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Chlamydial Respiratory Infection during
Allergen Sensitization Drives Neutrophilic
Allergic Airways Disease
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of June 17, 2017.
Jay C. Horvat, Malcolm R. Starkey, Richard Y. Kim,
Kenneth W. Beagley, Julie A. Preston, Peter G. Gibson, Paul
S. Foster and Philip M. Hansbro
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Copyright © 2010 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2010; 184:4159-4169; Prepublished online 12
March 2010;
doi: 10.4049/jimmunol.0902287
http://www.jimmunol.org/content/184/8/4159
The Journal of Immunology
Chlamydial Respiratory Infection during Allergen
Sensitization Drives Neutrophilic Allergic Airways Disease
Jay C. Horvat,* Malcolm R. Starkey,* Richard Y. Kim,* Kenneth W. Beagley,*,†
Julie A. Preston,* Peter G. Gibson,* Paul S. Foster,* and Philip M. Hansbro*
A
sthma is characterized by allergic airways inflammation
and recurrent episodes of wheezing, breathlessness, and
cough (1, 2). Aberrant CD4+ Th2 responses to environmental Ags play pivotal roles in the development of disease (3,
4). Upon exposure to specific stimuli, affected individuals typically mount strong Th2 responses with increased levels of IL-4,
IL-5, and IL-13 that promote the hallmark features of asthma,
which include mucus-secreting cell (MSC) hyperplasia, eosinophil
influx, and airways hyperresponsiveness (AHR) (5–7).
Respiratorychlamydialinfectionisassociated with the development
of asthma in children and adults (reviewed in Ref. 8). However,
*Centre for Asthma and Respiratory Disease and Hunter Medical Research Institute,
The University of Newcastle, Newcastle, New South Wales; and †Institute of Health
and Biomedical Innovation, Queensland University of Technology, Brisbane,
Queensland, Australia
Received for publication July 29, 2009. Accepted for publication February 12, 2010.
This work was supported by grants from the National Health and Medical Research
Council of Australia (project Grants 401238 and 569219), the Asthma Foundation of
New South Wales, and the Rebecca Cooper Medical Research Foundation, as well as
by The University of Newcastle project grants and the Hunter Medical Research
Institute infrastructure grants.
Address correspondence and reprint requests to Dr. Philip M. Hansbro, Centre for
Asthma and Respiratory Disease and Hunter Medical Research Institute, Level 3,
David Maddison Clinical Sciences Building, Newcastle NSW 2300, Australia. Email address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: AAD, allergic airways disease; AHR, airways
hyperresponsiveness; AL, airway lumen; BALF, bronchoalveolar lavage fluid; BM,
basement membrane; DC, dendritic cell; i.n., intranasal; KC, keratinocyte chemokine; mDC, myeloid dendritic cell; MIP-2, macrophage inflammatory protein-2;
MLN, mediastinal lymph node; MSC, mucus-secreting cell; pDC, plasmacytoid dendritic cell; TARC, thymus- and activation-regulated chemokine.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902287
chlamydial infections typically induce potent Th1-type immune responses (9–11), and it remains unknown how these infections influence Th2-mediated asthma. The association may be driven by
chlamydial infection-induced alterations in the inflammatory profile
and phenotype of asthma, such as a switch from eosinophil-dominated
inflammation to that of neutrophilia in the allergic lung.
Indeed, different inflammatory subtypes of asthma that may be
related to infection have recently been recognized. A proportion of
asthmatics (∼25%; neutrophilic asthmatics) have strong neutrophilic inflammation when stable or during asthmatic episodes (12–
16). Neutrophilic asthmatics also have reduced eosinophilic inflammation and AHR (15, 16). Furthermore, increased numbers of
neutrophils in the sputum of asthmatics correlate with increased Th1
and reduced Th2 responses in the airways (17). Neutrophilic inflammation in asthma has been associated with viral respiratory
infection (18) and correlates with increased activation of innate
immune factors, namely, increased IL-8 and TLR-2 and TLR-4
expression in sputum (16). Neutrophil levels are also elevated in the
sputum of exacerbating asthmatics with evidence of chlamydial
respiratory infection, compared with uninfected individuals (18, 19).
Furthermore, chlamydial infection of mice induces a robust pulmonary neutrophil influx that is mediated by the TLR-dependent
production of keratinocyte chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), the mouse orthologs of human IL-8
(20, 21). Taken together, these observations suggest that chlamydial
infection may be linked to asthma with a neutrophilic phenotype.
Other work has shown that Chlamydia muridarum infection
prior to the induction of allergic airways disease (AAD) in mice
suppresses pulmonary eosinophil influx, MSC hypersecretion, and
allergen-specific Th2 cytokine responses, suggesting that infection
may protect against Th2-mediated inflammation during AAD (22,
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Neutrophilic asthma is a prevalent, yet recently described phenotype of asthma. It is characterized by neutrophilic rather than
eosinophilic airway inflammation and airways hyperresponsiveness (AHR) and may have an infectious origin. Chlamydial respiratory infections are associated with asthma, but how these Th1-inducing bacteria influence Th2-mediated asthma remains unknown. The effects of chlamydial infection on the development of asthma were investigated using a BALB/c mouse model of
OVA-induced allergic airways disease (AAD). The effects of current and resolved Chlamydia muridarum infection during OVA
sensitization on AAD were assessed and compared with uninfected and nonsensitized controls. Current, but not resolved, infection
attenuated hallmark features of AAD: pulmonary eosinophil influx, T cell production of IL-5, mucus-secreting cell hyperplasia,
and AHR. Current infection also induced robust OVA-driven neutrophilic inflammation and IFN-g release from T cells. The
phenotype of suppressed but persistent Th2 responses in association with enhanced neutrophilia is reminiscent of neutrophilic
asthma. This phenotype was also characterized by increased pulmonary IL-12 and IL-17 expression and activation of APCs, as
well as by reduced thymus- and activation-regulated chemokine. Inhibition of pulmonary neutrophil influx during infection
blocked OVA-induced neutrophilic inflammation and T cell IFN-g production and reversed the suppressive effects on mucussecreting cell hyperplasia and AHR during AAD. These changes correlated with decreased IL-12 and IL-17 expression, increased
thymus- and activation-regulated chemokine and altered APC activation. Blocking IFN-g and IL-17 during OVA challenge had no
effect. Thus, active chlamydial respiratory infection during sensitization enhances subsequent neutrophilic inflammation and Th1/
Th17 responses during allergen exposure and may have a role in the pathogenesis of neutrophilic asthma. The Journal of
Immunology, 2010, 184: 4159–4169.
4160
CHLAMYDIAL RESPIRATORY INFECTION AND NEUTROPHILIC ASTHMA
Materials and Methods
Experimental models
Six-week-old female BALB/c mice were used throughout. We have previously shown that bacterial recovery (using both real-time PCR and tissue
culture) and histopathological status of chlamydial pulmonary infection in
adult mice peaks between 10 and 15 d postinoculation. Clearance of infection and the majority of inflammation occur within 21 and 45 d, respectively (9). So that infections were either resolved or at their peak
during OVA exposure, mice were infected 45 (D-45) or 7 (D-7) days before
OVA sensitization (Fig. 1A). Mice were infected via the intranasal (i.n.)
route with 100 inclusion-forming units C. muridarum (30 ml, sucrosephosphate-glutamate buffer). Mice were sensitized to OVA (50 mg [SigmaAldrich, Castle Hill, Australia], 1 mg Rehydrogel [Reheis, Berkeley
Heights, NJ], and 200 ml 0.9% w/v sterile saline) on day 0 by i.p. injection
and were challenged i.n. with OVA 12–15 d later (D-45/OVA and D-7/
OVA) (9). One day later, mice were euthanized and AAD was characterized. Controls were; uninfected and allergic (OVA), uninfected and nonallergic (Sal [sham-sensitized with saline]) or infected and nonallergic
(D-45/Sal and D-7/Sal). All controls received i.n. OVA challenge. We have
shown that the vehicle for chlamydial infection, sucrose-phosphate-glutamate,
has no effect on immunological or physiological responses examined (9).
All procedures were approved by the animal ethics committee, University
of Newcastle.
Chlamydial infection
Pulmonary Chlamydia numbers were determined by real-time PCR of
DNA extracted from lung homogenates (9).
Local and systemic leukocytes
Cytospins were prepared from 2 ml bronchoalveolar lavage fluid (BALF),
and blood smears were prepared from whole blood (9). Cytospins and blood
smears were stained with May-Grunwald-Giemsa, and differential leukocyte counts were determined using morphological criteria (200 cells by
light microscopy [340]) (9). All samples were coded and counts performed in a blinded fashion.
Histopathological findings and airway inflammation
Lungs were perfused (0.9% saline) and fixed by inflation with formalin (1.5
ml, 10% buffered formalin; Sigma-Aldrich). The trachea was tied off, and
the entire heart-lung block was removed from the thoracic cavity and
immersed in buffered formalin. The left lung was excised and embedded in
paraffin, sectioned (4–6 mm), and stained using chrome salt fixation (for
eosinophils) or periodic acid-Schiff (for MSCs). Average eosinophil and
MSC counts in 10 3 100-mm fields adjacent to the basement membrane
(eosinophils) and within the airway lumen (MSCs) were determined using
FIGURE 1. Study protocols. A, Groups were infected i.n. with C.
muridarum (100 inclusion-forming units, 30 ml sucrose-phosphate-glutamate
buffer) 45 [(i) Resolved] or 7 [(ii) Current] d prior to i.p. sensitization with
OVA (D-45/OVA and D-7/OVA, respectively). AAD was induced by i.n.
OVA challenges 12–15 d postsensitization. AAD was assessed 24 h after
the final challenge. Controls were 1) uninfected, allergic (OVA); 2) uninfected, nonallergic (Sal); or 3) infected, sham-sensitized (D-45/Sal and
D-7/Sal) groups. B, Groups were infected with C. muridarum 7 d prior to
OVA sensitization (day 0) and were treated with anti-KC and anti–MIP-2
mAbs (aKCMIP2) on 3, 5, 7, and 9 d postinfection (D-4, -2, 0 and 2,
aKCMIP2/D-7/OVA). AAD was induced by i.n. OVA challenges on days
12–15 postsensitization. The effect of treatment on pulmonary and systemic leukocyte numbers was assessed at the peak of infection (day 3) and
before OVA challenge (day 12). Chlamydial numbers in lung tissue were
determined at day 3. AAD was assessed 24 h after the final challenge.
Controls were 1) uninfected, isotype-treated, allergic (Iso/OVA); 2) uninfected, Ab-treated, allergic (aKCMIP2/OVA); and 3) infected, isotypetreated, allergic (Iso/D-7) groups. C, Groups were infected with C. muridarum 7 d prior to OVA sensitization (day 0). AAD was induced by i.n.
OVA challenges on days 12–15 postsensitization. Experimental groups
were treated with anti–IFN-g mAb (aIFN-g) on days 11, 13, and 15 or
anti–IL-17 mAb (aIL-17) on days 11 and 13 during OVA challenge (aIFNg/D-7/OVA or aIL-17/D-7/OVA, respectively). The effect of treatment on
BALF leukocyte numbers was assessed 24 h after the final challenge.
Controls were 1) uninfected, isotype-treated, allergic (Iso/OVA); 2) uninfected, Ab-treated, allergic (aIFN-g/OVA or aIL-17/OVA); and 3) infected, isotype-treated, allergic (Iso/D-7) groups.
light microscopy (9). All samples were coded and counts performed in
a blinded fashion.
T cell cytokines
Cytokine release from lung draining mediastinal lymph node (MLN) cells
was determined by ELISA (9). Cells (5 3 106) were cultured in RPMI (1
ml, 10% FBS, 20 mM HEPES, 10 mg/ml penicillin/streptomycin, 2 mM
L-glutamine, and 50 mM 2-ME) and restimulated with OVA (200 mg/ml)
for 6 d. Concentrations of IFN-g, IL-5, and IL-13 were determined in
supernatants using mouse IFN-g and IL-5 (BD Biosciences, North Ryde,
Australia) and IL-13 (R&D Systems, Gymea, Australia) Ab combinations
according to the manufacturers’ instructions (9).
Lung function
Lung function was measured using whole-body, invasive plethysmography
in restrained and anesthetized animals (9). Mice were anesthetized with
ketamine/xylazine (80–100 mg/kg and 10 mg/kg, respectively; Troy
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23). However, we have demonstrated that a C. muridarum lung infection in early life results in the generation of both allergen-specific
Th2 and Th1 responses. Although the generation of this mixed
phenotype results in a suppression of Th2 cytokine production and
eosinophil into the airways, the severity of MSC hyperplasia and
AHR is increased and inflammation prolonged in later life (9).
Clinical studies have also found that respiratory infection may be
related to strong IFN-g and Th1-type responses in moderate to severe asthmatics (24–27). Thus, Chlamydia-induced Th1 responses
in the asthmatic lung may enhance or modify disease, although Th2mediated inflammation may be suppressed. Indeed, the presence of
both allergen-specific Th1 and Th2 cells in the airways enhance
disease in a model of OVA-induced asthma (28).
In this study, we investigated whether Chlamydia-induced neutrophilic and Th1 responses during allergen sensitization may subsequently enhance or modify AAD, despite reducing Th2-mediated
inflammation. Because immune responses vary during chlamydial
lung infection (11, 29), the timing of infection relative to allergen
exposure may be important in determining the effects on AAD.
Thus, we employed murine models of resolved or current chlamydial lung infection and OVA-induced AAD to examine the effect
on neutrophils, Th1 responses, and Th2-mediated AAD. We demonstrate that current chlamydial infection induces the development
of AAD with a phenotype reminiscent of neutrophilic asthma.
The Journal of Immunology
4161
Laboratories, Smithfield, Australia), and the trachea was cannulated and
connected to an inline aerosol and ventilator, which were attached to
a preamplifier and computer (Buxco Electronics, Sharon, CT). Airways
resistance and dynamic compliance were determined in response to increasing doses of methacholine (Sigma-Aldrich).
Suppression of neutrophil influx
To assess the influence of neutrophil influx into the lungs during chlamydial
infection on infection-associated changes during AAD, mice were injected i.p.
with a combination of anti-KC and anti–MIP-2 (18 mg and 4 mg, respectively
[aKCMIP2, R&D Systems], in 200 ml PBS) or corresponding isotype control
Abs (18 mg and 4 mg, Rat IgG2a and IgG2b, respectively [Iso; R&D Systems]). Treatment occurred 3, 5, 7, and 9 d postinfection. On day 7, mice were
sensitized and subsequently challenged with OVA (Fig. 1B). One day later,
mice were euthanized and AAD was characterized. Controls were uninfected
and treated with aKCMIP2 or isotype control Abs.
Pulmonary cytokine expression
Lung tissue cells
Single-cell suspensions of collagenase D-digested lungs (1 3 106 cells) were
stained for surface markers (31). Cells were characterized as follows: neutrophils, CD11c2CD11bhiGr1hi, low-moderate forward scatter and moderate-high side scatter; plasmacytoid dendritic cells (pDC), CD11clowCD11b2
Gr1+, moderate forward scatter and low-moderate side scatter; and myeloid
dendritic cells (mDC), CD11c+CD11bhiGr12, moderate forward scatter and
low-moderate side scatter. Maturation and activation of dendritic cells (DCs)
were assessed by determination of MHC II expression. The functional capacity of pulmonary APCs (CD11c+) was also assessed by analyzing expression of the costimulatory markers CD80 and CD86.
Depletion of IFN-g and IL-17
Groups were infected with C. muridarum 7 d prior to OVA sensitization.
AAD was induced by i.n. OVA challenges on days 12–15 postsensitization.
To assess the role of IFN-g and IL-17 in infection-induced changes during
AAD, mice were injected i.p. with anti–IFN-g or anti–IL-17 (10 mg
[aIFN-g] or 50 mg [aIL-17], respectively [R&D Systems], in 200 ml PBS)
or corresponding isotype control Abs (10 mg and 50 mg, Rat IgG2a, respectively [Iso, R&D Systems]) on days 11, 13, and 15 (aIFN-g) or days
11 and 13 (aIL-17) during OVA challenge. One day after the final OVA
challenge, mice were euthanized and AAD was characterized (Fig. 1C).
Controls were uninfected and treated with aIFN-g, aIL-17, or isotype
control Abs.
Statistics
Results are presented as mean 6 SEM from between 4 and 8 mice, in duplicate or triplicate experiments. Statistical significance of whole data sets
was initially confirmed using one-way ANOVA. The Wilcoxon rank-sum test
was used for nonparametric tests (Mann-Whitney U test for two independent
samples). Comparison of airways resistance and dynamic compliance between groups was performed using one-way repeated measures ANOVA, and
significance was assessed for the entire dose-response curve.
Results
Development of AAD
Compared with uninfected, nonallergic (Sal) groups, allergic
groups without infection (OVA; Fig. 1) displayed hallmark features of Th2-driven AAD characterized by eosinophil influx into
the airways (Fig. 2) and tissue (Fig. 3), airways MSC hyperplasia
FIGURE 2. Airway inflammatory cell influx postinfection and AAD.
Experimental groups were infected 7 (D-7/OVA) or 45 (D-45/OVA) d prior
to OVA sensitization and challenge, as described in Fig. 1A. Total leukocyte (A), neutrophil (B), and eosinophil (C) numbers were determined in
BALF. Results are presented as mean 6 SEM from n $ 4, in duplicate,
with allergic groups represented by black bars and nonallergic controls by
white bars. Significant differences between groups are shown as pp , 0.05,
ppp , 0.01, and pppp , 0.001.
(Fig. 3), OVA-specific Th2 cytokine responses from MLN T cells
(Fig. 4), and AHR (Fig. 5).
Infection and AAD
To investigate the effect of current or resolved infection on the
development of AAD, groups were infected 7 or 45 d before
sensitization (Fig. 1A). There were no significant differences in
bacterial recovery from the lungs of infected allergic groups,
compared with infected, nonallergic controls (data not shown).
Current infection during sensitization alters cellular responses
during AAD
The effects of infection on cellular responses during AAD were
assessed. Current (D-7/OVA), but not resolved (D-45/OVA), infection during sensitization had substantial effects on immune cell
influx into the lungs during OVA-induced AAD.
Although current infection had minimal effects on the influx of
total leukocytes (Fig. 2A), macrophages, or lymphocytes (not
shown) into the BALF during AAD, the numbers of neutrophils were
significantly increased, whereas eosinophil influx was attenuated
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Pulmonary cytokine expression was evaluated by real-time PCR (30). Total
RNA was extracted from whole-lung tissue, using TRIzol according to the
manufacturer’s instructions (Invitrogen, Mount Waverley, Australia). Reverse transcription of RNA (1000 ng) was performed using Superscript III
and random hexamer primers (Invitrogen). Relative abundance of genes
was determined by comparison with the reference gene hypoxanthineguanine phosphoribosyltransferase, using a Prism7000 Sequence Detection
System (Applied Biosystems, Scoresby, Australia). Primers used were
IL-12, Fwd 59-ACC AGC TTC TTC ATC AGG GAC ATC-39, Rev 59-TCT
TCT CTA CGA GGA ACG CAC CTT-39; IL-17, Fwd 59-CAA ACA TGA
GTC CAG GGA GAG CTT-39, Rev 59-ACT GAG CTT CCC AGA TCA
CAG AGG-39; thymus- and activation-regulated chemokine (TARC), Fwd
59-AGT GGA GTG TTC CAG GGA TG-39, Rev 59- CTG GTC ACA GGC
CGT TTT AT-39; and hypoxanthine-guanine phosphoribosyltransferase,
Fwd 59- AGG CCA GAC TTT GTT GGA TTT GAA-39, Rev 59- CAA
CTT GCG CTC ATC TTA GGC TTT-39.
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CHLAMYDIAL RESPIRATORY INFECTION AND NEUTROPHILIC ASTHMA
compared with that in uninfected, allergic (OVA) controls (Fig. 2B,
2C). By contrast, resolved infection suppresses eosinophil numbers
only in the BALF. Current, but not resolved, infection also attenuated the numbers of tissue eosinophils and MSCs in the airways (Fig.
3A, 3B). Notably, the increased recruitment of neutrophils to the
airways correlated with OVA-induced mobilization of neutrophils
into the blood (Fig. 3C).
Current infection during AAD (D-7/OVA) also significantly
increased neutrophils in BALF and blood, compared with infection
in the absence of AAD (D-7/Sal; Figs. 2B, 3C). This indicates that
the influx of neutrophils during AAD occurs in response to OVA
sensitization and challenge, suggesting that infection primes for
the neutrophil influx during AAD.
Current infection during sensitization drives allergen-specific
Th1 T cell cytokine responses during AAD
The mobilization of neutrophils into the blood during allergen
challenge suggested that OVA-specific Th1 cells had been generated during sensitization. To assess the effect of infection on
FIGURE 4. OVA-specific MLN T cell responses postinfection and
AAD. Experimental groups were infected 7 (D-7/OVA) or 45 (D-45/OVA) d
prior to OVA sensitization and challenge, as described in Fig. 1A. MLN
production of OVA-specific IFN-g (A), IL-5 (B), and IL-13 (C) was determined in culture supernatants, using ELISA. Results are presented as
mean 6 SEM from n $ 4, in duplicate, with allergic groups represented by
black bars and nonallergic controls by white bars. Significant differences
between groups are shown as pp , 0.05, ppp , 0.01, and pppp , 0.001.
OVA-specific T cell cytokine responses, MLN T cells were cultured
with OVA. T cells from uninfected mice with AAD (OVA) produced
strong OVA-specific Th2 responses characterized by substantial
increases in the production of IL-5 and IL-13, compared with T cells
from uninfected, nonallergic controls (Sal; Fig. 4B, 4C). By contrast, T cells from mice that had a current infection during OVA
sensitization released substantially increased levels of OVA-specific IFN-g, suppressed IL-5 (Fig. 4A, 4B), but unaltered IL-13
release (p = 0.17; Fig. 4C), compared with T cells from uninfected,
allergic (OVA) controls. A resolved infection (D-45/OVA) had no
effect on IFN-g or IL-5 production but decreased IL-13 levels.
Current infection during sensitization suppresses AHR to
methacholine
The effects of infection on AHR during AAD were assessed. The
induction of AAD without infection (OVA) resulted in the development of AHR, characterized by increased airway resistance
and decreased compliance, compared with findings in uninfected,
nonallergic controls (Sal; Fig. 5). Current, but not resolved, infection suppressed AHR.
Current infection during sensitization (D-7/OVA) resulted in
reduced AHR with decreased resistance and increased compliance,
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FIGURE 3. Pulmonary inflammation and circulating neutrophil levels
postinfection and AAD. Experimental groups were infected 7 (D-7/OVA)
or 45 (D-45/OVA) d prior to OVA sensitization and challenge, as described
in Fig. 1A. Numbers of (A) airway tissue eosinophils within the basement
membrane and (B) airway MSCs in the airway lumen in lung sections were
determined by histology. C, Neutrophil levels were determined as a percentage of total leukocytes in blood smears from groups with infection
during OVA sensitization (D-7/OVA). Results are presented as mean 6
SEM from n $ 4, in duplicate, with allergic groups represented by black
bars and nonallergic controls by white bars. Significant differences between groups are shown as pp , 0.05, ppp , 0.01, and pppp , 0.001. AL,
airway lumen; BM, basement membrane.
The Journal of Immunology
4163
compared with findings in uninfected, allergic (OVA) controls (Fig.
5). By contrast, a resolved infection (D-45/OVA) had no effect.
Notably, infected, nonallergic groups, regardless of the timing of
infection (D-7/Sal and D-45/Sal), had increased AHR, compared
with uninfected, nonallergic (Sal) controls, indicating that infection
alone altered baseline lung function.
Suppression of neutrophil influx during infection
Chlamydial lung infections induce potent neutrophil influx into the
lungs (20, 32); therefore, the effects of these cellular responses to
infection on AAD were investigated. First, the effectiveness of
suppressing neutrophil influx during infection by treatment with
aKCMIP2 to reduce the levels of the chemotactic chemokines KC
and MIP-2 was assessed.
The effects of Ab treatment on leukocyte influx during infection
in the absence of AAD (i.e., without sensitization or challenge with
OVA) were determined. Pulmonary neutrophil, but not lymphocyte,
influx was reduced in infected, Ab-treated (aKCMIP2/D-7)
groups, compared with untreated (Iso/D-7) controls, 10 d postinfection (Supplemental Fig. 1A). This finding is equivalent to day
3 of the experimental model (Fig. 1B) and is the time of peak
infection. Neutrophil numbers were reduced by 52% in the BALF.
However, treatment had no effect on the percentage of neutrophils
in the blood (Supplemental Fig. 1B). Treatment did not affect
infection, with no change in the number of chlamydia recovered
from the lungs (Supplemental Fig. 1C).
Suppression of neutrophil influx during infection alters cellular
responses in Chlamydia-driven neutrophilic AAD
Next, the influence of neutrophil influx into the lungs during infection on AAD was assessed. This was achieved by suppressing
neutrophil influx during infection and analyzing the effects on the
subsequent development of OVA-induced AAD (Fig. 1B). Treatment
during infection reduced neutrophil influx into the lung and blood
in AAD (aKCMIP2/D-7/OVA), compared with isotype-treated (Iso/
D-7/OVA) controls. Neutrophil numbers were reduced by 48% in
BALF, 65% in lung tissue, and 63% in blood (Fig. 6A–C).
We investigated whether the reduction in neutrophils in AAD
resulted from removing the effects of neutrophils during infection or
from a direct influence of Ab treatment on neutrophil influx during the
challenge phase of AAD. To find out, neutrophil numbers in lungs and
blood were assessed on day 12 (D12; Fig. 1B) of the experimental
protocol, when groups had been infected and sensitized with OVA
but prior to OVA challenge. Ab treatment did not alter the numbers
of neutrophils in lungs or blood, compared with findings in isotypetreated controls prior to OVA challenge (not shown). Therefore, the
influence of treatment on neutrophil numbers during infectioninduced AAD resulted from suppression of the effects of neutrophils during infection and not from the prolonged effects of
Abs on neutrophil influx during the challenge phase of the model.
Taken together, these results show that suppression of neutrophil
influx during infection leads to suppression of neutrophil numbers
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FIGURE 5. AHR postinfection and AAD. AHR was determined in experimental groups infected (A) 7 (D-7/OVA) or (B) 45 (D-45/OVA) d before OVA
sensitization and challenge, as described in Fig. 1A. AHR is expressed in terms of airways resistance (upper panels) and dynamic compliance (lower
panels) in response to increasing doses of methacholine. Comparison of statistical differences across whole curves is shown in the tables. Results are
presented as mean 6 SEM from n $ 4, in duplicate. Significant differences between groups are shown as pp , 0.05, ppp , 0.01, and pppp , 0.001.
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CHLAMYDIAL RESPIRATORY INFECTION AND NEUTROPHILIC ASTHMA
in the airways and blood during AAD. This occurs despite neutrophil numbers being similar between infected groups prior to
OVA challenge.
The suppression of neutrophil influx during infection removed
the suppressive effects of infection on MSC numbers during AAD
(Fig. 6D). However, treatment did not alter the infection-induced
reduction of pulmonary eosinophil numbers during AAD (Supplemental Fig. 2A, 2B).
Reduction of pulmonary neutrophil influx during infection
alters T cell responses in Chlamydia-driven neutrophilic AAD
The suppression of neutrophil influx during infection reversed the
effects of infection on OVA-specific cytokine release from MLNs
during AAD. Ab treatment of infected and allergic (aKCMIP2/D7/OVA) groups substantially reduced OVA-specific IFN-g release,
compared with that in infected, isotype-treated (Iso/D-7/OVA)
controls (Fig. 6E). Treatment also increased IL-5 release by .2fold, compared with controls, however, these levels were still
significantly lower than those in Ab- (aKCMIP2/OVA) or isotypetreated, uninfected, allergic (Iso/OVA) groups (Supplemental Fig.
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FIGURE 6. Effect of suppression of neutrophil influx during infection on hallmark features of infectioninduced neutrophilic AAD. Experimental groups were
infected 7 d before OVA sensitization and treated with
anti-KC and anti–MIP-2 mAbs (aKCMIP2) during
infection. AAD was induced and assessed (aKCMIP2/
D-7/OVA) as described in Fig. 1B. Neutrophils were
enumerated in BALF (A), pulmonary tissue (B), and
blood (C). D, MSC numbers within the airway lumen
were determined in lung sections by histological
study. E, MLN production of OVA-specific IFN-g was
determined in culture supernatants, using ELISA. Results are presented as mean 6 SEM from n $ 4, in
duplicate, with aKCMIP2 treatment represented by
white bars and isotype treatment represented by black
bars. Significant differences between groups are
shown as pp , 0.05, ppp , 0.01, and pppp , 0.001.
AL, airway lumen.
2C). Treatment did not affect IL-13 levels in infected, allergic
(aKCMIP2/D-7/OVA) groups (Supplemental Fig. 2D).
Reduction of pulmonary neutrophil influx during infection
alters AHR in Chlamydia-driven neutrophilic AAD
Reduced neutrophil influx into the lungs during infection completely reversed the suppressive effect of infection on AHR during
AAD. Treatment (aKCMIP2/D-7/OVA) significantly increased
resistance (p , 0.01) and decreased compliance (p = 0.06),
compared with findings in isotype-treated (Iso/D-7/OVA) controls
(Fig. 7). No differences were found between treated, infected, and
allergic (aKCMIP2/D-7/OVA) groups and Ab- or isotype-treated,
uninfected, allergic (aKCMIP2/OVA and Iso/OVA) controls.
Current infection during sensitization affects the expression of
immune mediators in the lung during AAD, which are partially
reversed by reduction of pulmonary neutrophil influx during
infection
The mechanisms of how infection modulates the phenotype and inflammatory profile of OVA-induced AAD were further investigated.
The Journal of Immunology
4165
Chlamydial infections induce or alter the release of a variety of
immune factors, including IL-12 and IL-17 and TARC, that may
influence AAD. Current infection resulted in significant increases in
IL-12 and IL-17 and decreased TARC expression in the lungs during
AAD (Iso/D-7/OVA), compared with findings in uninfected, allergic
(Iso/OVA) controls (Fig. 8A–C). Significantly, suppression of neutrophil influx into the lungs during infection partially reversed these
changes. Ab-treated (aKCMIP2/D-7/OVA) groups had significantly decreased IL-12 and IL-17 and increased TARC expression in
the lungs, compared with isotype-treated (Iso/D-7/OVA) controls
(Fig. 8A–C).
Current infection during sensitization increases APC
activation in the lung during AAD, which is altered by
reduction of pulmonary neutrophil influx during infection
Infections may also alter the phenotype and function of APCs, which
may influence how infection modulates AAD. Current infection increased the numbers of MHC II+ mDCs and costimulatory factors
on APCs in lung tissue in AAD. Current infection (Iso/D-7/OVA)
increased the number of viable cells that were MHC II+ mDCs and
pDCs and APCs (CD11c+ cells) expressing CD80 and CD86 in the
lungs during AAD, compared with findings in uninfected, allergic
(Iso/OVA) controls (Fig. 8D–G).
Ab treatment during infection (aKCMIP2/D-7/OVA) increased
the number of MHC II+ pDCs in the lungs during AAD over that
seen in isotype-treated, infected (Iso/D-7/OVA) controls (Fig. 8D,
8E). This occurred despite treatment having no effect on the level
of infection and therefore on antigenic load in the lungs. Treatment reversed the effect of infection on the number of APCs expressing CD80 during AAD (Fig. 8F) but had no effect on CD86
expression (Fig. 8G). Ab treatment in the absence of infection
(aKCMIP2/OVA) did not substantially influence DCs or APCs,
compared with findings in uninfected isotype-treated (Iso/D-7/
OVA) controls (Fig. 8D–G).
Increased levels of IFN-g and IL-17 during OVA challenge do
not play a critical role in infection-induced neutrophilic AAD
We have shown that infection-induced changes in AAD are associated with increased IFN-g and IL-17 responses following OVA
challenge. To investigate the role of these cytokines in infectioninduced neutrophilic AAD, aIFN-g or aIL-17 mAbs were administered during OVA challenge (Fig. 1C). Infected, allergic groups
that were treated with aIFN-g (aIFN-g/D-7/OVA) had increased
numbers of neutrophils in BALF, compared with both uninfected,
Ab- and isotype-treated (aIFN-g/OVA and OVA) groups (Fig. 9).
Significantly, treatment of infected, allergic groups with aIFN-g
(aIFN-g/D-7/OVA), compared with infected, isotype-treated (D-7/
OVA) controls, did not affect neutrophil numbers (Fig. 9). Similar
results were observed with aIL-17 treatment (not shown). Therefore,
although infection-induced changes in AAD are associated with
augmented Th1/Th17 immunity, increased IFN-g and IL-17 responses during OVA challenge do not play a critical role in driving
neutrophilic inflammation in infection-induced neutrophilic AAD.
Discussion
In this paper, we show that chlamydial infection promotes neutrophilic inflammation and OVA-specific Th1 responses and suppresses Th2-mediated eosinophilic inflammation, resulting in
a phenotype reminiscent of that of neutrophilic asthma. This
phenotype was largely reversed by suppression of neutrophil influx
into the lung during infection.
These studies employed the natural mouse pathogen C. muridarum, which was originally isolated from a mouse with respiratory infection (33, 34). The time course, as well as the
immunological and histopathological progression, of C. muridarum infection of mice closely resembles that observed with
Chlamydophila pneumoniae infection in humans (8, 9). Therefore,
C. muridarum is the organism of choice for investigating natural
host–bacteria–allergen interactions in mice.
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FIGURE 7. Effect of suppression of neutrophil influx during infection on AHR in infection-induced neutrophilic AAD. Experimental groups were
infected 7 d before OVA sensitization and treated with anti-KC and anti–MIP-2 mAbs (aKCMIP2) during infection. AAD was induced and assessed
(aKCMIP2/D-7/OVA) as described in Fig. 1B. AHR was determined and expressed in terms of airways resistance (upper panels) and dynamic compliance
(lower panels) in response to increasing doses of methacholine. Comparison of statistical differences across whole curves is shown in the tables. Results are
presented as mean 6 SEM from n $ 4, in duplicate. Significant differences between groups are shown as pp , 0.05, ppp , 0.01, and pppp , 0.001.
4166
CHLAMYDIAL RESPIRATORY INFECTION AND NEUTROPHILIC ASTHMA
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 8. Effect of suppression of neutrophil influx
during infection on immune factors and APC activation
in infection-induced neutrophilic AAD. Experimental
groups were infected 7 d before OVA sensitization and
treated with anti-KC and anti–MIP-2 mAb (aKCMIP2)
during infection. AAD was induced and assessed
(aKCMIP2/D-7/OVA) as described in Fig. 1B. Pulmonary expression of IL-12 (A), IL-17 (B), and TARC
(C) was determined in lung tissue homogenates by realtime PCR. Percentages of viable MHC II+ mDCs (D),
pDCs (E), and APCs (CD11c+ cells) expressing CD80
(F) and CD86 (G) costimulatory molecules were determined in lung tissue by FACS. Results are presented
as mean 6 SEM from n $ 4, in duplicate, with
aKCMIP2 treatment represented by white bars and
isotype treatment represented by black bars. Significant
differences between groups are shown as pp , 0.05,
ppp , 0.01, and pppp , 0.001.
The current and previous studies have reported that a resolved
chlamydial infection during AAD either has no effect or suppresses
key features of asthma in AAD (22, 23). Although the effects of
resolved infection investigated in previous studies resulted in
a suppression of Th2, and an increase in Th1 responses, the au-
thors did not investigate the effects of an ongoing infection or
demonstrate an increase in neutrophilic inflammation or the effects of infection on AHR. We have extended these studies to
show that, although an active infection suppressed Th2 responses,
infection should not be considered protective; rather, it changes
The Journal of Immunology
the phenotype of AAD to one underpinned by neutrophilic inflammation that is associated with allergen-specific Th1 (IFN-g)
responses. Neutrophilic inflammation plays an important role
during acute exacerbations of asthma, and respiratory pathogens
may be implicated in these events (18, 19, 35–37). More than onethird of asthmatics presenting with acute severe asthma exacerbations had elevated levels of Chlamydophila pneumoniaespecific IgG or IgA, indicating an acute or reactivated infection
(19). These subjects exhibited more intense neutrophilic inflammation, compared with acute exacerbators without infection.
Our results show that AHR in groups with current infection in
AAD was reduced to the same level as that induced by infection
alone, but lung function was impaired in both groups, compared
with uninfected, nonallergic controls. This observation suggests
that infection suppresses AHR that is associated with AAD;
however, infection alone impairs lung function. Clinical studies
show that increased Chlamydophila pneumoniae Ab levels are
associated with persistent airflow limitation in adult-onset, nonatopic asthmatics (38). Therefore, infection may result in abnormal lung function in the absence of Th2-mediated allergic
responses in humans. Chlamydia-driven neutrophilic asthma may
also be a more protracted or chronic disease (39). Infection may
result in a perpetuating feedback loop in which the neutrophilic
phenotype is reinforced and prolonged, resulting in the induction
of chronic disease. Chlamydia is able to infect and grow in neutrophils (21, 40), and infected neutrophils have delayed apoptosis
and secrete greater amounts of IL-8 (40). This characteristic may
promote further neutrophil influx, resulting in the persistence of
neutrophils within the airways during infection (40) and neutrophildominated asthma.
The switch toward a neutrophil-dominated phenotype correlated
with increased pulmonary expression of IL-12 and IL-17, which
may contribute to the suppression of Th2 responses during AAD
(41). IL-12 is associated with Th1 polarization (42), whereas IL17 is important in driving neutrophilic inflammation (43, 44).
Recently, increased levels of IL-17 were detected in the airways of
asthmatic patients and correlated with elevated neutrophil numbers in sputum and more severe disease (45). Taken together, our
observations may help explain the clinical link between Chlamydia and asthma. Infection may promote neutrophilic inflammation that is associated with Th1 and IL-17 responses, rather
than Th2-dominated, eosinophilic responses, while maintaining
some of the clinical features of asthma, such as impaired lung
function. Th1 and Th17 responses have been previously shown to
be associated with neutrophilic inflammation in mouse models of
AAD; however, these studies used adoptive transfer of in vitro
polarized Th1 and Th17 cells to demonstrate the link (46, 47). In
this study, we show that chlamydial infection may drive Th1- and
Th17-biased immune responses against allergens during AAD.
Neutrophilic asthma is difficult to treat, as patients are resistant
to corticosteroids, which are the mainstay of asthma therapy (48,
49). Steroid treatment also decreases neutrophil apoptosis and
increases neutrophil numbers in serum and tissue (50–52).
Therefore, treatment may increase neutrophil numbers and perpetuate neutrophilia in the lung. Steroid treatment also promotes
chlamydial infection in vitro and reactivates persistent lung infection in vivo (53, 54), and asthmatic patients on high, as opposed
to low, doses of inhaled steroids are more likely to have evidence
of Chlamydophila pneumoniae infection (55). Most importantly,
asthmatics with evidence of infection are more resistant to steroid
treatment than are asthmatics without infection (56). These observations suggest that treatment may result in increased susceptibility to acute infection or reactivation of persistent chlamydial
lung infection and that infection induces a phenotype of asthma
that is more resistant to treatment. Therefore, alternative therapies
may need to be considered when treating asthmatics with evidence
of Chlamydia-driven neutrophilic inflammation.
Because an association exists between increased activation of
innate immune factors and neutrophilic asthma (16), we hypothesized that the influx of neutrophils into the lungs may drive the
development of neutrophilic AAD. To investigate this, the inflammatory chemokines KC and MIP-2 were inhibited during
infection. Treatment resulted in ∼50% reduction in neutrophils in
the lungs during infection. A lack of complete inhibition may be
explained by the existence of non–CXCR2-dependent redundancies in chemotactic signaling, including C5a and MCP-1,
which signals through CCR2 (57, 58). Systemic leukocyte numbers were not altered, indicating that only cellular influx into the
lung, and not mobilization from the bone marrow, was affected.
However, treatment during infection did suppress pulmonary and
circulating neutrophil responses during subsequent AAD. This
observation suggests that neutrophil accumulation during infection is necessary to prime for both local and systemic neutrophil responses during AAD. Importantly, treatment also reversed
infection-driven alterations in OVA-specific IFN-g levels, MSC
numbers, and AHR during AAD. Treatment ceased 10 d before
OVA challenge and had no significant effect on AAD in uninfected, allergic controls, demonstrating that Ab treatment had no
direct or prolonged influence on AAD. Furthermore, neutrophil
numbers were similar between Ab-treated and isotype control
groups prior to the induction of AAD by OVA challenge. This
finding shows that changes in the phenotype of AAD were related
to the effects of infection. This also confirms that the suppression
of neutrophil numbers by Ab treatment during infection results
from a diminished ability to recruit neutrophils upon exposure to
an allergen. Whether the effects of treatment result from lower
numbers of neutrophils infiltrating the lungs during sensitization
or from reduced de novo recruitment of neutrophils after allergen
challenge remains unknown. Taken together, these data demonstrate that neutrophil influx into the lungs during infection plays
an important role in driving the hallmark features of infection
associated neutrophilic AAD.
The effect of treatment on pulmonary levels of IL-12, IL-17, and
TARC and changes in APC activation during AAD demonstrates
that the influx of neutrophils during infection is necessary to induce
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FIGURE 9. Effect of suppression of IFN-g during OVA challenge on
infection-induced neutrophilic AAD. Experimental groups were infected
7 d before OVA sensitization and challenge and treated with anti–IFN-g
mAb (aIFN-g) during challenge. AAD was induced and assessed (aIFNg/D-7/OVA) as described in Fig. 1C. Neutrophils were enumerated in
BALF posttreatment. Results are presented as mean 6 SEM from n = 6,
with aIFN-g treatment represented by white bars and isotype treatment
represented by black bars. Significant differences between groups are
shown as pp , 0.05.
4167
4168
CHLAMYDIAL RESPIRATORY INFECTION AND NEUTROPHILIC ASTHMA
Acknowledgments
We thank Prof. Rakesh Kumar (Department of Pathology, University of
New South Wales, Sydney, Australia) for assistance in the analysis of
changes in lung histology.
Disclosures
The authors have no financial conflicts of interest.
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