The Journal of Immunology
Alternaria Induces STAT6-Dependent Acute Airway
Eosinophilia and Epithelial FIZZ1 Expression That Promotes
Airway Fibrosis and Epithelial Thickness
Taylor A. Doherty,*,†Naseem Khorram,* Kotaro Sugimoto,‡Dean Sheppard,‡
Peter Rosenthal,* Jae Youn Cho,* Alexa Pham,* Marina Miller,* Michael Croft,†and
David H. Broide*
The fungal allergen, Alternaria, is specifically associated with severe asthma, including life-threatening exacerbations. To better
understand the acute innate airway response to Alternaria, naive wild-type (WT) mice were challenged once intranasally with
Alternaria. Naive WT mice developed significant bronchoalveolar lavage eosinophilia following Alternaria challenge when ana-
lyzed 24 h later. In contrast to Alternaria, neither Aspergillus nor Candida induced bronchoalveolar lavage eosinophilia. Gene
microarray analysis of airway epithelial cell brushings demonstrated that Alternaria-challenged naive WT mice had a >20-fold
increase in the level of expression of found in inflammatory zone 1 (FIZZ1/Retnla), a resistin-like molecule. Lung immunostaining
confirmed strong airway epithelial FIZZ1 expression as early as 3 h after a single Alternaria challenge that persisted for ‡5 d and
was significantly reduced in STAT6-deficient, but not protease-activated receptor 2-deficient mice. Bone marrow chimera studies
revealed that STAT6 expressed in lung cells was required for epithelial FIZZ1 expression, whereas STAT6 present in bone
marrow-derived cells contributed to airway eosinophilia. Studies investigating which cells in the nonchallenged lung bind FIZZ1
demonstrated that CD45+CD11c+cells (macrophages and dendritic cells), as well as collagen-1–producing CD452cells (fibro-
blasts), can bind to FIZZ1. Importantly, direct administration of recombinant FIZZ1 to naive WT mice led to airway eosinophilia,
peribronchial fibrosis, and increased thickness of the airway epithelium. Thus, Alternaria induces STAT6–dependent acute airway
eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness. This may provide some insight
into the uniquely pathogenic aspects of Alternaria-associated asthma.
cockroach, and mold. Alternaria is an example of a common
fungal allergen that is associated with the development of asthma
(1). Sensitization to Alternaria alternata is a risk factor for per-
sistence of asthma and fatal/near-fatal asthma (2–8). The spores of
Alternaria are known to be a source of outdoor allergens for
sensitized individuals, and they were recently detected at high
levels indoors (9). Dispersion of the spores occurs during periods
of warm, dry weather, especially in late summer/early fall, and has
been associated with epidemic, severe asthma symptoms (2–8).
Such clinical associations with Alternaria and asthma are in-
The Journal of Immunology, 2012, 188: 2622–2629.
lthough asthma has many clinical, physiologic, and im-
munologic phenotypes, the majority of asthmatics have
environmental allergic triggers, including dust mite,
triguing, but the mechanisms contributing to the pathologic airway
responses are still incompletely understood.
Allergic disease, including asthma, has largely been character-
ized by dysregulation of adaptive immunity in response to aller-
gens, including Th2 cell differentiation and IgE sensitization. More
recently, it has become clear that innate immune responses to
allergensinthe airway help to shapesubsequent adaptiveresponses
(10, 11). For example, recent reports have suggested that allergens
with high protease activity, such as cockroach and fungal aller-
gens, induce innate inflammatory events and allergen sensitization
through a protease-activated receptor 2 (PAR-2)–mediated path-
way in the bronchial epithelium (12–14). Investigations into such
innate epithelial responses to inhaled allergens may provide im-
portant clues to the pathogenesis of asthma. In this study, we in-
vestigated whether Alternaria is able to induce an acute Th2-like
airway inflammatory response in naive wild-type (WT) mice via
activation of innate epithelial genes. We demonstrate that Alter-
naria induces a significant acute airway eosinophil response in
naive WT mice that is mediated by innate immune mechanisms
distinct from those triggered by protease allergens through PAR-2
on the epithelium. This innate proeosinophil inflammatory and
proremodeling effect of Alternaria in naive WT mice is not shared
with other common fungal allergens, such as Aspergillus and
Candida, suggesting that different allergens trigger distinct innate
airway epithelial pathways that contribute to asthma.
Materials and Methods
Mice and airway challenges
Six- to eight-week-old female naive C57BL/6 WT mice were administered
100 mg A. alternata (lot 130656), Candida albicans (lot 111797), or As-
*Department of Medicine, University of California San Diego, La Jolla, CA 92093;
†Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, La
Jolla, CA 92037; and‡Department of Medicine, Lung Biology Center, University of
California San Francisco, San Francisco, CA 94143
Received for publication June 3, 2011. Accepted for publication January 10, 2012.
This work was supported by National Institutes of Health Grants 1K08AI080938-
01A1 (to T.A.D.); AI 38425, AI 70535, and AI 72115 (to D.H.B.); and National
Institute for Allergy and Infectious Diseases Grant U19 AI077439 (to D.S.).
Microarray data presented in this article have been deposited in the Gene Expression
Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE34764.
Address correspondence and reprint requests to Dr. Taylor Doherty, Department of
Medicine, University of California San Diego, Biomedical Sciences Building, Room
5080, 9500 Gilman Drive, La Jolla, CA 92093-0635. E-mail address: tdoherty@ucsd.
Abbreviations used in this article: BAL, bronchoalveolar lavage; FIZZ1, found in
inflammatory zone 1; PAR-2, protease-activated receptor 2; rFIZZ1, recombinant
found in inflammatory zone 1; TSLP, thymic stromal lymphopoietin; WT, wild-type.
pergillus fumigatus (lot 118033) extracts (Greer, Lenoir, NC) intranasally
in 80 ml PBS; they were killed 24 h later, at which time bronchoalveolar
lavage (BAL) and lung specimens were processed. For selected experi-
ments, naive WT mice were analyzed 3 h and 5 d after challenge. Control
groups of naive WT micewere given intranasal challenges of 80 ml PBS. In
selected experiments, PAR-2–deficient or STAT6-deficient mice (Jackson
Laboratories) on a B6 background were administered 100 mg A. alternata
extracts intranasally with WT controls, as described above. Collagen-1
GFP reporter mice were a gift from Dr. David Brenner (University of
California San Diego, La Jolla, CA) and were described previously (15).
In some experiments, 5 mg recombinant FIZZ1 (rFIZZ1; PeproTech) or
vehicle (PBS) was given intranasally to naive WT mice every day for 5 d,
and mice were killed on day 8. The endotoxin level detected in rFIZZ1 was
0.0051 ng/ml by Limulus assay (Lonza). All experiments were approved by
the University of California San Diego Institutional Animal Care and Use
BAL cellular analysis, lung processing, and FACS
BAL and lung processing were performed as previously described (16).
BAL fluid was obtained by intratracheal insertion of a catheter and five
lavages with 0.8 ml 2% filtered BSA (Sigma). The right lung was tied off,
removed, and snap-frozen in liquid nitrogen for RNA isolation or ELISA.
The left lung was instilled with 0.4 ml 4% paraformaldehyde and placed in
paraformaldehyde for paraffin embedding and staining. To obtain lung
single-cell suspensions, lungs were minced and shaken vigorously in RPMI
1640 with 2 mg/ml collagenase and 1 mg/ml DNAse I for 40 min. Lung
cells were isolated using a 70-mm cell strainer.
to block FcRs and then stained with PE-conjugated Siglec-F, FITC-
conjugated CD11c, and allophycocyanin-conjugated Gr-1 (eBiosciences)
for 30 min. BAL cells were washed with FACS buffer, and eosinophils
were identified as the Siglec-F+CD11c2population (17). FACS was
performed using an Accuri C6 flow cytometer and analyzed with FlowJo
software (Tree Star, Ashland, OR).
ELISA for cytokines and total IgE
ELISAof lung homogenateIL-5 and IL-13 (R&DSystems) wasperformed,
as previously described (16). ELISA for IL-33 (R&D Systems) was per-
formed on BAL supernatant. Serum total IgE was performed with an IgE
ELISA kit (BD Biosciences), according to the manufacturer’s instructions,
and all ELISA plates were read with a BioRad Model 680 microplate
Isolation of airway epithelial cells
To study which epithelial genes were induced by Alternaria, we adapted
epithelial brushing, BAL was performed to remove BAL cells. The bronchial
brushing was performed using a sterile plastic feeding tube (Solomon Sci-
entific) modified by removal of the rubber bulb, sanding to create roughness,
and autoclaving. The tube was inserted into the right main and left main
Microarray and real-time RT-PCR analysis
Airway epithelial cells obtained by bronchial brushing were lysed by
multiple passages through an 18G needle. RNA was then extracted,
according to the manufacturer’s protocol (Qiagen). Isolated epithelial RNA
with sufficient purity (A260/280 = 1.97–2.12) and yield (8.99–12.66 ng/ml)
was used for microarray analysis (GeneChip Mouse Gene 1.0 ST; Affy-
metrix). Gene chip results were confirmed by quantitative real-time PCR
and analyzed with Vampire software. Microarray data have been de-
posited in the Gene Expression Omnibus database (http://www.ncbi.nlm.
nih.gov/geo/query/acc.cgi?acc=GSE34764) and given accession number
GSE34764. For whole-lung RNA extraction, the lung was homogenized in
TRIzol reagent (Invitrogen), and RNA was extracted according to the
manufacturer’s protocol (Qiagen). RNA yield and purity were measured
with the Nanodrop 1000 (Thermo Scientific). Single-strand cDNA was
prepared by reverse transcription of 1 mg total RNA with the SuperScript
III kit (Invitrogen). FIZZ1 was quantitated by amplification of cDNA in
SYBR Green Supermix (Applied Biosystems) using the following primer
pairs: forward, 59-CCC TTC TCA TCT GCA TCT C-39 and reverse, 59-
CAG TAG CAG TCA TCC CAG CA-39. Triplicates of samples were run
with the mean value used for quantification.
WT and STAT62/2bone marrow chimeras
WT CD45.1 and STAT62/2(CD45.2+) mice, purchased from Jackson
Laboratories, received Sulfatrim antibiotic (5 ml/200 ml; Med Vet Inter-
national) for 1 wk prior and 2 wk after receiving donor bone marrow.
Recipient mice were irradiated twice with 450 rad, separated by 2 h. Donor
bone marrow was isolated from both tibias and fibulas, and 15 million cells
were injected to recipient via tail veins. Mice were rested for 6 wk before
Alternaria challenge. Efficiency of chimerism was assessed by FACS for
congenic markers CD45.1 and CD45.2 on BAL cells.
FIZZ1 lung-binding assay
The FIZZ1 lung-binding assay was adapted from a previously described
method to detect FIZZ1 binding to mouse splenocytes (19). The strategy
involved incubating naive WT lung cells with rFIZZ1, followed by the
addition of primary and secondary Abs. Negative control lung samples
were not incubated with rFIZZ1 but were identically processed otherwise.
To determine whether FIZZ1 binding colocalized with lung fibroblasts,
single-cell suspensions from lungs of naive WT mice and collagen-1 GFP
reporter mice were incubated with 0.5 mg rFIZZ1 or FACS buffer alone for
60 min at 4˚C. Cells were washed and incubated with Fc blocking Ab for
30 min and stained with allophycocyanin-CD45 and polyclonal rabbit anti-
FIZZ1 (PeproTech) for 30 min, followed by the addition of PE-conjugated
anti-rabbit Fab fragment (eBiosciences). Cells from WT mice were also
stained with FITC-CD11c.
For collagen-1 and FIZZ1 staining, lung samples were deparaffinized by
sequential placement in xylene and ethanol (16). Staining for collagen-1
was performed with a polyclonal Ab (Millipore) at 1:500 concentration,
and staining for FIZZ1 was performed with rabbit polyclonal Ab (Pepro-
Tech) at 1:1000 concentration. Tyramide Signal Amplification Kit #41
(Invitrogen) was used for fluorescent signal amplification, with subsequent
DAPI staining (Vector Laboratories). Lung airways were visualized with
a DM2500 microscope (Leica Microsystems).
Remodeling analysis and epithelial thickness
Paraffinized lung sections were stained with Masson’s trichrome, and the
area of peribronchial fibrosis on trichrome-stained sections was quantified
by analysis with Image-Pro Plus software (16). All slides were blinded,
and results are expressed as the area of positive staining per micrometer
length of bronchiole basement membrane. H&E-stained lung sections were
used to evaluate the thickness of the epithelium in micrometers was
measured from the bottom of the basement membrane to the mucosal
surface of the bronchial epithelium. Six individual areas per airway were
measured, and a minimum of four airways was analyzed per slide. All
measurements were done with Image-Pro Plus software.
In some experiments, invasive pulmonary-function testing was performed
using the Flexivent system (Scireq, Montreal, Canada), and airway resis-
tance was analyzed by Scireq Flexivent 5.1 software, as previously de-
scribed (16). Briefly, mice were anesthetized, cannulated via the trachea,
and administered increasing doses of methacholine, and airway resistance
Statistical analysis was performed using Prism Software (GraphPad). The
Mann–Whitney U test or Student t test was used, as indicated.
Alternaria induces acute BAL airway eosinophilia and lung
Th2 cytokine production in naive WT mice
We examined the acute airway inflammatory response to fungal
allergens Alternaria, Aspergillus, and Candida in naive WT mice.
One day after a single intranasal administration of these allergen
extracts, only mice receiving Alternaria developed significant
airway eosinophilia (Fig. 1A). More than 30% of the BAL cells
were eosinophils (Siglec-F+CD11c2cells) after acute Alternaria
exposure compared with ,1% found after instillation of the same
dose of Aspergillus, Candida, or control PBS (Fig. 1A). The total
number of BAL eosinophils was significantly elevated only in the
Alternaria-treated mice (Fig. 1A). Alternaria exposure induced
acute airway eosinophilia in a dose-response manner. Naive WT
mice challenged with increasing doses of Alternaria (10, 50, and
100 mg) had increases in both the percentage and total airway
The Journal of Immunology 2623
eosinophilia with escalating doses (Fig. 1B). Thus, Alternaria
specifically induces airway eosinophilia within 24 h of a single
exposure in naive WT mice in a dose-dependent manner.
Cytokines, including IL-33, IL-5, and IL-13, contribute to Th2-
type responses. We measured levels of these cytokines in the BAL
and lung at 3, 6, and 24 h after Alternaria challenge (Fig. 1C). BAL
levels of IL-33 and lung levels of IL-5 and IL-13 were increased
after Alternaria challenge compared with PBS challenge. Levels
of Il-33 peaked at 3 h and trended down by 24 h. Lung levels of
IL-5 and IL-13 were increased maximally at 6 h. Total serum IgE
levels were not significantly different between Alternaria- and
PBS-challenged mice (mean, 27.9 and 17.5 ng/ml, respectively;
n = 3–4 mice/group). Overall, this suggests that Alternaria induces
rapid Th2 cytokine production in addition to innate eosinophilia.
Bronchial brushing to identify Alternaria-induced epithelial
To identify genes that may be upregulated in the epithelium during
the acute response to a single challenge with Alternaria, we uti-
lized a technique using a bronchial brush to obtain cells from the
airway (18). Cytospun cells from bronchial brushings had the
ultrastructural appearance of ciliated airway epithelial cells and
immunostained positive for the epithelial marker E-cadherin (Fig.
2A). Compared with whole-lung cell suspensions that contained
both CD45+and CD452cells, the cells from the airway brushing
were nearly all CD452by FACS (Fig. 2B). Additionally, nearly
all of the live airway brushing CD452cell population expressed
E-cadherin compared with control Ab staining (Fig. 2B). These
bronchial brushing airway epithelial cells were used to identify
genes induced by acute exposure of naive WT mice to Alternaria.
FIZZ1 is highly induced in the airway epithelium in naive WT
mice after acute Alternaria exposure
Using the technique from Fig. 2, we isolated epithelial RNA from
Alternaria- and PBS-challenged mice for microarray analysis. As
shown in Fig. 3A, FIZZ1 (Retnla) was induced ∼20-fold after
Alternaria exposure in the expression array and was subsequently
confirmed by quantitative PCR to be induced .200-fold compared
with PBS (Fig. 3A, 3B). Other genes with highly elevated tran-
script levels included some critically involved in mucus produc-
tion, including MUC5AC (8-fold) and Clca3 (9-fold). Because
mucus production occurs exclusively in the epithelium, the fact that
these genes were highly induced in Alternaria-challenged mice
supports our airway brushing characterizing an epithelial tran-
scriptome. Chitinase genes Ym1 (Chi3l3) and Chi3l1 are members
of a family of proteins increasingly associated with severe asthma,
and they were induced 5-fold (20, 21). Interestingly, many other
genes significantly upregulated in the epithelium of Alternaria-
exposed mice, compared with PBS-treated mice, included muscle-
related genes and structural proteins that may represent epithelial–
mesenchymal transition proteins, such as MYO18b, a marker for
myocyte differentiation (22).
To visualize FIZZ1 expression in the Alternaria-challenged
airway, we performed immunofluorescent staining of lung sec-
tions. As expected, FIZZ1 was highly expressed in the epithelium
of Alternaria-challenged mice compared with PBS-treated mice
(Fig. 3C). Some subepithelial cells also expressed FIZZ1, al-
though at a significantly lower frequency compared with epithelial
of Alternaria in naive WT mice induces early
eosinophilia and Th2 cytokine production.
Naive mice received a single intranasal chal-
lenge with 100 mg of Alternaria, Candida, or
Aspergillus extract or PBS. (A) BAL was per-
formed 24 h later for eosinophil percentage by
FACS (left panels) and absolute eosinophil
counts (right panels). Eosinophils were de-
fined as Siglec-F+CD11c2cells. (B) Naive
mice were given intranasal administrations of
10, 50, or 100 mg Alternaria extract, and BAL
was performed 1 d later. BAL eosinophil per-
centage (left panels) and total numbers (right
panels) were analyzed atthegivendose.FACS
plots are representative of four to seven mice
per group and dose. Eosinophil numbers for
individual mice are shown. (C) ELISA for
BAL IL-33 (top panel), lung IL-5 (middle
panel), and lung IL-13 (bottom panel) at 3 h, 6
h, and 1d after Alternaria challenge (n = 4
mice/group). PBS data are from the 3-h time
****p , 0.0001, compared with PBS, t test.
Single acute airway exposure
Cells collected by brushing of large airways in naive WT mice were an-
alyzed by morphology and immunostained for E-cadherin. Scale bar, 10
mm. (B) Airway brushing cells pooled from six mice were analyzed by
FACS (middle panel) and compared with lung cells derived from naive WT
mouse lung (left panel) and stained for E-cadherin (right panel). Shaded
graph represents isotype control.
Airway epithelial cells obtained by bronchial brushing. (A)
2624ALTERNARIA-INDUCED FIZZ1 PROMOTES AIRWAY FIBROSIS
expression. FIZZ1 expression was detected in the epithelium of
lung sections as early as 3 h after a single Alternaria challenge and
remained elevated for 5 d (Fig. 3D). In contrast, we detected
minimal expression of epithelial FIZZ1 in lung sections from WT
mice challenged one time with fungal allergens other than Alter-
naria, such as Aspergillus and Candida. Thus, airway epithelial
FIZZ1 is specifically and highly induced early after exposure of
mice to Alternaria and persists for days after one challenge.
Alternaria-induced epithelial FIZZ1 expression and BAL
eosinophilia are STAT6 dependent
The transcription factor STAT6 is critical to IL-4/IL-13 signaling
and Th2 cell development (23) but little is known about its acute
role in airway disease in naive WT mice. To investigate this, WT
and STAT6-deficient (STAT62/2) mice were challenged with
a single dose of Alternaria. Dramatically, FIZZ1 expression was
nearly absent in the epithelium of STAT62/2mice (Fig. 4A). Lung
FIZZ1 mRNA levels were also significantly reduced in acute
Alternaria-challenged STAT62/2mice (Fig. 4B). Additionally,
BAL eosinophilia was strongly reduced in STAT62/2mice com-
pared with WT mice (Fig. 4C). This suggests that both FIZZ1
expression and the acute airway inflammatory events induced by
Alternaria are dependent on STAT6.
Fungal allergens are known to possess significant protease ac-
tivity, and early inflammatory responses in the airway can be
impaired by the addition of protease inhibitors (12, 24). Addi-
tionally, Alternaria was shown in vitro to induce the proallergic
cytokine thymic stromal lymphopoietin (TSLP) in epithelial cells
through PAR-2, suggesting that other epithelial-derived factors,
such as FIZZ1, may be regulated by PAR-2 (14). To test this, we
challenged WT and PAR-2–deficient mice with a single dose of
Alternaria. In contrast to STAT62/2mice, PAR-2–deficient mice
had only a slight reduction in the level of FIZZ1 expression in the
epithelium of stained lung sections and no difference at the lung
transcript level (Fig. 4B). Additionally, the BAL eosinophilia was
unchanged in PAR-2–deficient mice compared with WT mice.
This suggests that PAR-2 is not critical for FIZZ1 epithelial ex-
pression or acute airway eosinophilia after Alternaria challenge.
Bone marrow-derived STAT6 contributes to Alternaria-induced
To address whether STAT6 expressed in lung structural cells or
bone marrow-derived cells contributed to airway eosinophilia and
FIZZ1 expression after Alternaria challenge, we performed bone
marrow chimera studies with WTand STAT62/2mice. Six weeks
after WT mice and STAT62/2mice were irradiated and received
either WT or STAT62/2bone marrow transplantation, mice were
challenged once with Alternaria and analyzed 24 h later. We used
congenic WT mice (CD45.1) to determine the efficiency of chi-
merism in the lung (Fig. 5A). FACS analysis of BAL cells
revealed 94–98% efficiency of chimerism in the lung in all
groups. We next determined the level of BAL eosinophils in these
mice (Fig. 5B). WT CD45.2 mice that received WT CD45.1 bone
marrow developed significant airway eosinophilia when mea-
sured 24 h after one Alternaria challenge. STAT62/2recipients
that received WT bone marrow had similar levels of airway eo-
sinophilia compared with WT mice that received WT bone
marrow. In contrast, WT mice that received STAT62/2bone
marrow had significantly reduced eosinophilia after Alternaria
challenge. This suggests that STAT6 expressed in hematopoietic
cells contributes to the innate eosinophilia after a single Alter-
time and underwent bronchial brushing 24 h later and subsequent RNA isolation for microarray analysis (n = 3 mice/group). (B) Confirmatory quantitative
PCR performed on bronchial brush samples for FIZZ1 (n = 3–4 mice/group). (C and D) Lung sections from mice challenged with Alternaria, Candida, or
Aspergillus extracts or PBS. Immunofluorescent staining was performed for FIZZ1 at 24 h after challenge (C) and 3 h (D, left panel) or 5 d (D, right panel)
after one challenge with Alternaria. Scale bars, 100 mm. *p , 0.01
Epithelial brushing microarray and lung FIZZ1 expression after acute Alternaria airway challenge. (A) Mice received Alternaria or PBS one
The Journal of Immunology 2625
Alternaria-induced epithelial FIZZ1 is dependent on STAT6
expression in lung cells
We performed immunofluorescent staining for FIZZ1 in lung
sections from WT and STAT62/2bone marrow chimeras that
received a single challenge with Alternaria. Stained lung sections
from WT CD45.2 mice that received WT CD45.1 bone marrow
revealed strong FIZZ1 staining in the airway epithelium (Fig. 5C).
Further, WT recipients that received STAT62/2bone marrow had
similar levels of FIZZ1 staining in the airway epithelium com-
pared with WT recipients that received WT bone marrow. How-
ever, STAT62/2mice that received WT bone marrow had
significantly reduced FIZZ1 staining in the airway epithelium after
Alternaria challenge. This suggests that STAT6 in lung cells is
required for Alternaria-induced FIZZ1 expression.
FIZZ1 binds to inflammatory and structural cells in the lung
The receptor for FIZZ1 is unknown, so we used a FIZZ1 lung-
binding assay (19) to identify cell types in the lung that bind to
FIZZ1 (Fig. 6A). Single-cell suspensions from digested lung were
gated on either leukocytes (CD45+) or structural cells (CD452).
Both lung CD45+cells (Fig. 6B) and lung CD452cells (Fig. 6C)
bound to FIZZ1. In the lung CD45+cell population, CD45+
CD11c+cells (consisting of both macrophages and dendritic cells)
displayed significant FIZZ1 binding (Fig. 6B). In contrast, anal-
ysis of CD45+CD11c2cells revealed a smaller population that
bound FIZZ1 (Fig. 6B). The CD452population also displayed
FIZZ1 binding (Fig. 6C). Because the lung CD452population is
composed of structural cells, including fibroblasts, we then per-
formed the same FIZZ1-binding assay using single-cell suspen-
sions from digested lungs from collagen-1 GFP reporter mice in
which lung fibroblasts strongly express GFP (15); we determined
that the CD452collagen-1+population bound to FIZZ1 (Fig. 6D).
These studies suggest that several cell types (macrophages, den-
dritic cells, fibroblasts) within the naive lung may bind and re-
spond to FIZZ1. However, not all cell types in the lung bind
FIZZ1, because only a small population of lung CD45+CD11c2
cells bound FIZZ1.
Intranasal rFIZZ1 administration leads to airway eosinophilia,
increased epithelial thickness, and fibrosis
To evaluate possible roles of FIZZ1 in the airway, we administered
rFIZZ1 to naive WT mice for 5 d and performed BAL and his-
tologic analysis 3 d later. Mice that received rFIZZ1 had elevated
levels of eosinophils in the airways compared with mice that re-
ceived only PBS (Fig. 7A). These mice also had evidence of in-
creased epithelial thickness (Fig. 7B), a feature associated with
severe asthma (25). Elevated levels of peribronchial fibrosis, de-
tected by trichrome staining, and increased collagen-1 immuno-
fluorescent staining were present in lung sections from mice that
received rFIZZ1 compared with those that received PBS (Fig.
7C). To determine whether these changes were associated with
increased airway reactivity, we performed invasive pulmonary
testing in mice receiving rFIZZ1 or PBS, but we did not detect
a difference in airway resistance after increasing doses of meth-
FIZZ1 expression and early eosinophilia
are dependent on STAT6 but not PAR-2.
(A) WT, STAT62/2, or PAR-22/2mice
were challenged with Alternaria, and
FIZZ1 immunofluorescent staining was
performed on lung sections. Scale bar,
100 mm. (B) FIZZ1 mRNA levels mea-
sured in whole-lung samples from WT,
STAT62/2, or PAR-22/2mice. (C) Per-
centage of BAL eosinophils by FACS
(left panel) and total numbers (right
panel) for WT, STAT62/2, or PAR-22/2
mice. n = 4–6 mice/group. *p , 0.005,
**p , 0.01, compared with WT mice, t
Alternaria-induced eosinophilia, but FIZZ1 expression
is dependent on STAT6 in lung structural cells. (A) WT
(CD45.1 and CD45.2) and STAT62/2mice were irra-
diated and injected i.v. with WT or STAT62/2bone
marrow and challenged once with Alternaria 6 wk
later. FACS analysis of BAL cells for percentage of
donor bone marrow cells in airway. (B) Total BAL
eosinophils in WT and STAT62/2bone marrow chi-
meric mice 24 h after single Alternaria challenge. (C)
FIZZ1 immunofluorescent staining of lung sections;
scale bar, 100 mm. Data are representative of 8–10
mice/group (A, C). *p , 0.05, Mann–Whitney test (n =
Bone marrow STAT6 contributes to
2626ALTERNARIA-INDUCED FIZZ1 PROMOTES AIRWAY FIBROSIS
acholine (Fig. 7D). These data suggest that FIZZ1 may have many
roles in the airway, including promoting eosinophilia, epithelial
changes, and peribronchial fibrosis.
Alternaria has been associated with the development, persistence,
and severity of asthma (4). Little is known about the reasons for
the unique pathogenicity of Alternaria. In this study, we demon-
strate that naive WT mice developed significant BAL eosinophilia
following Alternaria challenge. In contrast to Alternaria, nei-
ther Aspergillus nor Candida induced BAL eosinophilia. Gene
microarray analysis of airway epithelial cell brushings demon-
strated that Alternaria-challenged naive WT mice had a 20-fold
increase in the expression of FIZZ1/Retnla, a resistin-like mole-
cule whose increased expression in airway epithelium was con-
firmed by quantitative PCR. Additional genes that were highly
induced by Alternaria in airway epithelium included those in-
volved in mucus expression (MUC5AC, Clca3), as well as chiti-
nase genes (Yim1 and Chi3l1). Lung immunostaining confirmed
strong airway epithelial FIZZ1 expression present as early as 3 h
cytes and structural cells isolated from
naive WT mouse lung. (A) Single-cell
suspensions from two naive WT mouse
lungs were incubated with rFIZZ1,
followed by staining. (B) Lung cells
were stained with CD45 and CD11c.
CD45+cells (left panel) were gated,
and CD11c+and CD11c2populations
(right panels) were analyzed for FIZZ1
binding. (C) CD452cells were gated
and analyzed for FIZZ1 binding. (D)
CD452cells from Col-1 GFP mice
were gated on collagen-1+cells and
analyzed for FIZZ1 binding. Lung cells
were analyzed from pooled samples
from two mice and are the results of
two independent experiments.
rFIZZ1 binds to leuko-
(PBS) for five consecutive days, and BAL and lung were analyzed 3 d later. Total BAL eosinophils were enumerated (n = 4 mice/group). (B) Epithelial
thickness (left panel) was measured in H&E-stained lung sections from mice receiving rFIZZ1 (right panels) or PBS (middle panels) (n = 19–25 airways/
group). Scale bar, top row: 100 mm, bottom row: 50 mm. (C) Lung sections stained with trichrome (top row) and scored (left panel) and stained for collagen-1
(bottom row) (n = 23–25 airways/group). Scale bars, 100 mm. (D) BALB(c) mice were challenged with intranasal rFIZZ1 or PBS for 5 d, and invasive airway
resistance was measured after increasing doses of methacholine (n = 4 mice/group). *p , 0.05, t test; **p , 0.0001, ***p , 0.001, Mann–Whitney test.
Exogenous FIZZ1 induces airway eosinophilia, epithelial thickening, and fibrosis. (A) Naive WT mice received intranasal rFIZZ1 or vehicle
The Journal of Immunology2627
after a single Alternaria challenge that persisted for $5 d and was
significantly reduced in STAT6-deficient, but not PAR-2–deficient,
mice. Bone marrow chimera studies revealed that STAT6 ex-
pressed in lung cells was required for epithelial FIZZ1 expression.
In contrast, STAT6 present in bone marrow-derived cells con-
tributed to airway eosinophilia. Direct administration of rFIZZ1
to naive mice led to airway eosinophilia, peribronchial fibrosis,
and increased thickness of the airway epithelium. Studies inves-
tigating which cells in the lung bind FIZZ1 demonstrated that
both fibroblasts (CD452collagen-1+) and leukocytes (CD45+) in
the lung, in particular CD45+CD11c+macrophages and dendritic
cells, bound to FIZZ1. Thus, Alternaria induces STAT6-dependent
acute airway eosinophilia and epithelial FIZZ1 expression that
promotes airway fibrosis and epithelial thickness. This may pro-
vide some insight into the uniquely pathogenic aspects of Alter-
Our studies identified that the innate acute eosinophilic response
to Alternaria is mediated by STAT6 and not PAR-2. This is in
contrast to a report of mice challenged with intranasal purified
protease from a different fungal allergen (Aspergillus), which did
not require STAT6 for the development of early eosinophilic air-
way inflammation at 18 h but did require intact allergen protease
activity (24). In our studies, we used whole allergen extracts from
Alternaria and Aspergillus instead of purified protease from As-
pergillus, and this may have contributed to the different results.
Other investigators suggested that a PAR-2–mediated pathway
may drive inflammatory events induced by protease allergens,
including Alternaria (12–14, 26). We did not detect a reduction in
eosinophilic inflammation in PAR-2–deficient mice in response to
Alternaria exposure; instead, our data suggest a STAT6-mediated
pathway. In vitro studies showed that PAR-2 is important in
Alternaria-induced bronchial epithelial cell activation and pro-
duction of TSLP (12, 14); however, as demonstrated in our study,
in vivo Alternaria-challenged PAR-2–deficient mice may use al-
ternate STAT6-dependent pathways. Studies also examined the
role of PAR-2 in the adaptive immune response in mice chal-
lenged with a nonfungal cockroach allergen. These studies dem-
onstrated that cockroach challenge did not induce an acute innate
airway eosinophilia in WT mice (26). However, cockroach chal-
lenge in PAR-2–deficient mice resulted in reductions in airway
inflammation in response to cockroach after three challenges over
17 d (13). Thus, differences between the results of our studies
(which do not demonstrate dependence on PAR-2) and those of
other investigators may be related to our use of in vivo models (as
opposed to in vitro studies), the time points studied (acute innate
24 h versus late adaptive-immune response), and the allergen used
(Alternaria versus cockroach).
We detected early increases in IL-33, IL-5, and IL-13 in the lung
after one Alternaria challenge. This is consistent with a recent
report showing that Alternaria induces the release of the pro-Th2
cytokine IL-33 within a few hours in vivo and was dependent upon
extracellular ATP inducing calcium influx in epithelial cells (27).
The same study showed that mice receiving a single challenge
with Alternaria extract had elevated lung levels of Th2 cytokines
IL-5 and IL-13 that were nearly absent in MyD88 and ST2 (IL-
33R)–deficient mice when measured 12 h later. This work high-
lights the complexity of the innate response to Alternaria and the
multiple pathways involved, including TLR/IL-1 family signaling.
We chose a 24 h time point following Alternaria challenge for
gene microarray analysis, because we were interested in deter-
mining which genes expressed at this time point might be re-
sponsible for persistent airway inflammation and remodeling.
Indeed, we identified both FIZZ1 and members of the chitinase
gene family as being expressed at this time point, both candidate
genes for remodeling. Given the very early rise (within a few
hours) of Th2 cytokines detected in the lung in our studies, as well
as those of other investigators (27), the 24-h time point that we
used for microarray analysis was likely not optimal for detection
of cytokine and chemokine genes induced in the initial few hours
following Alternaria challenge (e.g., TSLP, IL-25, IL-33).
To identify genes that are upregulated in the airway epithelium
in vivo after a single Alternaria challenge, we obtained airway
epithelial cells using bronchial brushings, an adapted method ini-
tially developed by two of the authors (K.S. and D.S.) (18). The
airway epithelial cell morphology, positive E-cadherin staining,
absence of CD45, and transcript signature that includes mucus
genes strongly suggest that the predominant cell type obtained by
brushing was airway epithelium. The most highly expressed
transcript, FIZZ1, was also detected in airway epithelium using
a systemic adaptive-immune sensitization protocol with OVA in
alum and subsequent OVA challenge detecting FIZZ1 expression
2–3 wk later (28, 29), but FIZZ1 has not been reported to be in-
duced following an innate stimulus with Alternaria. FIZZ1 is
a resistin-like molecule that shares homology with human resistin
and is known to be induced during Th2-mediated inflammation
(30). FIZZ1 is expressed primarily in the inflamed airway epi-
thelium, as well as by alternatively activated macrophages after
allergen challenge or helminth infection (19, 28). The expression
of FIZZ1 in the lung is regulated by STAT6 following one ex-
posure to Alternaria in naive mice at 24 h, as demonstrated in this
study, as well as at later time points following exposure to allergen
(2 wk) or bleomycin (7 d) (29, 31). The receptors for FIZZ1 re-
main elusive, although reports suggested that signaling occurs
through Bruton’s tyrosine kinase and Notch1 (32, 33). Using
a previously published FIZZ1-capture assay (19), we identified
that both lung CD11+cells (including macrophages and dendritic
cells) and lung fibroblasts bound to FIZZ1. This is consistent with
a previous report that found that rFIZZ1 could bind to splenic
macrophages and dendritic cells (19), and it also extends the ob-
servation to lung fibroblasts, which have not been previously re-
ported to bind to FIZZ1. Although the FIZZ1-binding assay has
limitations in terms of sensitivity, the levels of FIZZ1 binding that
we detected in the lung are similar to those reported in splenic
macrophages and dendritic cells. Prior reports showed that FIZZ1
can induce myofibroblast differentiation, including collagen-1 and
a-smooth muscle actin production, in vitro, suggesting that struc-
tural cells can respond to FIZZ1 directly (34, 35).
We found that rFIZZ1 administered intranasally to the airways of
naive mice leads to eosinophil accumulation. This is consistent
with previous reports suggesting that FIZZ1 regulates eosinophil
chemotaxis in the gastrointestinal tract (36, 37). It is possible that
FIZZ1 participates in the early recruitment of eosinophils after
Alternaria challenge; however, given the relatively low magnitude
of the eosinophilic response induced by rFIZZ1 compared with
that induced by Alternaria, it is likely that other mediators, such as
IL-5 and eotaxin, play larger roles. Previous reports suggested that
FIZZ1 dampens helminth-induced Th2-type inflammation (19, 38)
but that it may promote features of remodeling (34, 35, 39). Air-
way remodeling is an important feature of asthma, and previous
reports suggested that FIZZ1 can induce lung collagen deposition
and myofibroblast differentiation (34, 35, 39). Additionally, re-
petitive intranasal administration of rFIZZ1 was noted to induce
fibrotic changes in a lung granuloma model (40). Thus, FIZZ1 may
have several roles in the lung, depending on the cell types involved
and stage of the inflammatory response. Our data, as well as those
from other investigators, suggested an early proinflammatory and
proremodeling role for FIZZ1 (35, 37, 39, 40); however, other
investigators suggested an anti-inflammatory role during more
2628 ALTERNARIA-INDUCED FIZZ1 PROMOTES AIRWAY FIBROSIS
chronic Th2 responses (19, 38). Further work will be required to
fully elucidate the multiple functions of FIZZ1 in the lung during
chronic inflammatory responses.
In summary, we characterized a unique acute eosinophilic air-
way response to Alternaria that is STAT6 dependent and associ-
ated with significant upregulation of FIZZ1 in airway epithelium.
Further, exogenous FIZZ1 induced airway eosinophilia, epithelial
changes, and airway fibrosis. This underscores the potential im-
portance of FIZZ1 in asthma and airway remodeling and might
translate to a role for related human resistin molecules in human
We thank the University of California San Diego microarray core for pro-
cessing the epithelial microarray.
The authors have no financial conflicts of interest.
1. Bateman, E. D., S. S. Hurd, P. J. Barnes, J. Bousquet, J. M. Drazen,
M. FitzGerald, P. Gibson, K. Ohta, P. O’Byrne, S. E. Pedersen, et al. 2008.
Global strategy for asthma management and prevention: GINA executive sum-
mary. Eur. Respir. J. 31: 143–178.
2. O’Hollaren, M. T., J. W. Yunginger, K. P. Offord, M. J. Somers, E. J. O’Connell,
D. J. Ballard, and M. I. Sachs. 1991. Exposure to an aeroallergen as a possible
precipitating factor in respiratory arrest in young patients with asthma. N. Engl.
J. Med. 324: 359–363.
3. Downs, S. H., T. Z. Mitakakis, G. B. Marks, N. G. Car, E. G. Belousova,
J. D. Leu ¨ppi, W. Xuan, S. R. Downie, A. Tobias, and J. K. Peat. 2001. Clinical
importance of Alternaria exposure in children. Am. J. Respir. Crit. Care Med.
4. Bush, R. K., and J. J. Prochnau. 2004. Alternaria-induced asthma. J. Allergy
Clin. Immunol. 113: 227–234.
5. Pulimood, T. B., J. M. Corden, C. Bryden, L. Sharples, and S. M. Nasser. 2007.
Epidemic asthma and the role of the fungal mold Alternaria alternata. J. Allergy
Clin. Immunol. 120: 610–617.
6. Lyons, T. W., D. B. Wakefield, and M. M. Cloutier. 2011. Mold and Alternaria
skin test reactivity and asthma in children in Connecticut. Ann. Allergy Asthma
Immunol. 106: 301–307.
7. Plaza, V., J. Serrano, C. Picado, J. Cosano, J. Ancochea, A. de Diego,
J. J. Martı ´n, and J. Sanchı ´s; Grupo de Investigadores del Estudio Multice ´ntrico
del Asma de Riesgo Vital. 2003. [Clinical characteristics of the fatal and near-
fatal asthma in Alternaria alternata sensitized patients]. Med. Clin. (Barc.) 121:
8. Neukirch, C., C. Henry, B. Leynaert, R. Liard, J. Bousquet, and F. Neukirch.
1999. Is sensitization to Alternaria alternata a risk factor for severe asthma? A
population-based study. J. Allergy Clin. Immunol. 103: 709–711.
9. Salo, P. M., S. J. Arbes, Jr., M. Sever, R. Jaramillo, R. D. Cohn, S. J. London, and
D. C. Zeldin. 2006. Exposure to Alternaria alternata in US homes is associated
with asthma symptoms. J. Allergy Clin. Immunol. 118: 892–898.
10. Barrett, N. A., and K. F. Austen. 2009. Innate cells and T helper 2 cell immunity
in airway inflammation. Immunity 31: 425–437.
11. Hammad, H., M. Chieppa, F. Perros, M. A. Willart, R. N. Germain, and
B. N. Lambrecht. 2009. House dust mite allergen induces asthma via Toll-like
receptor 4 triggering of airway structural cells. Nat. Med. 15: 410–416.
12. Boitano, S., A. N. Flynn, C. L. Sherwood, S. M. Schulz, J. Hoffman,
I. Gruzinova, and M. O. Daines. 2011. Alternaria alternata serine proteases
induce lung inflammation and airway epithelial cell activation via PAR2. Am. J.
Physiol. Lung Cell. Mol. Physiol. 300: L605–L614.
13. Page, K., J. R. Ledford, P. Zhou, K. Dienger, and M. Wills-Karp. 2010. Mucosal
sensitization to German cockroach involves protease-activated receptor-2.
Respir. Res. 11: 62.
14. Kouzaki, H., S. M. O’Grady, C. B. Lawrence, and H. Kita. 2009. Proteases in-
duce production of thymic stromal lymphopoietin by airway epithelial cells
through protease-activated receptor-2. J. Immunol. 183: 1427–1434.
15. Taura, K., K. Miura, K. Iwaisako, C. H. Osterreicher, Y. Kodama, M. Penz-
Osterreicher, and D. A. Brenner. 2010. Hepatocytes do not undergo epithelial-
mesenchymal transition in liver fibrosis in mice. Hepatology 51: 1027–1036.
16. Doherty, T. A., P. Soroosh, N. Khorram, S. Fukuyama, P. Rosenthal, J. Y. Cho,
P. S. Norris, H. Choi, S. Scheu, K. Pfeffer, et al. 2011. The tumor necrosis factor
family member LIGHT is a target for asthmatic airway remodeling. Nat. Med.
17. Stevens, W. W., T. S. Kim, L. M. Pujanauski, X. Hao, and T. J. Braciale. 2007.
Detection and quantitation of eosinophils in the murine respiratory tract by flow
cytometry. J. Immunol. Methods 327: 63–74.
18. Sugimoto, K., M. Kudo, A. Sundaram, X. Ren, K. Huang, X. Bernstein,
Y. Wang, W. W. Raymond, D. J. Erle, M. Abrink, et al. 2012. The avb6 integrin
modulates airway hyperresponsiveness in mice by regulating intraepithelial mast
cells. J. Clin. Invest. 122: 748–758.
19. Nair, M. G., Y. Du, J. G. Perrigoue, C. Zaph, J. J. Taylor, M. Goldschmidt,
G. P. Swain, G. D. Yancopoulos, D. M. Valenzuela, A. Murphy, et al. 2009.
Alternatively activated macrophage-derived RELM-alpha is a negative regulator
of type 2 inflammation in the lung. J. Exp. Med. 206: 937–952.
20. Chupp, G. L., C. G. Lee, N. Jarjour, Y. M. Shim, C. T. Holm, S. He, J. D. Dziura,
J. Reed, A. J. Coyle, P. Kiener, et al. 2007. A chitinase-like protein in the lung
and circulation of patients with severe asthma. N. Engl. J. Med. 357: 2016–
21. Ober, C., Z. Tan, Y. Sun, J. D. Possick, L. Pan, R. Nicolae, S. Radford,
R. R. Parry, A. Heinzmann, K. A. Deichmann, et al. 2008. Effect of variation in
CHI3L1 on serum YKL-40 level, risk of asthma, and lung function. N. Engl. J.
Med. 358: 1682–1691.
22. Salamon, M., C. Millino, A. Raffaello, M. Mongillo, C. Sandri, C. Bean,
E. Negrisolo, A. Pallavicini, G. Valle, M. Zaccolo, et al. 2003. Human MYO18B,
a novel unconventional myosin heavy chain expressed in striated muscles moves
into the myonuclei upon differentiation. J. Mol. Biol. 326: 137–149.
23. Chen, W., and G. K. Khurana Hershey. 2007. Signal transducer and activator of
transcription signals in allergic disease. J. Allergy Clin. Immunol. 119: 529–541;
24. Kiss, A., M. Montes, S. Susarla, E. A. Jaensson, S. M. Drouin, R. A. Wetsel,
Z. Yao, R. Martin, N. Hamzeh, R. Adelagun, et al. 2007. A new mechanism
regulating the initiation of allergic airway inflammation. J. Allergy Clin.
Immunol. 120: 334–342.
25. Cohen, L., X. E, J. Tarsi, T. Ramkumar, T. K. Horiuchi, R. Cochran,
S. DeMartino, K. B. Schechtman, I. Hussain, M. J. Holtzman, and M. Castro;
and the NHLBI Severe Asthma Research Program (SARP). 2007. Epithelial cell
proliferation contributes to airway remodeling in severe asthma. Am. J. Respir.
Crit. Care Med. 176: 138–145.
26. Day, S. B., P. Zhou, J. R. Ledford, and K. Page. 2010. German cockroach frass
proteases modulate the innate immune response via activation of protease-
activated receptor-2. J. Innate Immun. 2: 495–504.
27. Kouzaki, H., K. Iijima, T. Kobayashi, S. M. O’Grady, and H. Kita. 2011. The
danger signal, extracellular ATP, is a sensor for an airborne allergen and triggers
IL-33 release and innate Th2-type responses. J. Immunol. 186: 4375–4387.
28. Holcomb, I. N., R. C. Kabakoff, B. Chan, T. W. Baker, A. Gurney, W. Henzel,
C. Nelson, H. B. Lowman, B. D. Wright, N. J. Skelton, et al. 2000. FIZZ1,
a novel cysteine-rich secreted protein associated with pulmonary inflammation,
defines a new gene family. EMBO J. 19: 4046–4055.
29. Stu ¨tz, A. M., L. A. Pickart, A. Trifilieff, T. Baumruker, E. Prieschl-Strassmayr,
and M. Woisetschla ¨ger. 2003. The Th2 cell cytokines IL-4 and IL-13 regulate
found in inflammatory zone 1/resistin-like molecule alpha gene expression by
a STAT6 and CCAAT/enhancer-binding protein-dependent mechanism. J.
Immunol. 170: 1789–1796.
30. Nair, M. G., K. J. Guild, and D. Artis. 2006. Novel effector molecules in type 2
inflammation: lessons drawn from helminth infection and allergy. J. Immunol.
31. Liu, T., H. Jin, M. Ullenbruch, B. Hu, N. Hashimoto, B. Moore, A. McKenzie,
N. W. Lukacs, and S. H. Phan. 2004. Regulation of found in inflammatory zone 1
expression in bleomycin-induced lung fibrosis: role of IL-4/IL-13 and mediation
via STAT-6. J. Immunol. 173: 3425–3431.
32. Su, Q., Y. Zhou, and R. A. Johns. 2007. Bruton’s tyrosine kinase (BTK) is
a binding partner for hypoxia induced mitogenic factor (HIMF/FIZZ1) and
mediates myeloid cell chemotaxis. FASEB J. 21: 1376–1382.
33. Liu, T., B. Hu, Y. Y. Choi, M. Chung, M. Ullenbruch, H. Yu, J. B. Lowe, and
S. H. Phan. 2009. Notch1 signaling in FIZZ1 induction of myofibroblast dif-
ferentiation. Am. J. Pathol. 174: 1745–1755.
34. Dong, L., S. J. Wang, B. Camoretti-Mercado, H. J. Li, M. Chen, and W. X. Bi.
2008. FIZZ1 plays a crucial role in early stage airway remodeling of OVA-
induced asthma. J. Asthma 45: 648–653.
35. Liu, T., S. M. Dhanasekaran, H. Jin, B. Hu, S. A. Tomlins, A. M. Chinnaiyan,
and S. H. Phan. 2004. FIZZ1 stimulation of myofibroblast differentiation. Am. J.
Pathol. 164: 1315–1326.
36. Munitz, A., L. Seidu, E. T. Cole, R. Ahrens, S. P. Hogan, and M. E. Rothenberg.
2009. Resistin-like molecule alpha decreases glucose tolerance during intestinal
inflammation. J. Immunol. 182: 2357–2363.
37. Munitz, A., A. Waddell, L. Seidu, E. T. Cole, R. Ahrens, S. P. Hogan, and M. E.
Rothenberg. 2008. Resistin-like molecule alpha enhances myeloid cell activation
and promotes colitis. J. Allergy Clin. Immunol. 122: 1200–1207.e1.
38. Pesce, J. T., T. R. Ramalingam, M. S. Wilson, M. M. Mentink-Kane,
R. W. Thompson, A. W. Cheever, J. F. Urban, Jr., and T. A. Wynn. 2009. Retnla
(relmalpha/fizz1) suppresses helminth-induced Th2-type immunity. PLoS
Pathog. 5: e1000393.
39. Yamaji-Kegan, K., Q. Su, D. J. Angelini, A. C. Myers, C. Cheadle, and R. A. Johns.
2010. Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMalpha) increases
lung inflammation and activates pulmonary microvascular endothelial cells via an
IL-4-dependent mechanism. J. Immunol. 185: 5539–5548.
40. Ito, T., M. Schaller, T. Raymond, A. D. Joshi, A. L. Coelho, F. G. Frantz,
W. F. Carson, IV, C. M. Hogaboam, N. W. Lukacs, T. J. Standiford, et al. 2009.
Toll-like receptor 9 activation is a key mechanism for the maintenance of chronic
lung inflammation. Am. J. Respir. Crit. Care Med. 180: 1227–1238.
The Journal of Immunology2629