IL-33 drives biphasic IL-13 production for noncanonical
Type 2 immunity against hookworms
Li-Yin Hunga,1, Ian P. Lewkowichb,1, Lucas A. Dawsonb, Jordan Downeyb, Yanfen Yangb, Dirk E. Smithc,
and De’Broski R. Herberta,2
aDivision of Experimental Medicine, University of California, San Francisco, CA 94110;bDivision of Immunobiology, Cincinnati Children’s Hospital Medical
Center, Cincinnati, OH 45229; andcDepartment of Inflammation Research, Amgen, Seattle, WA 91320
Edited by Richard A. Flavell, Howard Hughes Medical Institute and Yale School of Medicine, New Haven, CT, and approved November 19, 2012 (received for
review April 25, 2012)
Parasitic helminths are a major cause of chronic human disease,
affecting more than 3 billion people worldwide. Host protection
against most parasitic helminths relies upon Type 2 cytokine
production, but the mechanisms that regulate interleukin (IL) 4
and 13 production from CD4+T helper 2 cells (TH2) and innate
lymphoid type 2 cells (ILC2s) remain incompletely understood.
The epithelial cell-derived cytokines IL-25 and IL-33 promote Type
2 responses, but the extent of functional redundancy between
these cytokines is unclear and whether Type 2 memory relies upon
either IL-25 or IL-33 is unknown. Herein, we demonstrate a pivotal
role for IL-33 in driving primary and anamnestic immunity against
the rodent hookworm Nippostrongylus brasiliensis. IL-33–deficient
mice have a selective defect in ILC2–derived IL-13 during both
primary and secondary challenge infections but generate stronger
canonical CD4+T helper 2 cells responses (IL-4, IgE, mast cells, and
basophils) than WT controls. Lack of IL-13 production in IL-33–de-
ficient mice impairs resistin-like molecule beta (RELMβ) expression
and eosinophil recruitment, which are two mechanisms that elim-
inate N. brasiliensis parasites from infected hosts. Thus, IL-33 is
requisite for IL-13 but not IL-4–driven Type 2 responses during
mucosal immunity|gastrointestinal nematode|inflammation
responses are characterized by interleukins (ILs) 4, 5, 9 13, 25,
and 33; expansion of CD4+T helper 2 (TH2) cells; IgE pro-
duction; eosinophilia; mastocytosis; basophilia; alternatively ac-
tivated macrophages; smooth muscle hypercontractility; and
goblet cell metaplasia (1, 3). Although CD4+TH2 cells were
previously considered central drivers of Type 2 immunity, recent
discoveries of Type 2 cytokine-producing innate lymphoid cells
(ILC2s) have brought new insight(s) to our global understanding
of inflammatory responses (4–6). Prevailing hypotheses suggest
that IL-25 and IL-33 release from damaged epithelia promotes
the rapid expansion of ILC2s following allergen challenge or
cytokine release from both TH2 cells and ILC2s remains unclear.
IL-33 is an IL-1 family cytokine that regulates a wide array of
pathological states associated with cardiovascular disease, rheu-
matoid arthritis, anaphylaxis, ulcerative colitis, and pathogen
infestation (9, 10). Like other IL-1 family members, IL-33 can be
cleaved by caspase-1, although it appears that this cleavage leads
to functional inactivation (11), leading to speculation that IL-33
primarily functions as a chromatin-associated nuclear factor (12).
However, bioactive IL-33 can be generated following pro–IL-33
cleavage by other proteases such as neutrophil elastase and
cathepsins, suggesting that bioactive IL-33 may be released into
the extracellular environment (13). Indeed, mucosal damage
caused by allergen or helminth infection elicits IL-33 production
from epithelial cells, macrophages, and inflammatory dendritic
cells (DCs) (14). Extracellular IL-33 exerts its biological activities,
at least in part, through the suppressor of tumorigenicity (TI/ST2)
receptor that signals through MyD88-mediated activation of
MAP kinases and NF-κb (15). This may partially explain how
ype 2 immunity underlies host protection against diverse
helminth species and most allergic disorders (1, 2). Type 2
rIL-33 administration to rodents promotes clearance of the
gastrointestinal helminth Trichuris muris (16). Although TI/ST2
was considered expressed only on TH2 cells and mast cells, IL-33
signaling via TI/ST2 regulates the function(s) of fibroblasts, DCs,
macrophages, eosinophils, and ILC2s/nuocytes (6) (15, 17). De-
spite clear evidence that IL-33 can promote Type 2 responses,
whether IL-33 is necessary for primary and/or secondary Type 2
responses remains controversial (18–21).
Mouse infection with the hookworm parasite Nippostrongylus
brasiliensis is widely used for understanding Type 2 immunity
(22). In this system, infectious larvae (L3) migrate from the skin
into the pulmonary tract and cause hemorrhagic lung injury
within 1–3 d postinfection. TH2 cell expansion and systemic Type
2 cytokines start to increase from 3 to 5 d postinfection, as worms
migrate from the lung into the small intestine (23). Between 6–
12 d postinfection, IL-4– and IL-13–dependent effects cause
intestinal epithelial cells (IECs) to expel adult worms from the
intestinal lumen through mechanisms that require resistin-like
molecule beta (RELMβ), a goblet cell-specific protein that
interferes with worm nutrition (23, 24). If previously infected
animals are rechallenged, STAT-6–dependent processes drive
rapid expulsion of worms, potentially via IL-4 and CD4+T cells,
but there is lack of consensus on the exact mechanism (25–28).
memory responses and high rates of reinfection (29), it is possible
that better understanding of host protection in rodents will lead
to novel approaches for reducing the burden of human disease.
These data show that IL-33 is necessary for immunity against
primary and secondary N. brasiliensis infection. IL-33–deficient
mice (IL-33KO) mice failed to clear worms despite a strong in-
duction of IL-4, IgE, basophil, and mast cell responses. Instead,
IL-33KO mice generated insufficient IL-13 production for IEC-
derived RELMβ and eosinophil recruitment. IL-33 was essential
for the early expansion of IL-13+ILC2s and was partially re-
sponsible for the increase of IL-13–producing CD4+T cells
within the lung tissue. Taken together, this suggests that IL-33
promotes in vivo IL-13 production that drives worm expulsion
independently of canonical TH2 responses.
IL-4 and IL-13 Are Differentially Induced and Regulated During N.
brasiliensis Infection. IL-4Rα–mediated STAT-6 activation is nec-
essary for immunity against N. brasiliensis infection. However,
IL-4KO mice have a moderate defect in host resistance, whereas
IL-13 deficiency severely impairs immunity (30, 31). To deter-
mine whether innate vs. adaptive lymphocytes differentially
Author contributions: L.A.D. and D.R.H. designed research; L.-Y.H., I.P.L., L.A.D., J.D., Y.Y.,
and D.R.H. performed research; D.E.S. contributed new reagents/analytic tools; L.-Y.H., I.P.L.,
L.A.D., and D.R.H. analyzed data; and L.-Y.H., I.P.L., L.A.D., J.D., and D.R.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1L.-Y.H. and I.P.L. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 2, 2013
| vol. 110
| no. 1 www.pnas.org/cgi/doi/10.1073/pnas.1206587110
produced IL-4 and IL-13 during primary N. brasiliensis infection,
we sorted lineage-negative lymphocytes and CD4+T cells from
the lung and mesenteric lymph nodes of wild-type C57BL/6
(WT) mice at d 3 and d 6 postinfection. ILC2s were defined as
B220−, F4/80−, CD5−, CD4−, CD3−, CD27−, NK1.1−, TCRβ−,
CD11b−, CD11c−, CD45+, c-kit+, CD90.2+, and CD127+, and
CD4+T cells were identified by TCRβ+and CD4+(32). cDNA
was generated from 1 × 104cells from each population and qRT-
PCR was used to determine IL-4 and IL-13 relative expression
levels. ILC2s did not show an infection-induced increase of IL-4
expression, but CD4+T cells increased IL-4 expression levels
from d 3 to d 6 (Fig. 1A). Conversely, ILC2s expressed slightly
higher IL-13 levels than CD4+T cells at d 3 but nearly fourfold
higher IL-13 levels than CD4+T cells by d 6 (Fig. 1B). To de-
termine whether ILC2-derived IL-13 required the presence of T
and B cells, systemic IL-13 levels at d 6 postinfection were
compared between WT and RAG-1–deficient mice using the in
vivo cytokine capture assay (IVCCA) (33). Both strains gener-
ated similar increases in IL-13 (Fig. 1C), which demonstrates
that T/B cells were not required for most of the IL-13 produced
up to d 6 following primary N. brasiliensis infection.
IL-25 and IL-33 can have redundant roles for Type 2 immunity
(6); therefore, IL-33KO mice were generated to determine
whether IL-33 was essential for any aspect of a Type 2 response.
This mouse strain had no differences in body weight, blood
chemistry, or organ development. Compared with WT controls,
IL-33KO mice did not show defects in thymic T-cell ratios, T-cell
activation status (as determined by CD25 expression), or myeloid
subsets within the lung tissue (Fig. S1).
To investigate whether IL-33 was necessary for immunity
against hookworms, WT and IL-33KO mice were inoculated s.c.
with 750 N. brasiliensis infectious stage larvae (L3). Although
worm numbers at d 3 postinfection were equivalent between
strains, by d 6 IL-33KO had threefold greater intestinal worm
numbers than WT mice (Fig. 1D). Moreover, IL-33KO mice
remained chronically infected for >21 d following a primary in-
oculation, whereas WT mice cleared their parasites by d 10 post-
infection (Fig. S2). Regarding cytokine production, IL-33KO mice
produced less IL-4 than WT at d 3, but more IL-4 than WT by d 6
(Fig. 1E). However, IL-33KO mice failed to increase their IL-13
levels over baseline at d 3 or d 6 postinfection (Fig. 1F). Therefore,
N. brasiliensis up-regulated IL-4 and IL-13 production from dis-
tinct lymphocyte populations, with IL-33 serving an essential role
for IL-13, but only a transient role for IL-4 production.
IL-33 Selectively Drives ILC2-Derived IL-13 for RELMβ and Eosinophil
Responses. To investigate whether IL-33 was requisite for dif-
ferent lineages of IL-13–producing lymphocytes, we compared
intracellular IL-13 levels between ILC2s and CD4+T cells of WT
and IL-33KO mice. In WT, the percentage of IL-13+ST2+ILC2s
within lung tissue changed from 1% (naïve) to 34% (d 3) and
21% (d 6), but in IL-33KO, the IL-13+ST2+ILC2 population
changed from 0.01% (naïve) to only 1.8% by d 6 postinfection
(Fig. 2A). Total numbers of IL-13+ST2+ILC2s in WT lung tissue
ranged between 5,000–6,000 cells/lung between d3–d6, whereas
in IL-33KO tissues, the numbers were <500 cells/lung up to
d 6 (Fig. 2B). On the other hand, the numbers of IL-13+CD4+T
cells that expanded in response to N. brasiliensis infection were
equivalent between strains at d 3 and d 6 postinfection (Fig. 2B).
Next, relative expression levels for IL-4 and IL-13 were de-
termined from ILC2s that were sorted from the lung and CD4+
sorted from the mesenteric lymph nodes at d 6 postinfection.
The gating strategy used to identify N. brasiliensis-induced ILC2
from the lung and and CD4+T cells from the mesenteric lymph
nodes is shown in Fig. S3. Interestingly, both ILC2 and CD4+T-
cell populations from IL-33KO mice expressed slightly higher
IL-4 levels than from WT, whereas IL-33KO ILC2s completely
lacked IL-13 expression (Fig. 2C). Moreover, by d 14 post-
infection, IL-33KO produced systemic levels of IL-4, IgE, and
mast cells (mast cell protease 1, MCPT-1) that were greater than
or equal to WT levels (Fig. S4). Hence, IL-33 was necessary for
ILC2-derived IL-13 but was not required for CD4+T-cell–de-
rived IL-4, IgE, or mast cells during the primary response.
IL-4/IL-13–dependent effects on epithelia drive expulsion of
N. brasiliensis adult worms from the intestine through RELMβ,
a cytokine that interferes with worm nutrition (23). In compar-
ison with WT mice, RELMβ mRNA transcripts in IL-33KO lung
and intestines were significantly reduced at both d 3 and d 6
postinfection (Fig. 3 A and B). Reduced RELMβ levels were
associated with fewer IL-13 mRNA transcripts in the lung and
intestines of IL-33KO compared with WT mice (Fig. 3 C and D).
Also, IL-33KO mice generated significantly fewer airway eosi-
nophils than WT at d 3 and d 6 postinfection (Fig. 3E).
Erythrocyte (RBC) numbers within bronchoalveolar lavage
(BAL) fluid were quantified to determine whether IL-33 re-
strained lung injury caused by N. brasiliensis infection. RBCs in
IL-33KO BAL fluid were threefold greater than WT BAL at d 3
postinfection, but no differences were observed between strains
by d6 (Fig. 3F). Therefore, IL-33 deficiency impaired IL-13–
dependent RELMβ expression, reduced eosinophil recruitment
to the lung, and increased the apex of N. brasiliensis-induced lung
injury following primary infection.
IL-33 Mediates Anamnestic Immunity Against Secondary Infection.
Given the profound importance for IL-33 in primary host re-
sistance, we postulated an additional role for IL-33 for secondary
immunity against reinfection. To investigate, WT and IL-33KO
mice were infected with 750 L3, treated with the antihelminthic
drug pyrantel pamoate on d 14, rechallenged with 500 L3on d 28,
and monitored for intestinal worms and immune responses at d
3 and d 6 following reinfection. Despite equivalent worm num-
bers between strains at d 3, there were 10-fold higher worms in
IL-33KO mice vs. WT by d 6 postinfection (Fig. 4A). Comparison
N. brasiliensis infection. 1 × 104CD4+T cells or ILC2s were sorted from lung
and mesenteric lymph nodes of WT C57BL/6 mice that were infected with
750 N. brasiliensis L3, and mRNA transcript levels for (A) IL-4 and (B) IL-13
were determined at the times indicated. Mean ± SE of five mice/group an-
alyzed from two independent experiments. (C) Serum IL-13 levels in WT and
RAG1KO mice as determined by IVCCA. (D) Numbers of N. brasiliensis worms
recovered from the intestinal lumen of WT and IL-33KO mice. Serum levels
of (E) IL-4 and (F) IL-13 in WT and IL-33KO mice determined by IVCCA. Data
shown represent the mean ± SE of 6–12 mice/group infected with 750 L3of
three independent experiments. *P < 0.05 and ***P < 0.001.
IL-33 is necessary for IL-13 but not IL-4 induction during primary
Hung et al. PNAS
| January 2, 2013
| vol. 110
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of worm lengths between strains revealed significantly longer par-
asites in IL-33KO mice compared with WT mice (Fig. 4B). Thus,
IL-33 deficiency impaired worm clearance during secondary chal-
lenge, most likely due to a failure in limiting parasite development.
RELMβ inhibits the ability of hookworms to feed upon their
hosts (23, 24); therefore, we investigated whether IL-33 was
necessary for inducing an IL-13/RELMβ axis upon rechallenge.
IL-33KO mice produced significantly less IL-13 than WT mice at
d 3 but not at d 6 postinfection (Fig. 4C). Both lung and in-
testinal RELMβ mRNA transcripts were significantly reduced
in IL-33KO compared with WT at d 3 postinfection, but only
intestinal RELMβ expression levels remained suppressed in
IL-33KO tissues at d 6 (Fig. 4 D and E).
To identify the cellular sources of IL-13 during secondary
responses to N. brasiliensis, we determined both the percentage
and number of lung IL-13+ST2+ILC2s and IL-13+CD4+T cells
at d 3 and d 6 following reinfection. At d 3, the percentage of
lung 13+ILC2s in WT mice was 14-fold higher than in IL-33KO
(Fig. 5A), and the percentage of IL-13+CD4+T cells in WT was
twofold higher than in IL-33KO (Fig. 5B). In total cell number,
the WT IL-13+ST2+ILC2 population was 10-fold greater than
in IL-33KO at d 3 (Fig. 5C), but these numbers decreased to
levels that were no different from IL-33KO by d 6. In addition,
IL-33KO lung tissues also had fewer IL-13+CD4+T cells than
WT lung tissues at d 6 postinfection (Fig. 5D).
Lastly, BAL fluid and intestinal tissues were examined to de-
termine whether IL-33 regulated granulocyte recruitment during
the secondary response. Lung eosinophil numbers were fourfold
lower in IL-33KO compared with WT mice at d 6 (Fig. 5E).
Conversely, lung mast cell numbers were 4–5-fold greater in IL-
33KO BAL fluid compared with WT (Fig. 5F). Also, IL-33KO
expressed higher intestinal mRNA levels for MCPT-8 (basophil-
specific granule protein) compared with WT (Fig. 5G) (25, 34).
Therefore, secondary immune responses were IL-33–dependent
and strongly associated with coordinated IL-13 production
from ILC2s and CD4+T cells. This coordinated IL-13 production
most likely contributed to intestinal RELMβ production and
eosinophil recruitment but was not essential for mast cell or
TH2-associated cytokines predominate the immune responses
generated against helminths and allergens, but mechanisms that
govern Type 2 response initiation, maintenance, and resolution
are incompletely understood (3, 29). Herein, the rodent-specific
hookworm, N. brasiliensis, was used to investigate how IL-33
Surprisingly, IL-33 was necessary for both phases of immu-
nity. Mice lacking IL-33 were unable to generate (i) primary and
secondary expansion of IL-13+ILC2s, (ii) secondary expansion
of IL-13+CD4+T cells, (iii) primary and secondary RELMβ ex-
pression, and (iv) primary and secondary eosinophil-recruitment.
On the other hand, canonical Type 2 responses were enhanced
in IL-33KO mice, such as (i) systemic IL-4 production, (ii) IgE
secretion, (iii) mast cell, and (iv) basophil responses. Taken to-
gether, these data imply that IL-33 preferentially instructs IL-13–
driven inflammation instead of IL-4–mediated immunity (35, 36),
which provides greater insight into the in vivo regulation of
Type 2 responses.
IL-33 was initially described as an epithelial/endothelial cell-
specific cytokine. More recently, we and others have shown that
IL-33 is also produced from myeloid lineage cells within hours of
pathogen exposure (8, 14), which is consistent with a hypothesis
that IL-33 functions as an “alarmin” that instructs adaptive im-
mune responses (12). Our demonstration that IL-33KO mice
were highly susceptible to both primary and secondary N. brasi-
liensis infection is consistent with reciprocal experiments that
demonstrated IL-33 administration promoted the rapid expulsion
of the gastrointestinal helminth T. muris (16). Our results are
similar to the phenotype of N. brasiliensis-infected mice lacking
IL-25, a cytokine functionally related to IL-33 and one that is also
produced by epithelia and professional antigen presenting cells
(APCs) (37). Whether IL-25KO mice also have defects in sec-
ondary responses against N. brasiliensis is presently unclear, but
our work suggests that IL-25 and IL-33 may not function in an
entirely redundant manner for Type 2 immunity (5, 7).
We focused on how IL-33 regulated IL-13–producing lym-
phocytes (ILC2 and CD4+T) within the lung, which is a major
target organ of hookworm infection in both mice and humans
duction from ILC2s and CD4+T cells during a primary response.
(A) TI/ST2 expression and percentage of intracellular IL-13
within lung ILC2s of WT and IL-33KO mice at the indicated
time points following infection with 750 N. brasiliensis L3.
Representative plots are shown. (B) Total numbers of lung
IL-13+ILC2s (Upper) and lung IL-13+CD4+TCRβ+cells (Lower)
from WT and IL-33KO mice at the indicated time points fol-
lowing infection with 750 N. brasiliensis L3. Data shown rep-
resent the mean ± SE of 4–6 mice/group analyzed from three
independent experiments. (C) IL-4 and IL-13 mRNA levels in
sorted ILC2s (Upper) and CD4 T (Lower) from WT and IL-33KO
mice 6 d following 750 L3infection. *P < 0.05, **P < 0.01, and
***P < 0.001.
IL-33 drives hookworm infection-induced IL-13 pro-
| www.pnas.org/cgi/doi/10.1073/pnas.1206587110 Hung et al.
(38). Migratory parasites cause hemorrhagic tissue injury within
this organ as they molt from L3to L4, break out of the alveoli,
and migrate into the gastrointestinal tract by 3 d postinfection
(23). Within 1 d of N. brasiliensis infection, IL-33 levels increased
dramatically within the lung (14). Within 3 d, the IL-13+ILC2
population increased >100-fold in number through an IL-33–
dependent mechanism. Importantly, ILC2s were present within
IL-33KO lung tissues but fail to produce IL-13, implying that IL-33
provided a “license” for IL-13 expression. Whether IL-33 drives
lymphocyte-specific IL-13 transcription and/or indirectly pro-
motes IL-13 production through effects on professional APC
remains unclear. IL-33–dependent IL-13 induction was important
for mucosal tissue repair because lung hemorrhage was exacer-
bated in IL-33KO compared with WT. This suggestion of IL-13–
dependent tissue repair is consistent with evidence that IL-13+
ILC2 expansion during influenza infection promotes regeneration
of lung epithelia via amphiregulin, an EGF family cytokine (32).
The lung is also a critical site for TH2 cell priming during N.
brasiliensis infection (39). At d 3 postinfection, IL-13–producing
CD4+T cells expand within the mediastinal lymph nodes (14),
but this population does not accumulate within lung tissues until
d 6 postinfection. IL-33 deficiency also significantly decreased
both the percentage and number of lung IL-13+CD4+T cells
during secondary immune responses, but IL-33 was not essential
for expanding IL-4–producing CD4+T cells. In fact, IL-4 ex-
pression in CD4+T cells and systemic IL-4 levels in the sera of
infected IL-33KO mice was generally higher than in WT animals.
Based on our cell-sorting experiments and experiments per-
formed with WT vs. RAG1KO mice, our interpretation is that
primary immune responses against N. brasiliensis are shaped by
the combined actions of IL-4–producing CD4+T cells and IL-13–
producing ILC2s. However, the IL-13–dependent effector func-
tions have a dominant role in host protection, because IL-33KO
mice remain chronically infected for >21 d despite elevated IL-4
responses. During the completion of this manuscript, Yasuda
et al. demonstrated that IL-33 from alveolar type 2 cells promoted
IL-13 production and host immunity against the gastrointestinal
(GI) nematode Strongyloides venenzuelensis, but whether IL-33 was
essential for Type 2 memory responses was not addressed (40).
Demonstration that IL-33 was essential for secondary immu-
nity against reinfection was particularly intriguing because an-
amnestic responses are considered mechanistically distinct from
primary immunity against N. brasiliensis (27, 41, 42). Consider-
able debate has centered upon the site of worm killing and the
types of granulocytes required for immunity against rechallenge
(26, 27, 39). Our data showed no differences in worm burdens
between WT and IL-33KO mice at d 3 postsecondary challenge.
However, by d 6 postinfection, WT mice eliminated >95% of
their worms, and those still present were pale and poorly de-
veloped. Conversely, worms recovered during secondary chal-
lenge of IL-33KO mice at d 6 were larger and were feeding upon
the host. Although a direct role for RELMβ in secondary im-
munity has not been demonstrated, impaired RELMβ expression
monary pathogenesis during primary infection with N. brasiliensis . RELMβ
mRNA transcript levels in the (A) lung and (B) jejunum at the indicated time
points following infection with 750 N. brasiliensis L3. IL-13 mRNA levels in the
(C) lung and (D) gut at indicated time points postinfection. Data shown
represent the mean ± SE of 6–12 mice/group analyzed from two in-
dependent experiments. (E) Total numbers of eosinophils and (F) red blood
cells within the BAL fluid in WT and IL-33KO mice at d 3 and d 6 following
750 L3infection. Data shown represent the mean ± SE of 4–6 mice/group
analyzed from three independent experiments. *P < 0.05 and **P < 0.01.
IL-33 is required for IL-13 production, RELMβ expression and pul-
RELMβ axis during rechallenge. (A) Numbers of intestinal worms recovered
from WT and IL-33KO mice at d 3 and d 6 following secondary challenge. (B)
Worm lengths at d 6 postinfection in experiment described in A. Each in-
dividual dot represents an individual parasite. (C) Systemic IL-13 production,
(D) lung RELMβ mRNA transcript levels, and (E) intestinal RELMβ mRNA levels
in WT and IL-33KO mice at d 3 and d 6 following secondary challenge. Data
shown represent the mean ± SE of 6–8 mice/group analyzed from three in-
dependent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.
IL-33 is required for worm expulsion and up-regulation of the IL-13/
Hung et al. PNAS
| January 2, 2013
| vol. 110
| no. 1
in the lung and intestines of IL-33KO mice during both primary
and secondary responses is entirely consistent with a central role
for RELMβ in IL-13–mediated immunity (23).
IL-33 deficiency reduced eosinophil recruitment, which was
another potential explanation for the impaired anamnestic re-
sponse. Immunity against N. brasiliensis rechallenge was im-
paired in mice made eosinophil deficient by targeted deletion of
the erythroid transcription factor (GATA-1) or IL-5 (27, 28, 43).
Mechanistically, IL-33 could promote eosinophilic lung in-
flammation through inducing IL-5 and IL-13 production from
CD4+T cells and ILC2s (19), or by directly activating eosinophils
through TI/ST2. On the other hand, IL-33KO mice generated
higher levels of mast cells and expressed higher intestinal MCPT-
8 mRNA transcripts than WT mice. Intact mast cell and basophil
responses in IL-33KO mice were associated with enhanced IL-4
and IgE levels. This dichotomy between IL-33–driven IL-13 versus
IL-4-driven inflammatory responses resolves the controversy over
whether signaling via T1/ST2 is necessary for Type 2 immunity
(18, 21, 44). The source of IL-4 within IL-33KO mice was
CD4+T cells but also could include mast cells and basophils.
Indeed, basophils are a major source of IL-4 during secondary
immune responses, and were shown to promote secondary im-
munity against N. brasiliensis (26, 45–47). Thus, while multiple
redundant mechanisms are functioning during secondary chal-
lenge infection, our data support a hypothesis that eosinophils
serve a predominant role in worm destruction during anamnestic
immunity against GI nematodes (48, 49).
Overall, our data indicate that within hours of a primary N.
brasiliensis infection, IL-33 release drives initial expansion of IL-
13+ILC2/nuocytes followed by IL-13+CD4+T cells within three
days. This accumulation of IL-13 production instructs IEC to
produce RELMβ and recruits eosinophils, which together result
in parasite destruction. Upon rechallenge, this process occurs
more rapidly, perhaps due to the progressive accumulation of
alternatively activated macrophages that can provide a larger
pool of IL-33 for IL-13–driven immunity (50).
basophil responses following rechallenge. (A) Flow cytometry contour plots show TI/ST2 expression and percentage of intracellular IL-13 within the lung ILC2/
nuocyte populations of WT and IL-33KO mice at d 3 and d 6 following secondary challenge. (B) Zebra plots show percentage of IL-13 within lung CD4+TCRβ+
cells at d 3 and d 6 following secondary challenge. (C) Total numbers of lung IL-13+ILC2s within gate shown in A. (D) Total numbers of lung IL-13+CD4+TCRβ+
cells within gate shown in B. (E) Total numbers of eosinophils and (F) mast cells within the BAL fluid and (G) intestinal MCPT8 mRNA transcript levels in WT
and IL-33KO mice at d 3 and d 6 following secondary challenge. Data shown represent the mean ± SE of 4–6 mice/group analyzed from two independent
experiments. *P < 0.05, **P < 0.01, and***P < 0.001.
IL-33 is necessary for the early expansion of IL-13+ILC2, eosinophil recruitment, and late accumulation of IL-13+CD4+T cells but not mast cell or
| www.pnas.org/cgi/doi/10.1073/pnas.1206587110Hung et al.
Materials and Methods Download full-text
Mice andParasites. A targeting construct that replaced exons 1–6 was injected
into 129 ES cells, and chimeras were backcrossed onto the C57BL/6 back-
ground >10 generations and confirmed using a 377 SNP panel for micro-
satellite analysis. All experiments were conducted with age- and sex-
matched WT or IL-33–deficient mice on a C57BL/6 background obtained
from Taconic and have been described elsewhere (51). N. brasiliensis was
maintained in the laboratory using established protocols (23). The In-
stitutional Animal Care and Use Committee at the Cincinnati Children’s
Hospital Medical Center and University of California at San Francisco ap-
proved all procedures.
qRT-PCR. RNA was DNase I–treated and cDNA prepared using SuperScript II
Reverse Transcriptase (Invitrogen). Real-time PCR was carried out on a Biorad
iCycler (Hercules) or CFX Connect (Bio-Rad) with the Syber Green detection
reagent. Cycle threshold (CT) values for genes evaluated were determined
and expressed using the 1/ΔΔctmethod, as described previously (52).
Flow Cytometry. Whole lung tissues were perfused with 1× PBS and minced
with scissors followed by digestion in serum-free RPMI containing Liberase
TL (0.25 mg/mL, Roche) and DNase I (0.5 mg/mL, Sigma) RPMI for 45 min at
37 °C. Samples were passed through a 70 μm cell strainer to obtain a single
cell suspension. Single cell suspensions of lung tissue were stained with one
or more of the following fluorescently labeled mAb: TCRβ (clone H57-597),
CD4 (clone GK1.5), F4/80 (clone BM8), CD5 (clone 53–7.3), CD27 (clone
LG.7F9), NK1.1 (clone PK136), CD45R/B220 (clone RA3-6B2), CD11c (clone
N418), CD11b (clone M1/70), CD3 (clone 17A2), CD127 (clone A7R34), CD117/
cKit (clone 2B8), Ly-6G/Gr-1 (clone RB-8C5), ST2L/IL-1R4 (clone 245707) and
anti–IL-13 (clone eBio13A), and isotype control (MOPC-173) (eBioscience).
Statistical Analysis. Statistical significance was assessed by either two-tailed
Student t test (two groups) or ANOVA for multiple groups with a post hoc
tukey test to determine significance; all were performed using Prism Graph
Pad 4.0 software (* P < 0.05, ** P < 0.01, ***P < 0.001).
ACKNOWLEDGMENTS. The authors thank Judy Appleton for critical reading
of this manuscript and Danielle Kellar and Charles Perkins, who provided
technical assistance. This work was supported by National Institutes of
Health Grants R01 GM083204 and AI095289 (to D.R.H.).
1. Finkelman FD, Urban JF, Jr. (2001) The other side of the coin: The protective role of
the TH2 cytokines. J Allergy Clin Immunol 107(5):772–780.
2. Locksley RM (2010) Asthma and allergic inflammation. Cell 140(6):777–783.
3. Wills-Karp M (2004) Interleukin-13 in asthma pathogenesis. Immunol Rev 202:
4. Barlow JL, McKenzie AN (2011) Nuocytes: expanding the innate cell repertoire in
type-2 immunity. J Leukoc Biol 90(5):867–874.
5. Price AE, et al. (2010) Systemically dispersed innate IL-13-expressing cells in type 2
immunity. Proc Natl Acad Sci USA 107(25):11489–11494.
6. Neill DR, et al. (2010) Nuocytes represent a new innate effector leukocyte that me-
diates type-2 immunity. Nature 464(7293):1367–1370.
7. Barlow JL, et al. (2012) Innate IL-13-producing nuocytes arise during allergic lung
inflammation and contribute to airways hyperreactivity. J Allergy Clin Immunol 129
8. Chang YJ, et al. (2011) Innate lymphoid cells mediate influenza-induced airway hyper-
reactivity independently of adaptive immunity. Nat Immunol 12(7):631–638.
9. Palmer G, Gabay C (2011) Interleukin-33 biology with potential insights into human
diseases. Nat Rev Rheumatol 7(6):321–329.
10. Milovanovic M, et al. (2012) IL-33/ST2 axis in inflammation and immunopathology.
Immunol Res 52(1-2):89–99.
11. Zhao W, Hu Z (2010) The enigmatic processing and secretion of interleukin-33. Cell
Mol Immunol 7(4):260–262.
12. Haraldsen G, Balogh J, Pollheimer J, Sponheim J, Küchler AM (2009) Interleukin-33
cytokine of dual function or novel alarmin? Trends Immunol 30(5):227–233.
13. Lefrançais E, et al. (2012) IL-33 is processed into mature bioactive forms by neutrophil
elastase and cathepsin G. Proc Natl Acad Sci USA 109(5):1673–1678.
14. Wills-Karp M, et al. (2012) Trefoil factor 2 rapidly induces interleukin 33 to promote
type 2 immunity during allergic asthma and hookworm infection. J Exp Med 209(3):
15. Chackerian AA, et al. (2007) IL-1 receptor accessory protein and ST2 comprise the IL-33
receptor complex. J Immunol 179(4):2551–2555.
16. Humphreys NE, Xu D, Hepworth MR, Liew FY, Grencis RK (2008) IL-33, a potent in-
ducer of adaptive immunity to intestinal nematodes. J Immunol 180(4):2443–2449.
17. Cherry WB, Yoon J, Bartemes KR, Iijima K, Kita H (2008) A novel IL-1 family cytokine,
IL-33, potently activates human eosinophils. J Allergy Clin Immunol 121(6):1484–1490.
18. Hoshino K, et al. (1999) The absence of interleukin 1 receptor-related T1/ST2 does not
affect T helper cell type 2 development and its effector function. J Exp Med 190(10):
19. Kurowska-Stolarska M, et al. (2008) IL-33 induces antigen-specific IL-5+ T cells and
promotes allergic-induced airway inflammation independent of IL-4. J Immunol
20. Rank MA, et al. (2009) IL-33-activated dendritic cells induce an atypical TH2-type re-
sponse. J Allergy Clin Immunol 123(5):1047–1054.
21. Townsend MJ, Fallon PG, Matthews DJ, Jolin HE, McKenzie AN (2000) T1/ST2-deficient
mice demonstrate the importance of T1/ST2 in developing primary T helper cell type 2
responses. J Exp Med 191(6):1069–1076.
22. Finkelman FD, et al. (1997) Cytokine regulation of host defense against parasitic
gastrointestinal nematodes: Lessons from studies with rodent models. Annu Rev
23. Herbert DR, et al. (2009) Intestinal epithelial cell secretion of RELM-beta protects
against gastrointestinal worm infection. J Exp Med 206(13):2947–2957.
24. Artis D, et al. (2004) RELMbeta/FIZZ2 is a goblet cell-specific immune-effector mole-
cule in the gastrointestinal tract. Proc Natl Acad Sci USA 101(37):13596–13600.
25. Ohnmacht C, et al. (2010) Basophils orchestrate chronic allergic dermatitis and pro-
tective immunity against helminths. Immunity 33(3):364–374.
26. Ohnmacht C, Voehringer D (2010) Basophils protect against reinfection with hook-
worms independently of mast cells and memory Th2 cells. J Immunol 184(1):344–350.
27. Voehringer D, Reese TA, Huang X, Shinkai K, Locksley RM (2006) Type 2 immunity is
controlled by IL-4/IL-13 expression in hematopoietic non-eosinophil cells of the innate
immune system. J Exp Med 203(6):1435–1446.
28. Knott ML, et al. (2007) Impaired resistance in early secondary Nippostrongylus bra-
siliensis infections in mice with defective eosinophilopoeisis. Int J Parasitol 37(12):
29. Bethony J, et al. (2006) Soil-transmitted helminth infections: Ascariasis, trichuriasis,
and hookworm. Lancet 367(9521):1521–1532.
30. Finkelman FD, Wynn TA, Donaldson DD, Urban JF (1999) The role of IL-13 in helminth-
induced inflammation and protective immunity against nematode infections. Curr
Opin Immunol 11(4):420–426.
31. Finkelman FD, et al. (2004) Interleukin-4- and interleukin-13-mediated host pro-
tection against intestinal nematode parasites. Immunol Rev 201:139–155.
32. Monticelli LA, et al. (2011) Innate lymphoid cells promote lung-tissue homeostasis
after infection with influenza virus. Nat Immunol 12(11):1045–1054.
33. Finkelman F, Morris S, Orekhova T, Sehy D (2003) The in vivo cytokine capture assay
for measurement of cytokine production in the mouse. Curr Protoc Immunol Chapter
34. Wada T, et al. (2010) Selective ablation of basophils in mice reveals their non-
redundant role in acquired immunity against ticks. J Clin Invest 120(8):2867–2875.
35. Liang HE, et al. (2012) Divergent expression patterns of IL-4 and IL-13 define unique
functions in allergic immunity. Nat Immunol 13(1):58–66.
36. Grünig G, et al. (1998) Requirement for IL-13 independently of IL-4 in experimental
asthma. Science 282(5397):2261–2263.
37. Fallon PG, et al. (2006) Identification of an interleukin (IL)-25-dependent cell pop-
ulation that provides IL-4, IL-5, and IL-13 at the onset of helminth expulsion. J Exp
38. Brooker S, Bethony J, Hotez PJ (2004) Human hookworm infection in the 21st century.
Adv Parasitol 58:197–288.
39. Harvie M, et al. (2010) The lung is an important site for priming CD4 T-cell-mediated
protective immunity against gastrointestinal helminth parasites. Infect Immun 78(9):
40. Yasuda K, et al. (2012) Contribution of IL-33-activated type II innate lymphoid cells to
pulmonary eosinophilia in intestinal nematode-infected mice. Proc Natl Acad Sci USA
41. Sullivan BM, Locksley RM (2009) Basophils: A nonredundant contributor to host im-
munity. Immunity 30(1):12–20.
42. Sullivan BM, et al. (2011) Genetic analysis of basophil function in vivo. Nat Immunol
43. Knott ML, Matthaei KI, Foster PS, Dent LA (2009) The roles of eotaxin and the STAT6
signalling pathway in eosinophil recruitment and host resistance to the nematodes
Nippostrongylus brasiliensis and Heligmosomoides bakeri. Mol Immunol 46(13):
44. Mangan NE, Dasvarma A, McKenzie AN, Fallon PG (2007) T1/ST2 expression on Th2 cells
negatively regulates allergic pulmonary inflammation. Eur J Immunol 37(5):1302–1312.
45. Min B, et al. (2004) Basophils produce IL-4 and accumulate in tissues after infection
with a Th2-inducing parasite. J Exp Med 200(4):507–517.
46. Seder RA, et al. (1991) Production of interleukin-4 and other cytokines following
stimulation of mast cell lines and in vivo mast cells/basophils. Int Arch Allergy Appl
47. Khodoun MV, Orekhova T, Potter C, Morris S, Finkelman FD (2004) Basophils initiate
IL-4 production during a memory T-dependent response. J Exp Med 200(7):857–870.
48. O’Connell AE, et al. (2011) Major basic protein from eosinophils and myeloperoxidase
from neutrophils are required for protective immunity to Strongyloides stercoralis in
mice. Infect Immun 79(7):2770–2778.
49. Herbert DR, et al. (2000) Role of IL-5 in innate and adaptive immunity to larval
Strongyloides stercoralis in mice. J Immunol 165(8):4544–4551.
50. Reece JJ, et al. (2008) Hookworm-induced persistent changes to the immunological
environment of the lung. Infect Immun 76(8):3511–3524.
51. Beamer CA, et al. (2012) IL-33 mediates multi-walled carbon nanotube (MWCNT)-in-
duced airway hyper-reactivity via the mobilization of innate helper cells in the lung.
52. Herbert DR, et al. (2010) Arginase I suppresses IL-12/IL-23p40-driven intestinal in-
flammation during acute schistosomiasis. J Immunol 184(11):6438–6446.
Hung et al. PNAS
| January 2, 2013
| vol. 110
| no. 1