INFECTION AND IMMUNITY, Aug. 2008, p. 3511–3524
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 8
Hookworm-Induced Persistent Changes to the Immunological
Environment of the Lung?†
Joshua J. Reece,1Mark C. Siracusa,1Teresa L. Southard,2Cory F. Brayton,2
Joseph F. Urban, Jr.,3and Alan L. Scott1*
The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health,
Johns Hopkins University, Baltimore, Maryland 212051; Department of Molecular and Comparative Pathobiology, School of
Medicine, Johns Hopkins University, Baltimore, Maryland 212052; and Beltsville Human Nutrition Research Center,
Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 207053
Received 12 February 2008/Returned for modification 18 March 2008/Accepted 15 May 2008
A number of important helminth parasites of humans have incorporated short-term residence in the lungs
as an obligate phase of their life cycles. The significance of this transient pulmonary exposure to the infection
and immunity is not clear. Employing a rodent model of infection with hookworm (Nippostrongylus brasiliensis),
we characterized the long-term changes in the immunological status of the lungs induced by parasite infection.
At 36 days after infection, alterations included a sustained increase in the transcription of both Th2 and Th1
cytokines as well as a significant increase in the number and frequency of alveolar macrophages displaying an
alternatively activated phenotype. While N. brasiliensis did not induce alternate activation of lung macrophages
in STAT6?/?animals, the parasite did induce a robust Th17 response in the pulmonary environment,
suggesting that STAT6 signaling plays a role in modulating Th17 immunity and pathology in the lungs. In the
context of the cellular and molecular changes induced by N. brasiliensis infection, there was a significant
reduction in overall airway responsiveness and lung inflammation in response to allergen. In addition, the N.
brasiliensis-altered pulmonary environment showed dramatic alterations in the nature and number of genes
that were up- and downregulated in the lung in response to allergen challenge. The results demonstrate that
even a transient exposure to a helminth parasite can effect significant and protracted changes in the immu-
nological environment of the lung and that these complex molecular and cellular changes are likely to play a
role in modulating a subsequent allergen-induced inflammatory response.
Historically, an infection with one or multiple helminth par-
asites has been essentially universal for human populations. It
is estimated that a majority of living humans have a history of
or are currently infected with a helminth parasite, with the
highest prevalence of exposure in developing areas (11). A
number of important helminth parasites such as hookworm,
Ascaris, and Schistosoma have incorporated a short-term resi-
dence in the lungs as an obligate phase of their life cycle. The
significance of the lung phase to the developmental biology of
these parasites has not been clearly defined. Equally unclear is
the impact that the pulmonary phase has on the immunobiol-
ogy and pathogenesis of infection and the nature and extent to
which the immunological environment of the lung is altered by
these transient exposures. If significant changes to the pulmo-
nary environment do ensue, to what extent do they influence
the level and nature of the responses to subsequent challenges
from bacteria, fungi, viruses, and allergens?
In recent decades, the incidence and prevalence of allergy
and asthma have increased dramatically in industrialized na-
tions, with no comparable increase in developing regions of the
world (3, 9, 10, 39). Although a number of factors have been
proposed to explain this difference in disease prevalence in
developed and developing regions, including vaccination rates,
differential exposure to certain environmental and pathogenic
microorganisms, antibiotic usage, and general levels of hygiene
(63), there is a particularly strong negative correlation between
helminth infection and allergy (52, 67). Upon initial inspection,
this inverse relationship appears to present a paradox. Hel-
minth infections, like allergens, induce a robust Th2 response
characterized by interleukin-4 (IL-4), IL-13, and IL-5 produc-
tion. However, instead of potentiating allergy, a previous or
ongoing helminth infection significantly reduces the level of
reactivity to allergen challenge (60, 64, 65). While a role for
helminth-induced regulatory T cells has been established as a
component of the mechanism that modulates allergic reactivity
(64), it is likely that there are additional mechanisms involved
in regulating pulmonary immunity.
To study the long-term changes in the lungs that ensue from
helminth infection, we employed a mouse model of human
hookworm infection. The rodent hookworm Nippostrongylus
brasiliensis has been used extensively to study the regulation of
immunoglobulin E (IgE) synthesis (21) and Th2 immunity in
general, as it is one of the strongest inducers of a polarized Th2
immune response (1, 29, 48). The life cycle of N. brasiliensis
parallels that of its human counterparts Necator americanus
and Ancyclostoma duodenale. Briefly, infectious third-stage lar-
vae (L3) penetrate the skin, enter the circulation, and within
hours arrive in the lungs, where they reside for 18 to 24 h. After
molting, larvae migrate from alveoli up the trachea, are swal-
* Corresponding author. Mailing address: Department of Molecular
Microbiology and Immunology, Bloomberg School of Public Health,
Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD
21205. Phone: (410) 955-3430. Fax: (410) 955-0105. E-mail: ascott
† Supplemental material for this article may be found at http://iai
?Published ahead of print on 27 May 2008.
lowed, and develop into adult nematodes in the small intestine,
where they attach, feed, and reproduce. Healthy BALB/c mice
expel N. brasiliensis at 10 to 11 days postinfection (p.i.) through
an IL-13/STAT6-dependent mechanism (56). Almost immedi-
ately upon entry into the lungs, the larvae induce a strong
innate immune response characterized by the rapid production
of IL-4 and IL-13 and the alternative activation of alveolar
macrophages (43). Although the structural damage and in-
flammation quickly resolve, a number of the cellular and mo-
lecular changes induced by the transient presence of larvae
endure through day 12 p.i. (43). In the work reported here, we
characterized the nature of the persistent N. brasiliensis-in-
duced immunological changes that define a permanent change
to the immunological status of the lungs. This work represents
the first account of the protracted molecular and cellular
changes that take place in the lungs as a consequence of a
helminth infection and the role that these changes might have
in altering responses to environmental antigens.
MATERIALS AND METHODS
Animal model. (i) Helminth infection. Male BALB/cJ and STAT6?/?(on a
BALB/cJ background) mice, 6 to 8 weeks of age, were obtained from the Jackson
Laboratory (Bar Harbor, ME). Mice were serologically negative for 53 bacterial,
viral, fungal, and parasitic agents as tested by Jackson Laboratory surveillance
and sentinel monitoring through Johns Hopkins University. All experimental
procedures described in this paper were performed under the approval of the
Johns Hopkins University Animal Care and Use Committee in accordance with
the guidelines set out by the Institute of Laboratory Animal Resources 1996
Guide for the Care and Use of Laboratory Animals. Mice were housed in filter
top microisolator cages, provided food and water ad libitum, and kept on a
12-hour light/dark cycle. Infectious Nippostrongylus brasiliensis L3 were harvested
from a fecal culture via a Baermann apparatus, washed multiple times in phos-
phate-buffered saline (PBS), and counted. Mice were infected subcutaneously
with 500 L3.
(ii) Allergen sensitization and challenge. Mice were sensitized with two intra-
peritoneal injections of 75 allergy units of house dust mite (HDM) Dermatopha-
goides pteronyssinus allergen extract (Greer, Lenoir, NC) bound to 1 mg of Imject
aluminum hydroxide (Pierce, Rockford, IL) on days 21 and 25 post-N. brasiliensis
infection. Challenge doses of 50 allergy units HDM in 50 ?l PBS were admin-
istered intranasally on days 35 and 36 post-N. brasiliensis infection to anesthe-
tized (isofluorane; Abbott Laboratories, North Chicago, IL) animals. Control
animals received 50 ?l of lipopolysaccharide-free, injectable PBS.
(iii) Anesthesia and euthanasia. Prior to tissue harvest or histological prepa-
ration of lungs, mice were anesthetized with 300 mg/kg of Avertin (2,2,2-tribro-
moethanol) administered intraperitoneally. Euthanasia was performed by an
overdose of Avertin.
Histology. (i) Histological preparation. Lungs, inflated and fixed as outlined
previously (43), were embedded in paraffin, and sagittal 5-?m sections were
obtained from four different levels of the lung. Sections were stained with
hematoxylin and eosin (H&E) or by the periodic acid-Schiff (PAS) method (28)
to identify mucus-producing goblet cells. Selected sections were stained with
Prussian blue to confirm the presence of Ferric (Fe3?) ion consistent with
hemosiderin pigment (28).
(ii) Immunohistochemistry. YM1 staining was performed using affinity-puri-
fied goat anti-mouse YM1 (1 ?g/ml; R&D Systems, Minneapolis, MN). Anti-
YM1 was detected using a biotinylated rabbit anti-goat IgG (Vector Laborato-
ries), peroxidase strepavidin, and 3,3?-diaminobenzidine (Vector Laboratories).
YM1-positive cells were counted in each of six contiguous lung histological fields
(10? magnification; 974 mm2/field), and the mean cell number per field was
calculated. For dual-fluorescence staining of macrophages, YM1 was resolved
with fluorescein-avidin DCS (Vector Laboratories), and, after an avidin-biotin
blocking step (Vector Laboratories), CD11c (eBioscience, San Diego, CA) bind-
ing was detected with Texas red-avidin DCS. Stained sections were mounted in
Vectashield containing 4?,6-diamidino-2-phenylindole (DAPI) (Vector Labora-
tories) and examined using Nikon Eclipse series microscopes (Nikon Inc.,
Melville, NY) equipped with a Spot Flex charge-coupled device camera (Diag-
nostic Instruments Inc., Sterling Heights, MI).
(iii) Histological scoring system. Scoring was performed under blinded con-
ditions for the number of eosinophils, goblet cells, pigmented macrophages, and
neutrophils from three sections representative of three different strata of the
lung for each of three mice per treatment group. The scoring scale is detailed in
the legend to Fig. 5. Statistical significance was determined using a two-tailed
paired Student t test.
Gene expression analysis. (i) Total RNA extraction. Lungs were harvested,
flash frozen in liquid nitrogen, and stored at ?80°C. Lung RNA was isolated as
outlined by Reece et al. (43). RNA quality was determined by RNA Nano
LabChip analysis on an Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA). An
RNeasy total RNA cleanup protocol (Qiagen) was performed, followed by spec-
trophotometric assessment of RNA concentration. The lungs from three animals
were independently processed for each treatment group and each time point.
(ii) Affymetrix GeneChip protocols. Processing of templates and hybridization
for the 430 2.0 array GeneChip (Affymetrix Inc, Santa Clara, CA) were in
accordance with methods described in the Affymetrix GeneChip expression anal-
ysis technical manual, revision Three, as previously described (20). Following
hybridization, the GeneChips were washed and stained in an automated fluidics
station (Affymetrix FS450) and then assessed using the GCS3000 laser scanner
(Affymetrix) at an emission wavelength of 570 nm at 2.5-?m resolution. The
intensity of hybridization for each probe pair was computed by GCOS 1.2
software (Affymetrix). (For more detailed methods, refer to the website of the
Malaria Research Institute Gene Array Core Facility at the Johns Hopkins
Bloomberg School of Public Health [http://jhmmi.jhsph.edu]).
(iii) Data analysis. Affymetrix CEL file data was preprocessed for use with the
probe-level GeneChip robust multiarray analysis (66) option in GeneSpring 7
software (Agilent Technologies). Initial filtering by probe intensity for raw levels
above 150 in at least 1 out of 16 conditions resulted in a list of 14,159 genes that
were then used as the basis for selecting differentially regulated genes. Raw
intensity values ranged from 150 to 51,838.
(iv) Real-time RT-PCR. From each treatment group and time point, 1 ?g of
lung RNA was reverse transcribed using the SuperScript first-strand synthesis
system for reverse transcription-PCR (RT-PCR) (Invitrogen) with an oligo(dT)
primer. The resultant cDNAs were amplified for real-time detection with flu-
orogenically labeled probes in assays specific for each target gene. Quantitative
real-time RT-PCR was performed using the Applied Biosystems 7500 real-time
PCR system, TaqMan Gene Expression Assays-on-Demand, and TaqMan Uni-
versal Master Mix (Applied Biosystems, Foster City, CA). The following assays
(Applied Biosystems product numbers) were used: Arg1 (Mm00475988_m1),
CD11c (Mm00498698_m1), FIZZ1 (Mm00445109_m1), gamma interferon (IFN-?)
(Mm00801778_m1), IL-10 (Mm00438616_m1), IL-12 (Mm00434165_m1), IL-13
(Mm00434204_m1), IL-17a (Mm00439619_m1), IL-1? (Mm00434228_m1), IL-21
(Mm00517640_m1), IL-4 (Mm00445259_m1), IL-5 (Mm00439646_m1), IL-6
(Mm00446190_m1), inducible nitric oxide synthase (Mm00440485_m1), Mrc1
(Mm00485148_m1), transforming growth factor ? (TGF-?) (Mm00441724_m1),
Ym1 (Mm00657889_mH), and Ym2 (Mm00840870_m1). Reactions were per-
formed using 1 ?l of cDNA in a 25-?l sample volume and the following thermal
and then 15 seconds of denaturation at 95°C. Analysis was performed using the 7500
system SDS software package (Applied Biosystems).
Lung monocyte preparations. (i) Lung digestions. Animals were deeply anes-
thetized by overdose and tracheotomized, followed by a bronchial alveolar lavage
three times with 1 ml of room-temperature PBS. Lungs were infused with 10 ml
of room-temperature PBS, removed, and suspended in 5 ml of 1-mg/ml collage-
nase type II (Gibco, Carlsbad, CA) with 30 ?g/ml DNase I (Roche, Indianapolis,
IN) in RPMI 1640 (Gibco). Tissue was minced thoroughly, incubated at 37°C for
30 min, and then forced through a 3-ml transfer pipette 10 times. A collagenase-
DNase solution (3 ml) was added, and samples were incubated for an additional
15 min at 37°C. The resulting suspensions were manually homogenized using a
syringe plunger and passed through a 100-?m nylon cell strainer (BD Falcon,
Franklin Lakes, NJ). Cell suspensions were spun for 3 min at 1,500 ? g (4°C), and
pellets were resuspended in 5 ml of ACK lysing buffer (Quality Biological Inc.,
Gaithersburg, MD) for 5 min at room temperature. Suspensions were then
passed through a fresh cell strainer, and cells were washed twice in fluorescence-
activated cell sorting (FACS) staining buffer (PBS, 2% heat inactivated fetal calf
(ii) CDllc?cell isolation. Lung-derived cells were suspended in 200 ?l of
staining buffer containing Fc Block (BD Pharmingen, San Diego, CA) and
incubated on ice for 5 min. The cells were incubated with allophycocyanin-
conjugated anti-CD11c (Miltenyi Biotec, Auburn, CA) for 30 min on ice and
then washed twice with staining buffer. The cells were resuspended 50 ?l of BD
IMag-DM antiallophycocyanin particle solution (BD Pharmingen) and incu-
bated on ice for 30 min. CD11c isolation was performed according to the
3512REECE ET AL.INFECT. IMMUN.
manufacturer’s protocol using the BD IMag Cell Separation System (BD Phar-
FACS staining and detection: surface phenotyping. Cell counts were per-
formed on a standard hemacytometer with dead cells excluded by trypan blue
stain (Gibco). Cells were incubated with anti-major histocompatibility complex
(MHC) class II–phycoerythrin (PE) (eBioscience), anti-CD206–PE (Serotec,
Raleigh, NC), or anti-F4/80 plus 2° PE (Abcam, Cambridge, MA) in FACS
staining buffer for 30 min on ice in the dark. Data were acquired by running
samples on a FACSCalibur flow cytometer using Cell Quest software (BD Bio-
sciences, Mountain View, CA). Data were analyzed using FloJo software (Tree
Star, Inc., Ashland, OR).
In vivo pulmonary function measurement. Mice were deeply anesthetized with
a mixture of 70 mg/kg Ketamine and 14 mg/kg Xylazine (both from Phoenix
Pharm., St. Joseph, MO) delivered intraperitoneally. The trachea was exposed by
dissection and cannulated using a 19-gauge blunt needle. Animals were venti-
lated with a FlexiVent (Scireq, Montreal, Quebec, Canada) at a rate of 150/min
with a delivered tidal volume of 8 ml/kg and a positive-end expiratory pressure
of 3 cm H2O. To eliminate measurement noise associated with small respiratory
efforts, deeply anesthetized mice were paralyzed with 1 mg/kg succinylcholine
(Hospira Inc., Lake Forest, IL) intraperitoneally. Respiratory resistance and
elastance were calculated during a 2-second sinusoidal oscillation controlled by
the FlexiVent 5 software. Measurements were made at baseline and during
cumulative aerosolized methacholine (Sigma, St. Louis, MO) dose-response
curves, continuing until resistance doubled (8). The methacholine doses were
delivered into the inspiratory line, each for a 10-s period using an Aeroneb Pro
micropump ultrasonic nebulizer (Nektar Therapeutics, Mountain View, CA).
Measurements of resistance were expressed in absolute terms (cm H2O/ml/s) and
as percentages of the baseline values.
Statistical analyses. Microarray data, numerical histological data, and real-
time RT-PCR data were analyzed using a two-way analysis of variance
(ANOVA) with variables of treatment group and time. In vivo pulmonary func-
tion measurements of dose responses to methacholine were analyzed using a
two-way ANOVA with variables of infection status and methacholine dose.
Differences in baseline resistance and doubling dose of methacholine were cal-
culated using one-way ANOVAs. Significant interactions were further analyzed
using Bonferroni posttesting for pairwise multiple comparisons. All other data
were analyzed using a two-tailed Student t test for pairwise comparisons of
infected and uninfected animals. Differences were considered statistically signif-
icant if the P value was ?0.05.
GEO accession number. The data discussed in this publication have been
deposited in the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm
.nih.gov/geo) and are accessible through GEO series accession number
N. brasiliensis induces persistent changes in the lung. The
results of previous studies demonstrated that N. brasiliensis
larvae induce molecular and cellular changes in the lung that
are still evident days after the parasites have exited the pul-
monary environment (30, 43, 65). These observations sug-
gested that the transient presence of N. brasiliensis could result
in lasting changes in the immunological status of the lungs. To
test this, we evaluated the phenotypic and immunological sta-
tus of the lungs at 36 days p.i. (34 to 35 days after the larvae exit
the lungs and 25 to 26 days after the adults are expelled from
the intestine). Histologically, N. brasiliensis larvae caused a
disruption of the alveolar architecture of the lungs resulting in
focal emphysema-like changes (Fig. 1A and B). Although the
perivascular inflammation that was visible in the first 72 h p.i.
(reference 43 and data not shown) had resolved, areas of
peribronchiolar mononuclear infiltration were still evident at
36 days p.i. (Fig. 1D). In contrast to the lungs from noninfected
animals (Fig. 1E), the lungs from N. brasiliensis-infected ani-
mals contained a large number of conspicuous, heavily pig-
mented macrophages (Fig. 1F). Approximately 10 to 20% of
the bronchiolar epithelial cells were goblet cells at 36 days p.i.
(Fig. 1H), which was significantly more than the 1 to 2%
observed in noninfected controls (Fig. 1G) but below the
?30% present at 4 days p.i. (data not shown).
To gain an idea of the type of immunological changes that
were induced by N. brasiliensis at 36 days p.i., real-time RT-
PCR was used to measure the steady-state expression levels of
selected cytokines from infected and noninfected lungs. The
constitutive expression levels of the Th2 cytokines IL-4, IL-5,
and IL-13 were elevated in the lungs from N. brasiliensis-in-
fected animals compared to those of noninfected controls (Fig.
2). By way of comparison, the IL-4 expression level at day
36 p.i. was sixfold lower than the level at day 8 p.i., the peak of
the initial larva-induced inflammatory response in the lungs
(43, 49, 65). IL-13 and IL-5 expression levels at days 8 and
36 p.i. were comparable (data not shown). Consistent with
these observations, eotaxin/CCL11 transcription was also sig-
nificantly increased compared to that in control lungs. Inter-
estingly, the baseline transcription levels of the Th1 cytokines
IFN-?, IL-12p40, and IL-6 were also elevated in the lungs from
N. brasiliensis-infected mice. These expressions levels for
IFN-? and IL-6 at day 36 p.i. were 20-fold and 4-fold higher
than those measured at day 8 p.i., respectively (reference 43
FIG. 1. Cellular and structural changes in the lungs at 36 days
post-N. brasiliensis (Nb) infection. Light microscopy of the lungs from
uninfected (control) and N. brasiliensis-infected (day 36 p.i.) BALB/c
mice is shown. Histological analysis illustrates focal emphysema-like
lesions resulting from parasite migration (A and B) (magnification,
?10; H&E stain), peribronchial infiltration (Panels C & D, 40x mag-
nification, H&E stain), the presence of large alveolar macrophages
containing pigmented granules (E and F) (magnification, ?100; H&E
stain), and an increase in the number of goblet cells (H) (magnifica-
tion, ?60; PAS stain).
VOL. 76, 2008 NIPPOSTRONGYLUS-INDUCED PULMONARY CHANGES3513
and data not shown). The expression levels of IL-12p40 did not
significantly differ between days 8 and 36 p.i. Although the
expression level of IL-1? was elevated, the increase was not
statistically significant. Expression levels of the regulatory cy-
tokines IL-10 and TGF-? and the proinflammatory cytokine
IL-17 (47) were not elevated as a consequence of N. brasiliensis
infection. In contrast, IL-21 transcription (Fig. 2) was signifi-
cantly elevated after N. brasiliensis infection compared to that
in noninfected control lungs. The histological, cellular, and
transcriptional changes demonstrate that the brief exposure to
N. brasiliensis larvae effects persistent and substantive changes
to the immunological status of the lungs.
N. brasiliensis alters the baseline of gene expression in the
lung. In order to gain a more comprehensive picture of the
overall effect of helminth infection on the immunological sta-
tus of the lung 36 days p.i., microarray technology was em-
ployed to characterize expression profiles in the lungs from
infected and control animals. Roughly 120 genes were signifi-
cantly (P ? 0.05) differentially expressed in the lungs at day
36 p.i. compared to the lungs from age-matched controls (Ta-
ble 1; see Table S1 in the supplemental material). There was a
significant increase in a number of genes associated with lung
remodeling and a persistent increase in the expression of genes
that encode molecules important for macrophage function and
macrophage regulation, including class II MHC, CD11c,
PPAR gamma, MRP-1/ccl6, MCP-2/ccl8 Mac-1, and eosino-
phil-associated RNase (Ear2). Notably, there was a significant
upregulation of genes associated with alternatively activated
macrophages (AAMs), i.e., fizz1, ym1, and ym2, which was
validated by real-time RT-PCR (Table 1; Fig. 3A). Two addi-
tional genes associated with AAMs, those for arginase 1 (arg1)
and the mannose receptor 1 (mrc1, CD206), while not signif-
icantly elevated in the microarray results, were significantly
upregulated in real-time RT-PCR analysis (Fig. 3A).
N. brasiliensis induces a long-term increase in lung AAMs.
The large number of macrophage-associated genes upregu-
lated in day 36 p.i. lungs along with the enhanced expression of
the genes encoding YM1, YM2, FIZZ1, and ARG1 predicted
an elevation in the number of AAMs. Employing immunohis-
tochemical analysis to identify YM1?cells, it was confirmed
that there was a significant (P ? 0.0001) increase in the num-
ber of AAMs at 36 days p.i. (Fig. 3C). In uninfected controls,
?5% of the alveolar macrophages were YM1?, while in day
36 p.i. lungs, ?75% were YM1?(data not shown). The YM1?
cells, which stained positive for CD11c (Fig. 3D) and F4/80?
(Fig. 3G), were distributed evenly throughout the parenchyma
of the lung (Fig. 3B). The increase in CD11c mRNA (Table 1;
Fig. 3A) correlated with an increase in CD11c protein levels on
the surface of lung cells (Fig. 3E). Also consistent with the
transcriptional data, N. brasiliensis infection increased the ex-
pression of class II MHC and the mannose receptor C type
lectin 1 (Mrc1/CD206) (Fig. 3F). The cellular and molecular
changes seen at day 36 p.i. show that pulmonary migration by
N. brasiliensis induces a persistent population of AAMs in the
STAT6 signaling and the Th1-Th2-Th17 axis. The persistent
production of IL-4 and IL-13 in the pulmonary environment by
N. brasiliensis infection (Fig. 2) suggests that sustained STAT6
signaling could play a key role in the maintenance of the
altered immunological phenotype of the day 36 p.i. lungs. We
compared the expression profile in the lungs from STAT6?/?
mice at day 36 p.i. to that in lungs from wild-type (WT) animals
to gain an insight into the role of STAT6 signaling in the
post-N. brasiliensis lung. In uninfected control STAT6?/?and
WT lungs, the expression levels of genes encoding AAM-as-
sociated and selected cytokines were equivalent (Fig. 4). Con-
sistent with previous reports, the mechanisms for the induction
and maintenance of the AAM phenotype (27, 59) and for
expression of eotaxin (31) were dependent on STAT6 signal-
ing. Interestingly, although the expression of ym1 and fizz1 in
STAT6?/?lungs was not significantly different from the levels
in uninfected control animals, arg1 expression was elevated
and essentially identical to the levels induced by N. brasiliensis
in WT lungs. The apparent division in the regulation of ym1/
fizz1 and arg1 suggests that other signaling pathways are im-
portant to the regulation of the full AAM phenotype.
The expression profiles of Th2 cytokines (IL-4, IL-13, and
IL-5), Th1 cytokines (IL-12 and IFN-?), and IL-10 and IL-1?
in STAT6?/?and WT lungs at day 36 p.i. were not significantly
different (Fig. 4). Importantly, in the absence of STAT6 sig-
naling, the post-N. brasiliensis lungs appeared to mount a
strong Th17 response. With the exception of TGF-? (Fig. 4)
and IL-23 (data not shown), which were both detectable but
FIG. 2. N. brasiliensis (Nb) induces an altered cytokine environ-
ment. Lungs from mice at 36 days p.i. were harvested and snap frozen
in liquid nitrogen, RNA was extracted using Trizol, and first-strand
DNA was synthesized. Real-time RT-PCR analysis of whole lungs for
Th1, Th2, and regulatory cytokines, as well as eotaxin (CCL11) and
IL-21, is shown. Bars represent the mean levels from five mice ?
standard errors of the means.*, P ? 0.05;**, P ? 0.01.
3514 REECE ET AL.INFECT. IMMUN.
not significantly elevated, the expression of genes encoding
Th17 effector (IL-17) and regulatory (IL-6 and IL-21) mole-
cules was significantly upregulated in infected STAT6?/?lungs
compared to infected WT lungs (Fig. 4). Thus, signaling
through STAT6 appears to contribute to the mechanism that
regulates expression of Th17 immunity in the post-N. brasil-
Histological analysis of lungs from day 36 p.i. STAT6?/?
animals revealed levels of alveolar destruction and peribron-
cular cellular infiltration similar to that observed in day 36 p.i.
WT animals (Fig. 1 and data not shown). The cellular infil-
trates were composed of lymphocytes, with no evidence of
neutrophil accumulation. The STAT6?/?animals had no evi-
dence of bronchial epithelial or goblet cell hyperplasia. Mac-
rophages with the granular morphology were also evident in
the day 36 p.i. STAT6?/?lungs, indicating that the develop-
ment of this phenotype is independent of alternative activa-
Allergen-induced inflammation is reduced in the post-N.
brasiliensis lung. Results of previous studies demonstrated that
helminth infection, including infection with N. brasiliensis, re-
sulted in a modulation in the inflammation resulting from a
subsequent allergen challenge (65). To determine if the N.
brasiliensis-mediated changes in the immunological status of
the lungs observed here also correlated with the capacity to
modulate responses to a subsequent challenge, day 36 p.i. mice
were sensitized and challenged with a clinically relevant aller-
gen, HDM antigen. Lungs were collected at 6, 24, and 72 h
after secondary HDM challenge (Fig. 5A) and processed for
either histology or RNA extraction. Pulmonary challenge of
sensitized BALB/c mice (referred to below as HDM mice) with
HDM resulted in a rapid and dramatic increase in eosinophil
infiltration (Fig. 5B and N) as previously reported (68). At 6
and 24 h after HDM challenge, the lungs from the HDM-
sensitized mice with a history of N. brasiliensis infection (re-
ferred to below as N. brasiliensis-HDM mice) had significantly
lower levels of eosinophil infiltration in perivascular regions
(Fig. 5 B to G and N). At 72 h, although the lungs from the N.
brasiliensis-HDM mice maintained lower levels of perivascular
eosinophils, the differences between HDM and N. brasiliensis-
HDM mice were no longer statistically significantly different
In contrast to the case for eosinophils, at 6 h after allergen
challenge, the large airways from the N. brasiliensis-HDM mice
had elevated levels of goblet cells compared to the airways
from HDM animals (Fig. 5B and E). A comparable increase in
goblet cells at 6 h was also seen in the N. brasiliensis-infected
animals given PBS (Fig. 5K). This increase in goblet cells was
not demonstrable 18 h later (Fig. 5L). Goblet cell numbers
increased in both N. brasiliensis-HDM and HDM lungs at 24 h
TABLE 1. N. brasiliensis alters baseline gene expression in the lung
Category and Affymetrix
Change in gene
Resistin like alpha
Chitinase 3-like 3
Chitinase 3-like 4
Complement component 1, q subcomponent
Itgax, integrin alpha X
Clecsf10, C-type lectin domain fam 4, member n
Eosinophil-associated, ribonuclease A
Fc receptor, IgG, low affinity IIb
Lipocalin 2, neutrophil gelatinase-assoc lipocalin
Itgb2, integrin beta 2
CD68, Mac marker; late endosomal
Mannose receptor, C type 2
Peroxisome proliferator activated receptor gamma
MHC class II, beta chain
B2M-associated MHC class I molecule
MRP-1, chemokine (C-C motif) ligand 6
MCP-2, chemokine (C-C motif) ligand 8
Eotaxin, chemokine (C-C motif) ligand 11
Actin, alpha 2, cytoskeleton biogenesis
Cathepsin K, extracellular matrix proteolysis
Procollagen, type XIV, alpha 1, extracellular
Gap junction membrane channel protein
Matrix metalloproteinase 2
a??, 2- to 3.9-fold change; ???, 4- to 9.9-fold change; ????, ?10-fold change.
VOL. 76, 2008 NIPPOSTRONGYLUS-INDUCED PULMONARY CHANGES3515
postchallenge to equivalent levels and remained elevated
through 72 h (Fig. 5C, D, F, and G). Only the lungs from N.
brasiliensis-infected mice contained large pigmented macro-
phages. Neutrophil infiltration into the lungs was minimal for
all of the test and control groups.
FIG. 3. The N. brasiliensis (Nb)-altered pulmonary environment is
characterized by a sustained increase in CD11c?AAMs. (A) Real-
time RT-PCR was used to measure the transcript levels of the genes
encoding the AAM-associated proteins ARG1, YM1, YM2, FIZZ1,
CD11c, and CD206 in RNA isolated from whole lungs of uninfected
control and day 36 p.i. (Nb?) BALB/c mice. Each bar represents the
mean of five mice ? the standard error of the mean. (B) Lung tissue
sections were immunostained with anti-YM1, and antibody binding
was detected using the chromogen 3,3?-diaminobenzidine and coun-
terstaining with H&E. YM1?cells are shown with arrowheads. Mag-
nification, ?10. (C) Quantification of YM1-expressing cells was calcu-
lated from six contiguous fields for triplicate mice and expressed as the
number of cells per unit area. (D) Colocalization of CD11c (Texas red)
and YM1 (fluorescein) in alveolar macrophages from N. brasiliensis-
infected animals. Nuclei were stained with DAPI, and images were
acquired using a SPOT charge-coupled device camera and software.
Cells were visualized at a magnification of ?100. (E) FLOW analysis
was used to measure the change in the levels of CD11c on the surface
of macrophages isolated from whole-lung digests from uninfected and
day 36 p.i. mice (see Materials and Methods). (F) FLOW analysis of
the changes in MHC class II and CD206 (Mrc1) expression on the
surface of CD11c?cells isolated from the lungs of control and day
36 p.i. mice. (G) FLOW analysis of F4/80 expression on the CD11c?
cells from the lungs of day 36 p.i. mice. The FLOW results are repre-
sentative of the results from five animals per group.
FIG. 4. N. brasiliensis (Nb)-induced changes in the pulmonary im-
mune environment is mediated partially by STAT6 signaling. RNA was
isolated from the lungs of STAT6?/?and WT mice at day 36 post-N.
brasiliensis infection, and real-time RT-PCR was used to assess the
expression levels of selected cytokines and genes associated with in-
flammation and alternative activation of macrophages. The results
were expressed in relative expression units, and each bar represents the
mean threshold cycle value from five animals (? standard error of the
mean). Horizontal bars represent the mean values of expression levels
obtained from uninfected STAT6?/?and WT lungs. Statistical com-
parisons were made using a two-tailed Student t test. Significance
between STAT6?/?and WT expression is designated by brackets, and
significance between expression in infected and uninfected STAT6?/?
or WT lungs is designated by asterisks within the bars (*, P ? 0.05;**,
P ? 0.01).
3516REECE ET AL.INFECT. IMMUN.
Altered gene expression in response to allergen challenge.
Lungs from mice at 6, 24, and 72 h post-HDM challenge were
processed for microarray-based gene expression analysis.
When the dynamics of gene expression at all three time points
are taken into consideration, the total numbers of genes sig-
nificantly upregulated in the lungs for at least one time point
from HDM and N. brasiliensis-HDM mice were virtually iden-
tical (486 versus 480) (Fig. 6A and B). However, examination
of the specific genes that were upregulated revealed that only
?20% (n ? 99) were common to both the HDM and the N.
brasiliensis-HDM gene lists (Fig. 6A). In contrast, lungs from
N. brasiliensis-HDM mice significantly downregulated over
twice the number of genes that were downregulated in the
lungs of the HDM-only mice (Fig. 6C). While 25% of the 226
genes significantly downregulated in HDM lungs were com-
mon to those in N. brasiliensis-HDM lungs, only ?12% (n ?
58) of the 464 genes significantly downregulated in N. brasil-
iensis-HDM lungs were in common with the HDM expression
profile. Thus, N. brasiliensis-induced modulation of the immu-
nological status of the lungs results in a dramatic change in the
character of the response to allergen challenge.
Utilizing a simplified gene ontology classification system, the
genes differentially expressed in HDM- and N. brasiliensis-
HDM-challenged lungs were grouped into functional catego-
ries (Fig. 6B and D). Approximately 30% of the genes signif-
icantly upregulated in the HDM lungs encoded proteins
associated with immune activity including genes encoding pro-
teins expressed by granulocytes, B cells, and T cells (Table 2;
see Table S2 in the supplemental material). In contrast, only
?10% of the genes upregulated in the N. brasiliensis-HDM
lungs were immunity associated. Conversely, significantly more
immunity-associated genes were downregulated in the N. bra-
siliensis-HDM lungs compared to the HDM lungs (Fig. 6D).
This pattern seen for the immunity-associated genes of upregu-
lation in HDM lungs and downregulation in N. brasiliensis-
HDM lungs was maintained for the other functional categories
except for genes encoding proteins predicted to bind to nucleic
acids (Fig. 6B and D).
increases in the transcription of genes encoding cytokines and
chemokines (Fig. 7) identified in the array results from HDM
challenge and control animals. At 6 and 24 h post-allergen chal-
the Th2 cytokines IL-4, IL-5, and IL-13 and the chemokine
CCL11 (eotaxin) were significantly higher in HDM lungs than in
the lungs from N. brasiliensis-HDM mice. The differences in the
expression of these cytokines in the HDM and N. brasiliensis-
HDM lungs were maintained through 72 h postchallenge, but the
differences were no longer statistically significant. N. brasiliensis-
HDM mice significantly downregulated the expression of IL-12,
FIG. 5. N. brasiliensis (Nb) infection dampens allergen-induced in-
flammation. (A) Schedule for HDM (Der p) sensitization and chal-
lenge of uninfected or N. brasiliensis-infected BALB/c mice. (B to M)
Lungs were harvested, fixed, and sectioned for histological examina-
tion at 6, 24, or 72 h after the second challenge dose of HDM. The
upper portions of the panels were stained with H&E, the lower parts
were stained with PAS, and both were photographed at a magnifica-
tion of ?60. HDM, uninfected mice sensitized and challenged with
Der p; NbHDM, day 36 p.i. mice sensitized and challenged with Der p;
PBS; uninfected mice exposed to PBS; NbPBS, day 36 p.i. mice ex-
posed to PBS. (N) Quantification of the eosinophil infiltrate. Eosino-
phil scoring was performed under blinded conditions from three sec-
tions representative of three different strata of the lung for each of
three mice per treatment group. Scoring was as follows: 0, no eosino-
phils; 0.5, scattered eosinophils throughout the lung; 1.0, 10 to 40% of
the perivascular infiltrate; 1.5, 50 to 60% of the perivascular infiltrate;
2.0, ?70% of the perivascular infiltrate.
VOL. 76, 2008 NIPPOSTRONGYLUS-INDUCED PULMONARY CHANGES 3517
IFN-?, and IL-1? (Fig. 7), whereas HDM mice showed two- to
expression was modestly elevated in the lungs of HDM animals
compared to the N. brasiliensis-HDM animals. There was no
significant change in the expression levels of TGF-? in any of the
test or control groups. IL-21 transcription was significantly in-
creased in the lungs from both uninfected and N. brasiliensis-
infected animals at all time points after allergen challenge. In
contrast to the nearly undetectable levels of IL-17 expression in
the lungs of the N. brasiliensis-HDM group at 6 h postchallenge,
there was a rapid, ?50-fold induction of IL-17 transcription in the
lungs of the HDM mice (Fig. 7). By 24 h post-HDM challenge,
the IL-17 expression levels for both groups were essentially equal.
Also of interest are the responses in the uninfected and previ-
ously N. brasiliensis-infected groups challenged with PBS. While
the control PBS challenges caused no change in transcription
levels of a majority of the cytokines and chemokines tested, the
transcription of genes encoding IL-17, IL-21, and IL-6 was signif-
icantly increased at 6 and 24 h after administering PBS (Fig. 7).
For the most part, this increase in gene transcription was ob-
served only in the uninfected mice. Those mice previously in-
fected with N. brasiliensis demonstrated no significant increase in
transcription of these cytokines. This trend for a rapid upregula-
tion of Th17-associated cytokines in the lungs of uninfected ani-
mals suggests that a Th17 response may be an important compo-
nent of the immediate reaction to lung damage or antigen
exposure. An antecedent N. brasiliensis infection appears to sig-
nificantly modulate the immediate Th17 response.
N. brasiliensis alters airway responsiveness. In vivo assess-
ment of the differences in pulmonary function between in-
fected and uninfected lungs was performed using anesthetized,
paralyzed, and intubated mice on a ventilator. At 36 days p.i.,
N. brasiliensis-infected animals showed a statistically signifi-
cantly higher baseline resistance (P ? 0.0006) (Fig. 8A), and
the amount of methacholine necessary to double the baseline
resistance in N. brasiliensis-infected mice was ?4-fold lower
than that required for the airways of uninfected control mice
(P ? 0.008) (Fig. 8B). Correspondingly, dose responses to
methacholine were significantly higher in parasitized lungs
(Fig. 8C). Thus, N. brasiliensis infection resulted in a significant
increase in airway hyperresponsiveness.
Paradoxically, N. brasiliensis infection resulted in a reduction in
airway responsiveness after challenge with an allergen (Fig. 8D to
F). Baseline resistance (no methacholine) in the lungs of N. bra-
siliensis-HDM mice was essentially identical to that in controls,
whereas HDM mice had an elevated baseline resistance (Fig.
8D). The mean dose of methacholine necessary for doubling
airway resistance in HDM mice was about half that of control
animals and only ?25% the dose needed for N. brasiliensis-HDM
mice (Fig. 8E). While N. brasiliensis-HDM mice clearly main-
tained lower airway resistance to methacholine throughout the
dose-response window compared to the HDM and control mice
(Fig. 8F), the differences were statistically different only at the
10-mg/ml dose of methacholine. Thus, although N. brasiliensis
infection resulted in heightened baseline airway reactivity,
there was a dramatic reduction in the level of airway re-
sponsiveness in N. brasiliensis-infected animals after a sec-
ondary challenge with HDM.
FIG. 6. N. brasiliensis (Nb) modifies global gene responses to aller-
gen challenge. Affymetrix chip-based whole-genome expression anal-
ysis was used to analyze the transcriptional responses of lungs at 6, 24,
and 72 h post-allergen challenge. Changes above or below twofold
were considered significant. Fold changes for the HDM and N. brasil-
iensis-HDM groups were calculated based on the transcriptional levels
measured in age-matched uninfected and N. brasiliensis-infected (day
36 p.i.) control lungs. Expression levels are represented by the means
for three mice/group/time point. (A and C) Venn diagrams showing
the total numbers of genes upregulated (A) and downregulated (C) at
6, 24, and 72 h post-allergen challenge in the lungs from N. brasiliensis-
HDM and HDM mice. (B and D) A simplified gene ontology break-
down of the upregulated genes (B) and downregulated genes (D) in
the lungs from N. brasiliensis-HDM and HDM mice. The graphs in
panels B and D include only the subset of up- and downregulated
genes that were clearly classifiable into the 10 GO terms used.
3518 REECE ET AL.INFECT. IMMUN.
The N. brasiliensis-induced alterations at 36 days p.i. in-
cluded an increase in the constitutive transcription of both Th2
(IL-4, IL-13, and IL-5) and Th1 (IFN-? and IL-12) cytokines
but not that of the traditional regulatory cytokines IL-10 and
TGF-?. The idea that this alteration in cytokine levels might be
a permanent feature in the lungs is supported by the fact that
it is still detectable at 100 days p.i. (data not shown). The exact
TABLE 2. Gene expression in HDM-challenged uninfected and N. brasiliensis-infected mice
Category and Affymetrix
Change in gene expression (fold)aat the indicated
time (h) postchallenge
N. brasiliensis-HDM HDM
6 24 726 24 72
Complement component 1q gamma
Eosinophil-associated, RNase 2
Eosinophil-associated, RNase 3
Complement factor B; C3/C5
Granzyme A; serine-type
1417898_a_at Gzma NM_010370
Surface markers and
Macrosialin, Mac marker; late
B-cell marker; antigen binding
DC marker; CD4 T-cell stimulator
Dectin 2, C-type lectin domain
Immunoreceptor with ITIM
CD115; macrophage colony-
stimulating factor receptor
colony-stimulating factor receptor
CD114; granulocyte colony-
stimulating factor receptor
High-affinity IgE receptor
Mac-2; IgE binding surface lectin
Mannose binding receptor 1
? ???? ???
1455660_atCsf2r?1 BB769628 NC NCNC
?? ?? ??
Cytokine and chemokine
?? ?? ??
RANTES; cytokine, chemokine
Secretoglobin; cytokine activity
Suppressor of cytokine signaling 3
IgG1 heavy chain
IgM heavy chain
Immunoglobulin joining chain
Immunoglobulin lambda chain,
CD300lf; polymeric immunoglobulin
Secreted phosphoprotein 1, T-cell
Schlafen 4; thymocyte regulatory gene
T-cell receptor alpha chain
T-cell receptor beta, joining region
T-cell receptor beta, variable 13
CD90, thymus cell antigen 1, theta
a????, ?10-fold change; ???, 4- to 9.9-fold change; ??, 2- to 3.9-fold change; ?, 1.5- to 1.9-fold change; NC, no change (0.6- to 1.4-fold change); ?, 0.5- to
0.66-fold change; ??, 0.25- to 0.49-fold change; ?, fluorescence signal of ?150.
VOL. 76, 2008 NIPPOSTRONGYLUS-INDUCED PULMONARY CHANGES 3519
significance of this altered balance between Th2 and Th1 cy-
tokines in the lungs is not clear. Aspects of the persistent
changes in cytokine production appear to be an extension of
the strong Th2-biased innate and early adaptive responses to
the presence of the larvae in the lungs (43). The constitutive
elevation of Th1 cytokine expression might be in response to
the presence of elevated levels of Th2 cytokines and reflect
efforts to control the extent of the Th2 reactivity or other
pathogenic mechanisms in the lung.
The results from infections in STAT6?/?animals suggest
that there is a mechanistic connection between persistent IL-4
and IL-13 expression and at least some aspects of the altered
immunological environment in the day 36 p.i. lung. In the
absence of STAT6 signaling, there was a significant decrease in
the transcription of ym1 and fizz1 (Fig. 4), signature molecules
of alterative macrophage activation. The development of the
alternatively activated phenotype has been shown previously to
be dependent on STAT6 (27, 59), and the genes encoding
YM1 and FIZZ1 have STAT6 response elements in their pro-
moter regions that combine with other transcription factors to
promote efficient expression (25, 53, 62, 69). Although the
results of studies indicate that arg1 transcription is also con-
trolled by a STAT6 response element (15, 69), arg1 expression
was significantly elevated in the day 36 p.i. STAT6?/?lungs to
a level comparable to that measured in the day 36 p.i. WT
lungs. The results presented here suggest that there is at least
one STAT6-independent pathway capable of regulating arg1
transcription in the lungs. The presence of IL-4 and IL-13 in
STAT6-deficient animals still allows for the possibility of sig-
naling through the IL-4 receptor (IL-4R) utilizing the STAT6-
independent insulin receptor substrate 1 and 2 pathway (37). It
is possible that insulin receptor substrate 1 and 2 signaling
alone or in conjunction with other dominant signals in the
infectious environment is sufficient to induce increased arg-1
expression in STAT6-deficient animals.
In the absence of STAT6 signaling, the post-N. brasiliensis
lung had a dramatic increase in the transcription of IL-6, IL-21,
and IL-17, an expression pattern consistent with the develop-
ment of a Th17 response (23, 38). Th17 differentiation from
naı ¨ve precursors is initiated by IL-6, TGF-? and IL-21 and
reinforced by IL-23 (38, 57), and transcripts for all four of
these cytokines were present in the day 36 p.i. STAT6?/?lungs
(Fig. 4 and data not shown). In the lungs from WT animals,
only low levels of IL-17 could be detected (Fig. 2). This sup-
pression of Th17 development in WT mice was presumably
due to the elevated production of IL-4 and IFN-? (Fig. 2 and
4). The presence of both IL-4 and IFN-? is required to sup-
press the development of Th17 cells from naı ¨ve precursor cells
and may represent a key mechanism by which N. brasiliensis
infection modulates allergic responsiveness in the pulmonary
FIG. 7. N. brasiliensis (Nb) alters immune responses to allergen challenge and reduces airway resistance. Real-time RT-PCR was used to
measure gene expression in the whole lungs removed from control or N. brasiliensis-infected mice at 6, 24, or 72 h postchallenge with either HDM
or PBS. Fold change calculations for the N. brasiliensis-HDM and N. brasiliensis-PBS groups were made based on the gene expression levels of N.
brasiliensis-infected lungs at day 36 p.i. Fold change calculations for the HDM and PBS groups were based on expression levels of age-matched
uninfected controls. Points represent the mean expression levels for five mice/group/time point. Error bars represent the standard errors of the
means. Statistical comparisons were generated by a two-way ANOVA followed by Bonferroni posttesting. Comparison of HDM versus N.
brasiliensis-HDM:*, P ? 0.05;**, P ? 0.01;***, P ? 0.001. Comparison of HDM versus PBS: †, P ? 0.05; ††, P ? 0.01; †††, P ? 0.001.
Comparison of PBS versus N. brasiliensis-PBS: §, P ? 0.05.
3520REECE ET AL.INFECT. IMMUN.
environment. (41). The development of a Th17 response in
the N. brasiliensis-infected STAT6?/?lungs despite elevated
IFN-? transcription is likely due to the inability to send the
necessary second, IL-4-mediated signal to T cells. Thus, it is
possible that the functional significance of the increase in both
Th1 and Th2 cytokines in the post-N. brasiliensis lung is to
regulate the level of Th17 development and thus minimize
tissue damage during subsequent pulmonary responses.
IL-17 transcription was robustly induced by challenge with
HDM allergen. In model systems, IL-17 is strongly associated
with tissue damage in the brain, heart, synovium, skin, and
intestines (reviewed in reference 51). The role that IL-17 plays
in the lungs during allergic responses is still not clear. While
constitutive overexpression of IL-17 in lung endothelial cells
result in a peribronchiolar cellular infiltration and mucus pro-
duction (41), IL-17 administered during the chronic phase of
an allergic response results in a reduction in eosinophilia and
a drop in the reactivity of the large airways (46). The over-50-
fold induction of IL-17 at 6 h post-HDM challenge suggests
that IL-17 can also play a significant role during the immediate/
early response after allergen challenge. However, it is still
unclear whether the HDM-induced Th17 response contributes
to the pathology or if it is part of the mechanism to control the
level of damage. This IL-17 induction appears to occur inde-
pendent of discernible increases in IL-23 transcription (data
not shown). Th17 cells induced in the absence of IL-23 have
been shown to express IL-10 and to restrain cell-mediated
pathology in the central nervous system (33). It is possible that
the IL-17-producing cells were also the source of the elevation
in IL-10 transcription in the HDM-challenged animals (Fig. 7).
IL-10/IL-17-expressing cells could also account for the limited
pathology observed in day 36 p.i. STAT6?/?lungs.
The specific cellular source(s) of the transcripts encoding
Th2 and Th1 cytokines at day 36 p.i. remains to be defined. The
results for day 36 p.i. lungs from the bicistronic IL-4 reporter
(4get ) mice indicate that the IL-4 was produced primarily
by T cells with some contribution from granulocytic cells (data
not shown). Infection with N. brasiliensis is known to recruit
IL-4- and IL-13-producing cells into the lungs, including
eosinophils, basophils, and CD4?Th2 cells (13, 32, 34, 58), and
these cells are the most logical sources of the elevated tran-
scription of Th2 cytokine genes. The demonstrable elevation in
the constitutive transcription of both eotaxin and IL-5 at day
36 p.i. compared to that in uninfected, age-matched controls
suggested that eosinophils might be a major source of IL-4 and
IL-13. However, histological analysis of day 36 p.i. lungs indi-
cated no obvious increase in the number of eosinophils. It has
been reported previously that several days after N. brasiliensis
larvae exit the lungs there is significant population of IL-4-
producing, partially degranulated eosinophils (49). It is possi-
ble that these partially degranulated cells persist through to
day 36 p.i. but were not readily identifiable in the histological
While it has been demonstrated that transcription of Th2
cytokines IL-4 and IL-13 can be uncoupled from protein pro-
duction (13), for Th1 cytokines, including IFN-? (32), tran-
scription appears to be a more reliable surrogate of protein
synthesis. Thus, it is possible that the increases in Th2 cytokine
transcription measured in the post-N. brasiliensis lungs do not
reflect a commensurate increase in the production of the active
FIG. 8. N. brasiliensis (Nb) infection alters airway responsiveness. (A to
resistance was calculated and expressed as cm H2O ? s/ml. (B) Methacholine
resistance was measured. The amount of MCh required to double the base-
line resistance is shown. (C) Dose responses to MCh were normalized to a
baseline resistance of 100. (D to E) N. brasiliensis-infected and control mice
were sensitized and challenged with HDM as outlined in Materials and
Methods. At 24 h after the second challenge, mice were sedated, intubated,
and placed on a ventilator to test their responsiveness to MCh. (D) Baseline
resistance in the lung before MCh challenge, expressed as cm H2O ? s/ml.
(E) Dose of MCh required to double the baseline resistance in the lungs.
the means. Two-way ANOVA analysis followed by Bonferroni posttesting
VOL. 76, 2008 NIPPOSTRONGYLUS-INDUCED PULMONARY CHANGES3521
cytokine. Attempts to measure differences in the protein levels
of the Th1 and Th2 cytokines in lung homogenates and in
bronchial alveolar lavage fluids from control and day 36 p.i.
animals proved to be problematic. The maintenance of the
AAM phenotype, a phenotype that is dependent on Th2 cyto-
kines (14), suggests that biologically active IL-4 and/or IL-13 is
being produced. It has been shown that mature mast cells,
eosinophils, and basophils are programmed for IL-4 and IL-13
transcription early in development, but these cells do not pro-
duce protein until they are properly activated (13). It is possi-
ble that the elevated transcription described here represents, in
part, a heightened capacity for the lung to rapidly respond to
N. brasiliensis infection resulted in a significant and persis-
tent upregulation of the gene encoding IL-21 (in the absence
of IL-17 and IL-23) in the lungs at day 36 p.i. IL-21 is a
member of the IL-2/IL-4/IL-15 family of cytokines (16), which
bind to a class 1 cytokine receptor that utilizes the common ?
chain for functional signaling (40). IL-21 is produced mainly by
antigen-activated CD4?T cells and the IL-21R; while found
on a broad spectrum of cell types, is preferentially expressed
on T cells, B cells, NK cells, keratinocytes, and cells of the
myeloid lineage (reviewed in reference 24). Although IL-21 is
known to contribute to the induction of Th17 responses, the
specific role that IL-21 plays in the post-N. brasiliensis lung
remains unclear. However, IL-21 has been reported to have
several functions that are independent of Th17 development,
and these suggest that it might contribute to the regulation of
allergic responsivenss in the post-N. brasiliensis lung. IL-21 has
been shown to modulate the levels of IgE produced by B cells
(54) and also appears to have a direct effect on the develop-
ment of AAMs. There was a marked reduction in the AAM-
associated genes encoding YM1, FIZZ1, and acidic mamma-
lian chitinase in the lungs and lung-associated lymph nodes in
IL-21R?/?mice (42). Mechanistically, it appears that signaling
through the IL-21R is required to upregulate IL-4R? and
IL-13R?1 so that macrophages can efficiently take on the
AAM phenotype. The constitutive upregulation of IL-4, IL-13,
and IL-21 in post-N. brasiliensis lungs could explain the persis-
tently elevated levels of AAMs. The cellular source of IL-21 in
the post-N. brasiliensis lung is yet to be defined.
At day 36 p.i., approximately 75% of the CD11c?, F4/80?
alveolar macrophages displayed an alternatively activated phe-
notype characterized by transcription of ym1, ym2, fizz1, and
arg1 and increased expression of mrc1 and the genes encoding
class II MHC. A previous report documented a nearly total
conversion of the alveolar macrophage population to the al-
ternatively activated phenotype within hours of the larvae en-
tering the lungs, which was maintained through day 12 p.i. (43).
The demonstration that the AAM phenotype remains domi-
nant at day 36 p.i. and that it is still readily detectable through
to day 100 p.i. (reference 30 and data not shown) suggests that
this phenotypic skewing is a fixed consequence of N. brasiliensis
It is likely that the AAMs resulting from N. brasiliensis in-
fection contribute to the regulatory environment in the lungs.
AAMs outside the lung have been assigned roles in debris
scavenging, tissue remodeling during wound healing, and the
promotion of Th2 immune responses (14, 36, 50). The results
of several studies indicate that AAMs play both direct and
indirect roles in the pathogenesis of infectious diseases. The
presence of AAMs in the lungs of Cryptococcus neoformans-
infected mice was accompanied by a switch from a chronic to
a progressive pulmonary fungal infection (4). In the Leishma-
nia major-mouse model of cutaneous leishmanisis, where Th1
responses are required for effective immunity, AAMs contrib-
ute to parasite persistence and disease progression (19). The
functional significance of the AAMs induced during schistoso-
miasis is complex. On one hand, the development of AAMs
appears to be critical to dampen the destructive potential of
the acute egg-induced inflammation that leads to oxidative
damage to liver tissue (17, 18). On the other hand, the Arg1
produced by the granuloma-associated AAMs mediates the
fibrosis that is characteristic of the chronic pathology in schis-
tosomiasis (18, 42, 44). AAMs induced in the peritoneal cavity
by the filarial nematode Brugia malayi are capable of inhibiting
T cell proliferation through a contact-mediated mechanism
(27). Another filarial species, Litomosoides sigmodontis, in-
duced F4/80?AAMs in the pericardial cavity that potently
inhibit antigen-specific CD4?T-cell proliferation (55). AAMs
have been shown to function as key effector cells in the
protective memory response to the elimination of Heligmo-
somoides polygyrus from the gut (2). Collectively, the data
strongly suggests that AAMs play an important role in reg-
ulating Th2-biased inflammation.
Recently, Loke and colleagues showed that mechanical
damage was capable of inducing alternative activation of mac-
rophages without the presence of an infectious agent (26). The
level of larval challenge used in this study (500 L3s) results in
obvious mechanical damage to the lungs (30, 43). Pulmonary
damage inflicted by helminth migration through the paren-
chyma is thus likely to be a contributor to the generation and
maintenance of AAMs in the lung.
A number of important helminth parasites of humans have
incorporated a short-term residence in the lungs as an obligate
phase of their life cycle. While the significance of the lung
phase to the developmental biology of the parasite is not clear,
this short-term exposure has a long-lasting impact on the im-
munobiology of the lung. Given this direct contact, it is per-
haps not surprising that helminths induce cellular and/or mo-
lecular changes that include regulatory responses in the lungs.
Interestingly, the strictly enteric helminth H. polygyrus, which
has no apparent direct impact on the lung (22), also induces
regulatory responses that are capable of modulating allergen-
induced pulmonary inflammation (64). A major mediator of
this downregulation of pulmonary inflammation was shown to
be H. polygyrus-induced CD4?CD25?Foxp3?regulatory T
cells. The CD4?CD25?Foxp3?phenotype and its ability to
work in an organ that presumably has not seen H. polygyrus
antigen suggests that the regulatory T cells induced by H.
polygyrus are natural Tregs that recognize self antigen (6). If H.
polygyrus does indeed have the ability to induce a population of
natural Tregs (possibly a population that is focused on mucosa-
associated self antigens), this would provide a mechanism for
bridging the gap between the gut and the lung. An important
common element in the cellular responses induced by both H.
polygyrus and N. brasiliensis is the strong induction of AAMs (2,
43). It is possible that AAMs have a direct role in the induction
and maintenance of a Treg-mediated regulatory environment.
Given the proven capacity of AAM as effector cells and ho-
3522 REECE ET AL.INFECT. IMMUN.
meostatic regulators in both mucosal and nonmucosal sites, the
substantial and persistent increase of AAMs in the lungs might
represent a heretofore-unappreciated cellular component in
the overall mechanism that modulates inflammation in the
post-N. brasiliensis lung.
The infection-mediated elevation in airway responsiveness
to methacholine challenge reported here (Fig. 8) was also
observed by Marsland and colleagues (30). This N. brasiliensis-
induced chronic hyperresponsiveness was shown to persist be-
yond 100 days p.i. (30). In stark contrast, the reactivity of the
large airways from N. brasiliensis-HDM animals was signifi-
cantly below that of the controls (Fig. 8). The mechanistic basis
for this dramatic transition in the level of airway reactivity in N.
brasiliensis-infected animals has yet to be fully defined. It is
possible that the 48-hour window between the first intranasal
HDM antigen exposure and the methacholine challenge pro-
vided a sufficient amount of time to activate the N. brasiliensis-
induced regulatory mechanisms that control airway reactivity.
It is likely that the mechanisms that function to rapidly repress
the reactivity of the large airways in the post-N. brasiliensis lung
are reflected in the distinctive expression profile observed post-
A number of studies have used animal models to address the
mechanistic issues that underlie the inverse relationship be-
tween the epidemiologies of helminth infection and allergic
disease in human populations (5, 61, 64, 65) and the immuno-
logical observations that preexisting helminth infection alters
the magnitude and character of the immune responses to sub-
sequent pathogen or antigen challenges (7, 12, 45). Results
from studies where infection with the nematode N. brasiliensis,
H. polygyrus, or Strongyloides stercoralis was superimposed
upon the ovalbumin allergy model demonstrated that reduc-
tions in the responses to allergen challenge were characterized
by downregulation of eotaxin production, a commensurate de-
crease in eosinophil infiltration, and a decrease in lung-asso-
ciated IgE levels (60, 64, 65). In addition to confirming this
helminth-induced decrement in responsiveness to subsequent
antigen challenge, the work presented here defines the persis-
tent molecular and cellular alterations that accompany this
reduction in response in the post-N. brasiliensis lung. These
long-term pulmonary changes provide an immunological con-
text to understand the mechanisms involved in the modulated
immune responses observed post-helminth infection.
We thank Anne Jedlicka and Meg Mintz (Johns Hopkins Malaria
Research Institute Genearray Core Facility) for assistance in the mi-
croarray experiments. For their assistance in the pulmonary function
testing, we thank Andrea Keane-Myers (NIH, Rockville, MD) and
Wayne Mitzner and Jon Fallica (JH BSPH, Baltimore, MD).
This work was supported by grants from NIH NHLBI (U01
HL66623) and NIH training grant T32AI007417.
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