al. (22). On day 32, airway responsiveness of mice was
assessed by barometric whole body plethysmography in response to a
methacholine (Mch) challenge. In brief, spontaneously breathing, nonanes-
thetized mice were placed in the plethysmograph (EMKA Technologies)
and PBS aerosol was administered to establish baseline readings over 3
min. Mch (acetyl ?-methylcholine chloride; Sigma-Aldrich) was then ad-
ministered by nebulization in increasing, serial 2-fold concentrations from
3.125–50 mg/ml for 3 min each dose to induce bronchoconstriction. In this
model, the extent of airway reactivity of individual mice was quantified as
Penh. Airway responsiveness of mice was expressed as the fold increase
for each concentration of Mch compared with Penh values after PBS chal-
lenge. Twenty-four hours later, mice were sacrificed by terminal anesthesia
and samples were taken for further immunological analysis.
Airway conductance and dynamic compliance studies
Pulmonary conductance (GL) and compliance (Cdyn) in response to Mch
were measured as described previously (23). In brief, mice were assessed
on day 31 after OVA sensitization and challenge using the same protocol
as outlined above. Mice were anesthetized with 105 mg/kg pentobarbitone,
tracheostomized and then mechanically ventilated (EMKA Technologies)
for airway assessment. A two-way connector was attached to the tracheal
cannulation tube to allow inspiration and expiration via the ventilator.
Breaths were stabilized at a rate of 60 breaths/min and the tidal volume was
fixed at 250 ?l. Increasing doses of Mch (3.125–250 ?g/kg) were admin-
istered i.p. and readings were taken continuously for 4 min after injection.
Maximum pulmonary resistance (GL is the inverse of resistance) and com-
pliance (Cdyn) values were taken after each dose for statistical analysis to
express changes in these functional parameters as compared against saline
Bronchoalveolar lavage (BAL)
BAL fluids were collected by cannulating the trachea and lavaging lungs
with 1 ml of cold PBS. Supernatant was removed for measurement of
cytokines by ELISA. BAL cells were pelleted, washed, and counted for
immunofluorescence staining for surface marker expression and cytospins.
The numbers of eosinophils, neutrophils, lymphocytes, and macrophages
was determined by performing a differential count on at least 400 cells/
slide of Giemsa-stained cytospins.
Lung processing and histology
For analysis of cells by FACS, lungs were removed from the mice and
digested in 1 mg/ml collagenase D (Roche Diagnostics) in RPMI 1640 with
FCS for 30 min at 37°C with shaking. Lung digests were then filtered
through 100-?m Falcon cell strainers (BD Biosciences) followed by three
washes and further filtering through 40-?m Falcon cell strainers. In some
experiments, the left lobes of the lungs, stored snap-frozen, were removed
for assessment of tissue cytokines and chemokines (IL-4, IL-5, IL-13, IL-
10, IFN-?, and eotaxin). Each lung lobe was homogenized using an Ultra-
Turrax homogenizer (IRK-WERKE) in 800 ?l of homogenization buffer
(2% FCS, 0.5% cetyltrimethylammonium bromide; Sigma-Aldrich). The
homogenate was microcentrifuged at 13,000 rpm for 15 min at 4°C. The
supernatant was aliquoted and the tissue extract was stored at ?20°C for
protein estimation, cytokine analysis, and eosinophil peroxidase (EPO) as-
say. The protein content of tissue extracts was determined by the BCA
protein estimation kit. EPO levels were determined based on a method
previously described (24). The remaining lung lobes were fixed in Forma-
lin (10% formaldehyde in 0.9% saline solution) for histological analysis.
Lung sections were stained with H&E, Giemsa for eosinophils, periodic
acid-Schiff (PAS) for goblet cell counts and Martius Scarlet Blue for col-
lagen. Pulmonary collagen was quantified by differential staining, and is
expressed as micrograms of collagen per milligram of lung protein, as
described (25). Goblet cells were counted on PAS-stained lung sections
using an arbitrary scoring system (26, 27). PAS-stained goblet cells in
airway epithelium were measured double-blind using a numerical scoring
system (0: ?5% goblet cells; 1: 5–25%; 2: 25–50%; 3: 50–75%; 4:
?75%). The sum of airway scores from each lung was divided by the
number of airways examined, 20–50 airways/mouse, and expressed as mu-
cus cell score in arbitrary units.
Spleens and the lung draining mediastinal lymph nodes were removed and
cells were isolated for cell culture and reactivation for cytokine measure-
ments. Single cell suspensions were prepared from spleens and mediastinal
lymph nodes and depleted of erythrocytes by lysis with 0.87% ammonium
chloride solution. For in vitro experiments, cells were cultured in RPMI
1640 (Invitrogen Life Technologies) supplemented with 10% (v/v) heat-
inactivated FCS (Labtech), 100 mM L-glutamine (Invitrogen Life Tech-
nologies), 100 U/ml penicillin, and 100 ?g/ml streptomycin (Invitrogen
Life Technologies). Mediastinal lymph node or spleen cells were plated at
5 ? 106cells/ml. Cells were unstimulated (media) or stimulated with plate-
bound anti-CD3 (clone 145-2C11) plus anti-CD28 (4 ?g/ml), OVA (5–200
?g/ml), in a 24-well plate (Greiner) at 37°C for 72 h. Plates were precoated
with anti-CD3 mAb at 10 ?g/ml for 2 h at 37°C and then washed in sterile
DPBS before addition of cells. Supernatants were harvested after 72 h and
cytokine levels (IL-4, IL-5, IL-13, IFN-?, IL-10, and TGF-?) were mea-
sured by ELISA. For cell proliferation analysis, cells were exposed to a
range of concentrations of OVA, in triplicate wells on 96-well plates. Cul-
tures were pulsed with 1 ?Ci/well [3H]thymidine (Amersham) for the last
14 h of culture. Cells were harvested with a Tomtec cell harvester and
[3H]thymidine incorporation was measured by a Wallac beta counter.
Surface marker expression and intracellular phenotyping of cells was as-
sessed by flow cytometry as described (15, 28). Cells were counted and
resuspended in ice-cold FACS buffer (2% FCS, 0.05% sodium azide in
PBS) at 2 ? 106cells/ml on a 96-well plate. Cells were stained with surface
139 The Journal of Immunology
Abs for 30 min on ice at the recommended concentration and then washed
three times in FACS buffer. For intracellular cytokine staining, unstimu-
lated cells were incubated with Brefeldin A (10 ?g/ml; Sigma-Aldrich) for
4 h. Following surface staining, cells were fixed and permeabilized using
the Fix and Perm Cell Permeabilization kit (Caltag Laboratories) with the
anti-cytokine Ab added upon permeabilization. Data were collected on a
FACScan flow cytometer (BD Biosciences) and analyzed using CellQuest
software. In all experiments, appropriate isotype controls were used to set
gates and were plotted on logarithmic scales.
Various cell populations in the lung digests were identified by flow
cytometry, as described (29). Lungs cells were first gated on CD19, CD4,
and CD8 vs forward side scatter (FSC). Lymphocytes were identified as
FSClow, side scatter (SSC)lowCD19?, CD4?, CD8?. Eosinophils distin-
guished as SSChigh, CD19?, CD4?, CD8?nonautofluorescent granulo-
cytes that stained positive for CCR3. Alveolar macrophages were charac-
terized as CD19?, CD4?, CD8?mononuclear cells that were highly
autofluorescent and F4/80?.
Ab and cytokine ELISA
OVA-specific serum Abs were detected by direct ELISA (25). Total serum
IgE was measured using Pharmingen Abs (BD Pharmingen). Sandwich
ELISAs were performed to quantify levels of specific cytokines in the
supernatants from lung tissue homogenates, BAL fluid, and in vitro cell
stimulation cultures. Reagents for quantification of IL-4, IL-5, IL-13, from
BD Pharmingen and IL-10 and IFN-? were purchased as a DuoSet ELISA
development system from R&D Systems. Total TGF-? (acidified samples)
was measured by ELISA according to the manufacturer’s instructions (Pro-
mega). Eotaxin detection reagents were also purchased from R&D Sys-
tems. Cytokines in lung homogenates are expressed as nanograms of cy-
tokine per milligram of lung protein.
GraphPad Prism and GraphPad Instat software was used to analyze the
data. Differences were considered significant when p ? 0.05.
S. mansoni worm ? egg infection of mice exacerbates AHR
To experimentally investigate whether schistosome infection mod-
ulates immune responses following exposure to an allergen schis-
tosome, infected mice were immunized with alum-adsorbed OVA,
a model-type cytokine 2-inducing allergen, and OVA-specific im-
munity was analyzed. Mice were exposed to a conventional S.
mansoni male and female worm infection, where eggs are laid, and
thus called here worm ? egg infections. Worm ? egg-infected
mice were sensitized with OVA systemically and in the lungs dur-
ing the acute (between 7 and 11 wk of infection) and chronic
(between 12 and 16 wk of infection) stages of infection (Fig. 1).
Spleen cells from worm ? egg-infected mice immunized with
OVA during acute or chronic infection both had elevated in vitro
production of allergen-specific type 2 cytokines (IL-4, IL-5, and
IL-13) and IL-10 compared with production of these cytokine by
cells from uninfected OVA-immunized mice (Fig. 2A). The in-
creased type 2 cytokine response in worm ? egg-infected mice
was associated with greater levels of OVA-specific IgE (Fig. 2B).
To analyze whether the increased allergen-induced immune re-
sponse in worm ? egg-infected mice altered pulmonary inflam-
mation, we used whole body plethysmography on conscious and
unrestrained mice to determine airway function, which was quan-
tified as Penh (22). In this model, uninfected mice sensitized with
OVA develop AHR, demonstrated by a dose-dependent elevation
in Penh in response to Mch when compared with uninfected PBS-
treated mice (Fig. 3A). Unexpectedly, both PBS- and OVA-sensi-
tized worm ? egg-infected mice developed severe respiratory dis-
tress following exposure to the lowest dose of Mch (3.125 mg/ml)
aerosol (Fig. 3A). This distress was manifested by significant el-
evated Penh values at all doses of Mch when compared with un-
infected OVA mice (p ? 0.001), with all worm ? egg-infected
mice dying in the plethysmograph chamber at doses of 12.5–25
mg/ml Mch (Fig. 3A). There was no difference in the rate of mor-
talities between acute vs chronically worm ? egg-infected mice,
but in both groups, sensitization with OVA accelerated the death of
mice, with acute and chronically infected OVA-sensitized animals
having elevated AHR and dying after exposure to 12.5 mg/ml Mch
(Fig. 3A). Therefore, following allergen challenge schistosome
induction of AHR. Infected mice were sensitized in the acute, starting week
7, or chronic, starting week 12, stages of infection. Mice were systemically
sensitized with an i.p. injection of 20 ?g of OVA (plus 2 mg of alum) on
days 0 and 14. Mice were challenged via the airways from days 28–30 with
an aerosol of OVA (1% OVA for 20 min). On day 32, lung function in
response to methacholine (Mch) was tested using either restrained or un-
restrained plethysmography, as described in Materials and Methods. Cell
depletion regimes were performed on days 27 and 30. Mice were killed on
day 33 (†).
Protocol for OVA sensitization and challenge in mice for
type 2 responses to OVA sensitization. OVA was administered to unin-
fected mice and to animals during the acute or chronic stages of a worm ?
egg-infection, as described in Fig. 1. A, Detection of in vitro production of
cytokines from spleen cells stimulated with OVA (200 ?g/ml). Spleens
from three mice were pooled. B, Levels of OVA-specific IgE. All data are
mean ? SD. Data are representative of two to three separate experiments.
S. mansoni worm ? egg-infected mice produce elevated
140HELMINTH-MODIFIED PULMONARY RESPONSE PREVENTS AHR
worm ? egg-infected mice develop elevated type 2 cytokine re-
sponses and, independent of allergen sensitization, are also pre-
disposed to fatal AHR.
During a schistosome worm ? egg infection of mice, eggs that
are laid by the female worms are swept to various organs where
they evoke granulomatous inflammation. When schistosome eggs
are injected i.v. into naive mice, they are trapped in the lung and
stimulate pulmonary inflammation. Therefore, an initial explana-
tion for the increased susceptibility of worm ? egg-infected mice
to AHR was the presence of eggs in the lungs inducing pathology.
However, we have not detected any eggs present in digests of
lungs from acute or chronically worm ? egg-infected mice (data
not shown). The lungs by histology sections from uninfected and
worm ? egg-infected mice were largely comparable (Fig. 3B),
with the notable exception of substantial subepithelial and paren-
chymal collagen deposition in acute and chronic worm ? egg-
infected mice (Fig. 3C; data not shown). Quantification of pulmo-
nary collagen showed that mice with an acute worm ? egg
infection have a significant, p ? 0.001 vs uninfected mice, 2- to
3-fold elevation in collagen (Fig. 3D). Previously, we have shown
schistosome worm ? egg-infected IL-13?/?mice do not develop
the elevated levels of hepatic collagen seen in comparably infected
wild-type mice (30), which is consistent with the role of IL-13 in
fibrosis (31). As schistosome worm ? egg-infected IL-13?/?mice
also do not develop pulmonary fibrosis (Fig. 3D), we analyzed
AHR in these mice during acute stages of infection. Worm ?
egg-infected IL-13?/?mice did not develop AHR and had com-
parable pulmonary function as observed in uninfected mice (Fig.
3E). Therefore, during acute worm ? egg infection there is ele-
vated IL-13-dependent pulmonary fibrosis that contributes to the
exacerbated lung pathology in these mice. In the chronic stages of
a worm ? egg infection, pulmonary fibrosis is reduced relative to
acutely infected mice, but the levels of lung collagen remain sig-
nificantly elevated above age-matched uninfected mice (data not
shown). However, although the increased susceptibility of chronic
worm ? egg-infected mice to AHR (Fig. 3A) is associated with
elevated pulmonary collagen, we have not addressed the involve-
ment of IL-13 in AHR and lung fibrosis in these chronically
Schistosome worm-infected mice are resistant to OVA-induced
Previously, we have shown that mice infected with schistosome
male worms are completely refractory to anaphylaxis, whereas
worm ? egg-infected mice were only partially resistant (15). We
immunized worm-infected mice with OVA, using the protocol de-
scribed in Fig. 1, to address OVA-induced AHR in these animals.
It is important to note that infection of mice with schistosome male
worms has been shown to induce a bias toward type 2 cytokine
responses (15, 18, 32); thus, worm-infected mice have elevated
basal levels of IL-4, IL-5, and IL-13 before OVA challenge. De-
spite this type 2 cytokine bias in worm-infected mice, spleen cells
from these animals, and also from uninfected mice, that were in-
jected with PBS and not OVA, did not produce OVA-specific cy-
tokines in vitro (Fig. 4). In contrast, spleen cells from OVA-im-
munized worm-infected mice had greater allergen-stimulated type
and worm ? egg-infected (acute and chronic) mice were unsensitized (PBS) or sensitized with OVA, as described in Fig. 1. A, Penh responses of acute
and chronically worm ? egg-infected mice. Data represent the mean change from baseline PBS values for each group. †, Denotes death of infected mice.
Penh is presented as mean ? SEM from three separate experiments (n ? 12–15 mice). B, Absence of inflammation in lungs of a 10 wk worm ? egg-infected
mouse. Representative H&E-stained sections of lungs from uninfected and worm ? egg-infected mice. C, Increased pulmonary fibrosis (blue stain) in worm
? egg-infected mice relative to uninfected mice (Martius Scarlett Blue stain). D, Quantification of elevated pulmonary collagen levels in worm ?
egg-infected wild-type (WT) mice relative to uninfected WT mice, and reduced fibrosis in worm ? egg-infected IL-13-deficient (?/?) mice. Data are
mean ? SEM from 6 to 8 mice per group. Statistical differences between levels of collagen in uninfected vs infected WT or IL-13?/?mice were tested
by Student’s t test. E, Worm ? egg-infected IL-13?/?mice do not develop spontaneous AHR, whereas worm ? egg-infected WT mice have dose-
dependent elevations in Penh. Penh values are mean ? SEM from 7 to 12 mice from two separate experiments. Data in B–D are from mice infected with
a worm ? egg infection for 8–10 wk.
S. mansoni worm ? egg-infected mice have increased susceptibility to AHR via increased IL-13-dependent pulmonary fibrosis. Uninfected
141The Journal of Immunology
2 cytokine (IL-4, IL-5, and IL-13) production, and also had mark-
edly elevated sera levels of OVA-specific IgE (data not shown),
compared with OVA-immunized uninfected mice (Fig. 4). Inter-
estingly, there was greater relative allergen-specific IL-10 release
by spleen cells from OVA-sensitized worm-infected mice com-
pared with IL-10 production from spleen cells from comparable
treated worm ? egg-infected mice (p ? 0.05; Figs. 2A and 4),
which is consistent with previous studies (15, 18).
Mice infected with a worm infection, but not exposed to OVA,
had no spontaneous AHR (Fig. 5), which is in marked contrast to
what was observed in worm ? egg-infected animals (Fig. 2). Strik-
ingly, OVA-sensitized worm-infected mice did not develop AHR,
with no alterations in Penh following Mch challenge, whereas
OVA-sensitized uninfected mice had dose-dependent increases in
Penh (Fig. 5A). As the accuracy of Penh as a measure of murine
lung function has been questioned (33), we also tested pulmonary
elevated allergen-induced IL-10 and type 2 cytokines following OVA sen-
sitization. Uninfected and worm-infected mice were sensitized with PBS or
OVA as described in Fig. 1. Spleen cells were stimulated with OVA (200
?g/ml) in vitro, and cytokines in supernatants were detected by ELISA.
Data are mean ? SD from pools of spleens from three mice, and are
representative of three experiments.
Spleen cells from S. mansoni worm-infected mice produce
duced AHR. Uninfected and worm-infected mice were exposed to OVA or
PBS and AHR was determined as (A) Penh, or (B) pulmonary resistance
(GL) or compliance (Cdyn). Data are presented as mean ? SEM. Penh
values are from four separate experiments, n ? 16–21 mice per group. GL
and Cdyn were from two separate experiments, n ? 8–10 mice per group.
S. mansoni worm-infected mice are refractory to OVA-in-
OVA-sensitized worm-infected mice. A, Levels of cytokines in BAL from
OVA-sensitized uninfected and worm-infected mice. Data are mean ?
SEM from five to eight individual mice, and are representative from two
separate experiments. The Student t test was used to determine the differ-
ence between uninfected and infected mice. B, Levels of cytokines in lung
homogenates from OVA-sensitized uninfected and worm-infected mice.
Cytokine values were adjusted to nanograms per milligram of lung protein.
Data are mean ? SEM from six to nine individual mice, and are repre-
sentative from three separate experiments. The Student t test was used to
determine difference between uninfected and infected mice. C, OVA-spe-
cific cytokine responses from mediastinal lymph node cells from OVA-
sensitized uninfected and worm-infected mice. Lymph nodes from four to
eight mice were pooled and cells were cultured in duplicate or triplicate
with OVA (200 ?g/ml). D, Cell proliferation of mediastinal lymph node
cells to different concentrations of OVA was measured by [3H] incorpo-
ration and are expressed as cpm. Data are representative of two separate
Modified type 2 response and elevated pulmonary IL-10 in
142 HELMINTH-MODIFIED PULMONARY RESPONSE PREVENTS AHR
function by analysis of pulmonary resistance (GL) and compliance
(Cdyn) in unconscious and restrained animals (23). Similar to the
Penh data, mice with worm infections had reduced GL and Cdyn
responses to Mch challenge compared with resistance/compliance
responses of sensitized uninfected mice (Fig. 5B).
During schistosome infection there is a larval migration phase
that passes through the lung (34) that may alter pulmonary func-
tion. To address this point, worms were removed from infected
mice by drug (praziquantel) treatment, after larval migration, and
the mice then became fully susceptible to OVA-induced AHR
(data not shown). These data demonstrate that mice infected with
S. mansoni worm infections do not develop OVA-induced AHR.
Modified pulmonary type 2 response in OVA-sensitized
We have addressed lung-specific responses in worm-infected mice,
as in the OVA pulmonary challenge model used, the elevated AHR
in sensitized mice is associated with elevations in pulmonary type
2 cytokines, eosinophil infiltration, and goblet cell hyperplasia
(35). OVA-sensitized worm-infected mice had significantly ele-
vated levels of both IL-4 and IL-13 in BAL fluid and lung homog-
enates (p ? 0.05) compared with OVA-sensitized uninfected
mice, with no differences in IFN-? levels (Fig. 6, A and B). In
contrast, IL-5 levels in BAL and lung homogenates from worm-
infected mice were lower than uninfected mice, with BAL IL-5
significantly reduced (p ? 0.05; Fig. 6, A and B). Thus, worm-
infected mice stimulated a modified type 2 cytokine response in
the lungs, with IL-4 and IL-13 levels being significantly elevated
whereas IL-5 was reduced. Sensitized worm-infected mice had a
striking ?3-fold increase in total IL-10 in both homogenates of
lungs and BAL fluid, which was significantly elevated above IL-10
levels detected in lungs of sensitized but uninfected mice (p ?
0.01–0.001; Fig. 6, A and B). OVA-specific cytokine production
by the lung mediastinal lymph node cells also demonstrated the
modified Th2 response, elevated IL-4 and IL-13 but reduced IL-5,
in sensitized worm-infected mice compared with sensitized unin-
fected mice, with an associated pronounced increase in OVA-in-
duced IL-10 (Fig. 6C). There were no differences in OVA-specific
proliferation of mediastinal lymph node cells from OVA-sensi-
tized uninfected and worm-infected mice, indicating normal Ag-
induced T cell responsiveness (Fig. 6D). Similarly, lung TGF-?
levels in worm-infected and uninfected OVA-sensitized mice were
not different (data not shown).
Histology sections of lungs of OVA-sensitized uninfected mice
showed normal airway inflammation and peribronchial infiltrating
eosinophils, and increased eosinophils in BAL and lung digests
and goblet cell hyperplasia (Fig. 7, A–D). In sensitized worm-
infected mice, there was no lung inflammation or peribronchial
eosinophilia (Fig. 7A). Enumeration of cells in the BAL showed
that worm-infected mice did not have the normal increase in eo-
sinophils that occurred in OVA-sensitized uninfected mice, with
significantly (p ? 0.005) fewer eosinophils recovered in BAL
from infected vs uninfected mice (Fig. 7B) and also reduced lung
eosinophils (FSC/SSChigh, CD19?, CD4?, CD8?, CCR3?cells)
but not in sensitized worm-infected mice (top panel). Lower panels are higher magnifications of sections of lungs from OVA-sensitized uninfected and
worm-infected mice showing peribronchial eosinophilia in uninfected mice (bracket) but not infected mice (H&E-stained). B, Differential cell counts of
cytospins of BAL cells from uninfected and worm-infected mice exposed to OVA or PBS. The Student test was used to test for statistical differences in
the percentage of BAL eosinophils. C, Percentage of granulocytes that are eosinophils in the lungs of OVA-sensitized uninfected and worm-infected mice.
Eosinophils were phenotyped as SSChigh, CD19?, CD4?, CD8?, CCR3?by flow cytometry. D, Detection of eotaxin in lung homogenates from OVA-
sensitized uninfected and worm-infected mice. E, OVA-induced goblet cell hyperplasia in uninfected mice but not in worm-infected mice (PAS-stained).
Graph shows quantification of goblet cell hyperplasia, expressed as mucus score. All data are mean ? SEM from four to nine individual mice and are
representative of two to three separate experiments. Statistical differences between infected mice and uninfected OVA-sensitized mice was tested by
Student’s t test.
Absence of pulmonary inflammation in OVA-sensitized worm-infected mice. A, Airway inflammation in OVA-sensitized uninfected mice
143The Journal of Immunology
detected by flow cytometry of lung digests (Fig. 7C). In view of
the deficit in eosinophils in the lungs of worm-infected mice, we
measured eotaxin. The levels of eotaxin were significantly lower in
lungs recovered from sensitized worm-infected mice relative to
uninfected mice (p ? 0.01; Fig. 7D). Worm-infected mice also
had limited goblet cell hyperplasia, with significantly reduced mu-
cus scores compared with uninfected mice (Fig. 7E).
IL-10 mediates resistance of worm-infected mice to AHR
We have previously demonstrated worm-infected mice are resis-
tant to anaphylaxis through an IL-10-dependent mechanism (15).
As there was elevated pulmonary IL-10 in OVA-sensitized worm-
infected mice, we used an anti-IL-10R mAb to block IL-10 activity
and analyzed lung function. OVA-sensitized worm-infected mice
were fully susceptible to AHR when IL-10 was blocked, with sig-
nificantly greater Penh values in these animals than OVA-sensi-
tized uninfected mice at doses ?12.5 mg/ml Mch (p ? 0.05–
0.001; Fig. 8A). The increase in AHR in infected mice with IL-10
blocked was associated with a restoration of OVA-induced eosin-
ophilia in the BAL (Fig. 8B). This increase in susceptibility to
AHR and development of pulmonary eosinophilia in worm-in-
fected mice treated with anti-IL-10R mAb was associated with
significant elevation (p ? 0.01) in lung IL-5 levels in the mice;
IL-10R mAb-treated mice had 0.46 ? 0.19 ng of IL-5/mg lung
protein, vs 0.14 ? 0.08 ng of IL-5/mg lung protein in control
Ig-treated mice (data are mean ? SD, n ? 5; Student’s t test).
These data show that schistosome worm infection prevents AHR
in a mouse model of allergen-induced pulmonary inflammation via
IL-10-dependent suppression of pulmonary eosinophilia.
Resistance of worm-infected mice to AHR is independent of
CD4?, CD25?cells and macrophages
In mice with S. mansoni worm ? egg or worm-only infections, a
number of IL-10-producing cells have being identified, including
CD4?cells, CD4?CD25?regulatory cells, macrophages, and B
cells (15, 36). As we were addressing a pulmonary allergic phe-
notype, we characterized by flow cytometry which of these differ-
ent potential cellular sources of IL-10 preferentially infiltrated the
lungs of worm-infected mice. We found no major difference in the
percentages of CD4?cells, CD25?cells, or macrophages infil-
trating the lungs of sensitized worm-infected vs sensitized unin-
fected mice (Fig. 9A). The absence of a role for CD4?and CD25?
cells in the resistance of worm-infected mice to AHR was further
corroborated by in vivo depletion studies whereby depletion of
either CD4?or CD25?cells in worm-infected mice demonstrated
that each cell population has no role in affording protection from
OVA-induced AHR (Fig. 9B). Furthermore, depletion of pulmo-
nary macrophages had no effect on the worm infection-mediated
airway protection, with OVA-sensitized worm-infected mice hav-
ing lower Penh values compared with uninfected mice that devel-
oped AHR (Fig. 9B).
AHR is independent of CD4?and CD25?cells and macrophages. A, Flow
cytometry of different potential cellular sources of IL-10 preferentially in-
filtrated the lungs of uninfected and worm-infected mice. Cells were char-
acterized as described in Materials and Methods. B, No alteration in the
Penh responses of worm-infected mice with CD4?, CD25?cells or mac-
rophages depleted. OVA-sensitized mice were administered depletion
treatments on days 27 and 30 of OVA sensitization and challenge protocol:
anti-CD4 (0.5 mg/mouse), anti-CD25 (0.25 mg/mouse), or clodronate-li-
posomes (50 ?l of liposomes in on day 27 and 0.1 ml i.p. on day 30). All
depletions were confirmed to be effective by flow cytometry. Data repre-
sent the mean change from baseline Penh values for each group. Data are
from two separate experiments; n ? 8–10 mice per group.
Schistosome worm infection-mediated protection from
IL-10. A, Penh responses of OVA-sensitized uninfected mice and sensitized-
infected mice treated with anti-IL-10R or control IgG. The Student t test was
used to determine the differences between uninfected mice and anti-IL-10R-
treated infected mice. Data represent the mean change from baseline Penh
values for each group. B, Percentage of eosinophils in the BAL of OVA-
sensitized mice. All data are presented as group mean ? SEM (n ? 8–10) and
are representative of at least two separate experiments.
Resistance of worm-infected mice from AHR is dependent on
144 HELMINTH-MODIFIED PULMONARY RESPONSE PREVENTS AHR
Resistance of worm-infected mice to AHR is dependent on B cells
Previously, we have shown that worm-infected mice are resistant
to anaphylaxis via a schistosome-induced splenic IL-10-producing
B cell subpopulation (15). Significantly, there was a ?30% in-
crease in the number of infiltrating B cells in the lungs of sensi-
tized worm-infected mice when compared with sensitized unin-
fected mice (Fig. 10A). Following partial depletion of B cells, via
anti-IgM treatment, OVA-sensitized worm-infected mice were
rendered fully susceptible to AHR (Fig. 10B), suggesting that a B
cell population is intrinsic in the mechanism of resistance of
worm-infected mice to AHR. In fact, the airway reactivity of these
mice was even more enhanced than in uninfected mice indicating
the significance of B cells in the protection from pulmonary in-
flammation. The susceptibility of worm-infected mice to AHR af-
ter B cell depletion was associated with a restoration of eosinophil
infiltration of the lungs (Fig. 10C).
In this study, we have experimentally investigated the role of a
helminth worm vs worm and egg infection in modulating allergen-
induced pulmonary airway hyperreactivity. We have demonstrated
both the protective and the exacerbating roles of schistosome infec-
tions in a murine model of allergen-induced pulmonary inflammation.
Schistosome worms alone stimulated a modified pulmonary type 2
response that prevents OVA-induced AHR, with protection involving
B cell and IL-10-dependent suppression of pulmonary eosinophil
infiltration. In contrast, infection of mice with S. mansoni egg-laying
male and female worms exacerbated AHR.
The failure of schistosome worm-infected mice to develop
OVA-induced AHR was not associated with an inability of in-
fected mice to respond to the allergen. On the contrary, cells from
infected mice had normal in vitro cell proliferation to OVA and
their spleen and mediastinal lymph nodes cells produced greater
levels of both spleen and lung IL-4 and IL-13 than uninfected
mice. Strikingly, worm-infected mice had reduced levels of total
and OVA-specific IL-5 in the lungs compared with uninfected an-
imals, suggesting a unique selective Th2 defect in the lungs. Re-
cently, Platts-Mills et al. (37) have suggested that a “modified Th2
response” to cat allergens may explain the reduced levels of
asthma in children exposed to cats. We now describe for the first
time that schistosome worms induce what we have termed “a hel-
minth-modified pulmonary type 2 response” to OVA that renders
mice refractory to allergen-induced AHR. The helminth-modified
pulmonary type 2 response is characterized by elevated pulmonary
allergen-specific IL-4, IL-13, but reduced IL-5 and elevated IL-10.
An important question is why would a worm selectively suppress
pulmonary immunity? The answer may relate to the biology of the
parasite infection. Humans are repeatedly reinfected with schisto-
somes, with new infections requiring migration of larvae through
the lungs. However, established schistosome infections evoke con-
comitant immunity, whereby immune responses against the adult
worms and the eggs cross-react with larval Ags and thereby invading
new larvae killed are killed in the lungs. Interestingly, this schisto-
IgG Ab (38), which is similar to what is proposed in the modified Th2
response to cat allergens (37). Therefore, the schistosome parasite
may induce a modified pulmonary type 2 response to suppress in-
flammation in the lung induced by new invading larvae.
There is already evidence from field studies in Africa that schis-
tosome infection of humans can reduce allergic responses. Schis-
tosoma hematobium-infected school children in The Gabon have
lower prevalence of skin reactivity to house dust mites than those
free of this infection (39). Strikingly, when worms are removed
from patients by chemotherapy there is an increase in atopy, di-
rectly establishing a link between the presence of worms and sup-
pression of allergic responses (40). In the experimental study de-
scribed here, when adult schistosome worms were killed by drug
treatment the previously resistant mice became susceptible to
OVA-induced AHR. Therefore, there is a requirement for the con-
tinual presence of the worm during infection to sustain a helminth-
modified type 2 pulmonary response to suppress allergic inflam-
mation. Therefore, our experimental data support the argument
that the chronic down-regulation of the immune system during
helminth infections evokes a regulatory environment (41), called
here a helminth-modified pulmonary type 2 response, that may
impart protection from allergies. However, it is important to stress
that schistosome worm-only infection is a laboratory model that
ated protection of mice from AHR. A, Flow cytometry of increased infil-
tration of B cells (CD19?) into lungs of worm-infected mice relative to
uninfected mice. B, Increased Penh responses worm-infected mice with B
cells depleted. Mice were sensitized against OVA and treated with anti-
IgM (?-chain specific; 0.5 mg/mouse) before OVA aerosol challenge. Data
represent the mean change from baseline Penh values for each group. C,
BAL eosinophil counts in OVA-sensitized mice and sensitized-infected
mice treated with anti-IgM. Data are presented as group mean ? SEM (n ?
8–10) and are representative of two separate experiments.
B cells have an integral role in schistosome worm-medi-
145The Journal of Immunology
facilitates intimate functional analysis of modulation of immunity
by the worm, in the absence of eggs. Such worm-only infections
may not occur in infected humans.
In S. hematobium-infected school children, the reduced mite-
specific allergic response was associated with production of par-
asite-specific IL-10 (42). We have shown that worm-infected mice
are resistant to allergen-induced AHR via suppression of pulmo-
nary eosinophilia via IL-10. IL-10 is a potent regulatory cytokine
suppressing a range of immune-mediated responses (43). In mouse
AHR models, there are various data showing a role for IL-10 in
suppressing airway inflammation and AHR (44–47). One of these
recent studies showed that when IL-10 was administered in vivo
by gene delivery it suppresses OVA-induced AHR and airway eo-
sinophilia (47). Although our data showing worm infection-in-
duced IL-10 also blocks AHR and airway eosinophilia unlike the
gene delivery of IL-10 in worm-infected OVA-sensitized mice, there
is elevated, not reduced, allergen-specific IL-4 and IL-13 cytokines
and IgE, and also no alteration in cellular response to OVA. IL-10
may be a regulatory component of the helminth modified pulmonary
type 2 response we describe, as pulmonary IL-5 levels are restored in
worm-infected mice with IL-10 blocked in vivo.
The production of IL-10 in conventional S. mansoni male and
female egg-laying infections of mice has been shown to have a
central role in preventing infection-induced pathology (36, 48, 49).
Indeed, IL-10 also mediates resistance of tapeworm-infected mice
to experimental colitis (50). Despite worm ? egg-infected mice
having elevated IL-10, these animals were highly susceptible to
AHR, with worm ? egg-infected mice dying, even without aller-
gen sensitization, when exposed to Mch-induced bronchoconstric-
tion (Fig. 2). The predisposition of worm ? egg-infected mice to
AHR was evident in both the acute stages of infection, which is the
peak of Th2 cytokine induction, and also during the chronic stages,
when the parasite has down-modulated host immunity. Although
worm-infected mice produce relatively more IL-10 than compara-
bly infected worm ? egg-infected mice (15, 18), the discrepancy
between infection with male worm alone causing mice to be re-
sistant to allergen-induced inflammation whereas a male and fe-
male worm and egg-laying infection exacerbated AHR is unlikely
to be solely due to IL-10 levels. Worm ? egg-infected mice also
have the modified type 2 response in the lungs that is observed in
worm-infected mice (Fig. 6; data not shown). However, the pres-
ence of marked IL-13-dependent fibrosis in the lungs (Fig. 3) of
worm ? egg-infected mice, and not in mice infected with male
worms (data not shown), is relevant due to the effects of IL-13-
induced fibrosis on lung inflammation (31). Indeed, the contradic-
tory exacerbating or suppressive influences of schistosomes on
AHR, described here, is comparable to the potential negative or
positive outcomes from disease following infections with a range
of other pathogens (51).
Previous experimental studies have shown that infection with
various parasitic worms causes reduced allergic responses (11–14).
Using the rodent gastro-intestinal nematode Nippostrongylus bra-
siliensis, it was shown that infection suppressed allergen-induced
airway eosinophilia via IL-10 from an unidentified cell source
(14). It has been argued that CD4?cells, either Th2 or regulatory,
are the potential source of the worm-induced IL-10 (14). As CD4?
IL-10-producing cells generated by pathogens or genetically pre-
pared suppress airway inflammation (52, 53), they are an attractive
possible source for helminth-induced IL-10. Indeed, we have pre-
viously shown that schistosome worm infections of mice induce
elevated frequencies of natural CD4?CD25?regulatory cell and
IL-10-producing CD4?cells (15). However, depletion of CD4?or
CD25?cells did not alter the resistance of worm-infected mice to
OVA-induced AHR. Similarly, depletion of alveolar macrophages,
which also produce IL-10, did not alter the worm-induced protec-
tion against AHR. Previously, we have shown B cell-IL-10 levels
were significantly enhanced in worm-infected mice, with B cells
having a crucial role in schistosome worm infection-mediated re-
sistance to anaphylaxis (15) and AHR (this study). Earlier studies
have already proposed that B regulatory cells or IL-10-producing
B cells may function in immune-mediated inflammatory reactions
(54–56). For example, in murine experimental autoimmune en-
cephalomyelitis and collagen-induced arthritis IL-10-producing B
cells have been shown to have protective function in ameliorating
disease (54, 55), and such cells may also suppress intestinal in-
Our initial studies have found no major alteration between worm
? egg vs worm-only infected mice in different B cell subpopula-
tions we have examined (B1: CD19?CD5?; B2: CD19?CD5?;
germinal center B cells: CD19?GL7?; Ab-containing B cells
(plasma cells and plasmablasts): syndecan-1?CD19int(57)), al-
though both groups had elevated numbers of B cells in the lungs in
comparison to uninfected mice. Worm-infected mice have in-
creased frequencies of IL-10-producing B-1 cells in the perito-
neum, an observation originally reported in worm ? egg-infected
mice (58). However, using worm-infected xid mice that have de-
fective B-1 cells, we have shown that these B cells have no role in
affording protection from anaphylaxis (15) or from OVA-induced
AHR (data not shown). We are currently addressing the specific B
cell subpopulation that is evoking this IL-10-mediated protection
from pulmonary insult in worm-infected mice. Nonetheless, it is of
significance that depletion of B cells disrupts the fine signaling bal-
ance in immune regulation and thus exacerbates OVA-induced pul-
monary inflammation by removal of a potentially critical regulatory
cell. Further studies are required to address the interplay between B
cells, IL-10, and the helminth-modified pulmonary type 2 response.
In this study, we demonstrate both protective and antagonistic
roles for schistosome infections of mice in an experimental test of
the hygiene hypothesis (51). In marked contrast, S. mansoni worm-
only infection of mice diminishes the disease effects in a model of
OVA-induced AHR via induction of what we have termed a hel-
minth-modified pulmonary type 2 response. These data highlight
the important influence of the helminth parasite S. mansoni and its
significance in allergic disorders. Importantly, this is the first for-
mal demonstration of protection by a pathogen of humans in a
mouse model of allergic pulmonary disease.
We are grateful for the assistance of Rosie Fallon, Philip Smith, and
The authors have no financial conflict of interest.
1. Busse, W., and W. Neaville. 2001. Anti-immunoglobulin E for the treatment of
allergic disease. Curr. Opin. Allergy Clin. Immunol. 1: 105–108.
2. Marsh, D. G., J. D. Neely, D. R. Breazeale, B. Ghosh, L. R. Freidhoff,
E. Ehrlich-Kautzky, C. Schou, G. Krishnaswamy, and T. H. Beaty. 1994. Linkage
analysis of IL4 and other chromosome 5q31.1 markers and total serum immu-
noglobulin E concentrations. Science 264: 1152–1156.
3. Cookson, W. 1999. The alliance of genes and environment in asthma and allergy.
Nature 402: B5–B11.
4. Girolomoni, G., S. Sebastianai, C. Albanesi, and A. Cavani. 2001. T-cell sub-
populations in the development of atopic and contact allergy. Curr. Opin. Im-
munol. 13: 733–737.
5. Umetsu, D. T., J. J. McIntire, O. Akbari, C. Macaubas, and R. H. DeKruyff. 2002.
Asthma: an epidemic of dysregulated immunity. Nat. Immunol. 3: 715–720.
6. Shirakawa, T., T. Enomoto, S. Shimazu, and J. M. Hopkin. 1997. The inverse asso-
ciation between tuberculin responses and atopic disorder. Science 275: 77–79.
7. Stroffolini, T., F. Rosmini, L. Ferrigno, M. Fortini, R. D’Amelio, and
P. M. Matricardi. 1998. Prevalence of Helicobacter pylori infection in a cohort of
Italian military students. Epidemiol. Infect. 120: 151–155.
146HELMINTH-MODIFIED PULMONARY RESPONSE PREVENTS AHR
8. Matricardi, P. M., F. Rosmini, S. Riondino, M. Fortini, L. Ferrigno, M. Rapicetta,
and S. Bonini. 2000. Exposure to foodborne and orofecal microbes versus air-
borne viruses in relation to atopy and allergic asthma: epidemiological study.
BMJ 320: 412–417.
9. Yazdanbakhsh, M., P. G. Kremsner, and R. van Ree. 2002. Allergy, parasites, and
the hygiene hypothesis. Science 296: 490–494.
10. Fallon, P. G. 2000. Immunopathology of Schistosomiasis: a cautionary tale of
mice and men. Immunol. Today 21: 29–34.
11. Wang, C. C., T. J. Nolan, G. A. Schad, and D. Abraham. 2001. Infection of mice
with the helminth Strongyloides stercoralis suppresses pulmonary allergic re-
sponses to ovalbumin. Clin. Exp. Allergy 2 31: 495–503.
12. Negrao-Correa, D., M. R. Silveira, C. M. Borges, D. G. Souza, and
M. M. Teixeira. 2003. Changes in pulmonary function and parasite burden in rats
infected with Strongyloides venezuelensis concomitant with induction of allergic
airway inflammation. Infect. Immun. 71: 2607–2614.
13. Bashir, M. E., P. Andersen, I. J. Fuss, H. N. Shi, and C. Nagler-Anderson. 2002.
An enteric helminth infection protects against an allergic response to dietary
antigen. J. Immunol. 169: 3284–3292.
14. Wohlleben, G., C. Trujillo, J. Muller, Y. Ritze, S. Grunewald, U. Tatsch, and
K. J. Erb. 2004. Helminth infection modulates the development of allergen-in-
duced airway inflammation. Int. Immunol. 16: 585–596.
15. Mangan, N. E., R. E. Fallon, P. Smith, N. van Rooijen, A. N. McKenzie, and
P. G. Fallon. 2004. Helminth infection protects mice from anaphylaxis via IL-
10-producing B cells. J. Immunol. 173: 6346–6356.
16. Boyce, J. A., and K. F. Austen. 2005. No audible wheezing: nuggets and conun-
drums from mouse asthma models. J. Exp. Med. 201: 1869–1873.
17. McKenzie, G. J., C. L. Emson, S. E. Bell, S. Anderson, P. Fallon, G. Zurawski,
R. Murray, and A. N. J. McKenzie. 1998. Impaired development of Th2 cells in
IL-13-deficient mice. Immunity 9: 423–432.
18. Smith, P., C. M. Walsh, N. E. Mangan, R. E. Fallon, J. R. Sayers,
A. N. McKenzie, and P. G. Fallon. 2004. Schistosoma mansoni worms induce
anergy of T cells via selective up-regulation of programmed death ligand 1 on
macrophages. J. Immunol. 173: 1240–1248.
19. Van Rooijen, N., and A. Sanders. 1994. Liposome mediated depletion of mac-
rophages: mechanism of action, preparation of liposomes and applications. J. Im-
munol. Methods 174: 83–93.
20. Watanabe, J., Y. Miyazaki, G. A. Zimmerman, K. H. Albertine, and
T. M. McIntyre. 2003. Endotoxin contamination of ovalbumin suppresses murine
immunologic responses and development of airway hyper-reactivity. J. Biol.
Chem. 278: 42361–42368.
21. Valentinis, B., A. Bianchi, D. Zhou, A. Cipponi, F. Catalanotti, V. Russo, and
C. Traversari. 2005. Direct effects of polymyxin B on human dendritic cells
maturation: the role of I?B-?/NF-?B and ERK1/2 pathways and adhesion.
J. Biol. Chem. 280: 14264–14271.
22. Hamelmann, E., J. Schwarze, K. Takeda, A. Oshiba, G. L. Larsen, C. G. Irvin,
and E. W. Gelfand. 1997. Noninvasive measurement of airway responsiveness in
allergic mice using barometric plethysmography. Am. J. Respir. Crit. Care Med.
23. Martin, T. R., N. P. Gerard, S. J. Galli, and J. M. Drazen. 1988. Pulmonary
responses to bronchoconstrictor agonists in the mouse. J. Appl. Physiol. 64:
24. Schneider, T., and A. C. Issekutz. 1996. Quantitation of eosinophil and neutrophil
infiltration into rat lung by specific assays for eosinophil peroxidase and myelo-
peroxidase: application in a Brown Norway rat model of allergic pulmonary
inflammation. J. Immunol. Methods 198: 1–14.
25. Fallon, P. G., and D. W. Dunne. 1999. Tolerization of mice to Schistosoma
mansoni egg antigens causes elevated type 1 and diminished type 2 cytokine
responses and increased mortality in acute infection. J. Immunol. 162:
26. Grunig, G., M. Warnock, A. E. Wakil, R. Venkayya, F. Brombacher,
D. M. Rennick, D. Sheppard, M. Mohrs, D. D. Donaldson, R. M. Locksley, and
D. B. Corry. 1998. Requirement for IL-13 independently of IL-4 in experimental
asthma. Science 282: 2261–2263.
27. Townsend, M. J., P. G. Fallon, D. J. Matthews, P. Smith, H. E. Jolin, and
A. N. McKenzie. 2000. IL-9-deficient mice establish fundamental roles for IL-9
in pulmonary mastocytosis and goblet cell hyperplasia but not T cell develop-
ment. Immunity 13: 573–583.
28. Fallon, P. G., P. Smith, and D. W. Dunne. 1998. Type 1 and type 2 cytokine-
producing mouse CD4?and CD8?T cells in acute Schistosoma mansoni infec-
tion. Eur. J. Immunol. 28: 1408–1416.
29. van Rijt, L. S., H. Kuipers, N. Vos, D. Hijdra, H. C. Hoogsteden, and
B. N. Lambrecht. 2004. A rapid flow cytometric method for determining the
cellular composition of bronchoalveolar lavage fluid cells in mouse models of
asthma. J. Immunol. Methods 288: 111–121.
30. Fallon, P. G., E. J. Richardson, G. J. McKenzie, and A. N. McKenzie. 2000.
Schistosome infection of transgenic mice defines distinct and contrasting patho-
genic roles for IL-4 and IL-13: IL-13 is a profibrotic agent. J. Immunol. 164:
31. Wynn, T. A. 2003. IL-13 effector functions. Annu. Rev. Immunol. 21: 425–456.
32. Grzych, J. M., E. Pearce, A. Cheever, Z. A. Caulada, P. Caspar, S. Heiny,
F. Lewis, and A. Sher. 1991. Egg deposition is the major stimulus for the pro-
duction of Th2 cytokines in murine schistosomiasis mansoni. J. Immunol. 146:
33. Adler, A., G. Cieslewicz, and C. G. Irvin. 2004. Unrestrained plethysmography
is an unreliable measure of airway responsiveness in BALB/c and C57BL/6 mice.
J. Appl. Physiol. 97: 286–292.
34. Pearce, E. J., and A. S. MacDonald. 2002. The immunobiology of Schistosomi-
asis. Nat. Rev. Immunol. 2: 499–511.
35. Wills-Karp, M. 2000. Murine models of asthma in understanding immune dys-
regulation in human asthma. Immunopharmacology 48: 263–268.
36. Hesse, M., C. A. Piccirillo, Y. Belkaid, J. Prufer, M. Mentink-Kane, M. Leusink,
A. W. Cheever, E. M. Shevach, and T. A. Wynn. 2004. The pathogenesis of
schistosomiasis is controlled by cooperating IL-10-producing innate effector and
regulatory T cells. J. Immunol. 172: 3157–3166.
37. Platts-Mills, T. A., J. A. Woodfolk, E. A. Erwin, and R. Aalberse. 2004. Mechanisms
of tolerance to inhalant allergens: the relevance of a modified Th2 response to aller-
gens from domestic animals. Springer Semin. Immunopathol. 25: 271–279.
38. Butterworth, A. E., R. Bensted-Smith, A. Capron, M. Capron, P. R. Dalton,
D. W. Dunne, J. M. Grzych, H. C. Kariuki, J. Khalife, D. Koech, et al. 1987.
Immunity in human schistosomiasis mansoni: prevention by blocking antibodies
of the expression of immunity in young children. Parasitology 94: 281–300.
B. Migombet, S. Borrmann, D. Luckner, P. G. Kremsner, and M. Yazdanbakhsh.
2001. The prevalence of parasite infestation and house dust mite sensitization in
Gabonese schoolchildren. Int. Arch. Allergy Immunol. 126: 231–238.
40. Van Den Biggelaar, A. H., L. C. Rodrigues, R. Van Ree, J. S. Van Der Zee,
Y. C. Hoeksma-Kruize, J. H. Souverijn, M. A. Missinou, S. Borrmann,
P. G. Kremsner, and M. Yazdanbakhsh. 2004. Long-term treatment of intestinal
helminths increases mite skin-test reactivity in Gabonese schoolchildren. J. In-
fect. Dis. 189: 892–900.
41. Maizels, R. M., and M. Yazdanbakhsh. 2003. Immune regulation by helminth
parasites: cellular and molecular mechanisms. Nat. Rev. Immunol. 3: 733–744.
42. van Den Biggelaar, A. H., J. L. Grogan, Y. Filie, R. Jordens, P. G. Kremsner,
F. Koning, and M. Yazdanbakhsh. 2000. Chronic schistosomiasis: dendritic cells
generated from patients can overcome antigen-specific T cell hyporesponsive-
ness. J. Infect. Dis. 182: 260–265.
43. Moore, K. W., R. de Waal Malefyt, R. L. Coffman, and A. O’Garra. 2001. In-
terleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19: 683–765.
44. Stampfli, M. R., M. Cwiartka, B. U. Gajewska, D. Alvarez, S. A. Ritz,
M. D. Inman, Z. Xing, and M. Jordana. 1999. Interleukin-10 gene transfer to the
airway regulates allergic mucosal sensitization in mice. Am. J. Respir. Cell. Mol.
Biol. 21: 586–596.
45. Makela, M. J., A. Kanehiro, A. Dakhama, L. Borish, A. Joetham, R. Tripp, L,
Anderson, and E.W. Gelfand. 2002. The failure of interleukin-10-deficient mice
to develop airway hyper-responsiveness is overcome by respiratory syncytial
virus infection in allergen-sensitized/challenged mice. Am. J. Respir. Crit. Care
Med. 165: 824–831.
46. Hernandez Y., S. Arora, J. R. Erb-Downward, R. A. McDonald, G. B. Toews, and
G. B Huffnagle. 2005. Distinct roles for IL-4 and IL-10 in regulating T2 immu-
nity during allergic bronchopulmonary mycosis. J. Immunol. 174: 1027–1036.
47. Nakagome K, M. Dohi, K. Okunishi, Y. Komagata, K. Nagatani, R. Tanaka,
J. Miyazaki, and K. Yamamoto. 2005. In vivo IL-10 gene delivery suppresses
airway eosinophilia and hyperreactivity by down-regulating APC functions and
migration without impairing the antigen-specific systemic immune response in a
mouse model of allergic airway inflammation. J. Immunol. 174: 6955–6966.
48. Flores-Villanueva, P. O., X. X. Zheng, T. B. Strom, and M. J. Stadecker. 1996.
Recombinant IL-10 and IL-10/Fc treatment down-regulate egg antigen- specific
delayed hypersensitivity reactions and egg granuloma formation in schistosomi-
asis. J. Immunol. 156: 3315–3320.
49. Hoffmann, K. F., A. W. Cheever, and T. A. Wynn. 2000. IL-10 and the dangers
of immune polarization: excessive type 1 and type 2 cytokine responses induce
distinct forms of lethal immunopathology in murine schistosomiasis. J. Immunol.
50. Hunter, M. M., A. Wang, C. L. Hirota, and D. M. McKay. 2005. Neutralizing
anti-IL-10 antibody blocks the protective effect of tapeworm infection in a murine
model of chemically induced colitis. J. Immunol. 174: 7368–7375.
51. Kamradt, T., R. Goggel, and K. J. Erb. 2005. Induction, exacerbation and inhi-
bition of allergic and autoimmune diseases by infection. Trends Immunol. 26:
52. Oh, J. W., C. M. Seroogy, E. H. Meyer, O. Akbari, G. Berry, C. G. Fathman,
R. H. Dekruyff, and D. T. Umetsu. 2002. CD4 T-helper cells engineered to pro-
duce IL-10 prevent allergen-induced airway hyperreactivity and inflammation.
J. Allergy Clin. Immunol. 110: 460–468.
53. Zuany-Amorim, C., E. Sawicka, C. Manlius, A. Le Moine, L. R. Brunet,
D. M. Kemeny, G. Bowen, G. Rook, and C. Walker. 2002. Suppression of airway
eosinophilia by killed Mycobacterium vaccae-induced allergen-specific regula-
tory T-cells. Nat. Med. 8: 625–629.
54. Fillatreau, S., C. H. Sweenie, M. J. McGeachy, D. Gray, and S. M. Anderton.
2002. B cells regulate autoimmunity by provision of IL-10. Nat. Immunol. 3:
55. Mauri, C., D. Gray, N. Mushtaq, and M. Londei. 2003. Prevention of arthritis by
interleukin 10-producing B cells. J. Exp. Med. 197: 489–501.
56. Mizoguchi, A., E. Mizoguchi, H. Takedatsu, R. S. Blumberg, and A. K. Bhan. 2002.
Chronic intestinal inflammatory condition generates IL-10-producing regulatory B
cell subset characterized by CD1d upregulation. Immunity 16: 219–230.
57. Achtman, A. H., M. Khan, I. C. MacLennan, and J. Langhorne. 2003. Plasmodium
chabaudi chabaudi infection in mice induces strong B cell responses and striking but
temporary changes in splenic cell distribution. J. Immunol. 171: 317–324.
58. Velupillai, P., and D. A. Harn. 1994. Oligosaccharide-specific induction of interleu-
kin 10 production by B220?cells from schistosome-infected mice: a mechanism for
regulation of CD4?T-cell subsets. Proc. Natl. Acad. Sci. USA 91: 18–22.
147The Journal of Immunology