Toll-Like Receptor-9 Agonist Inhibits Airway
O riginal Paper I nt Arch Allergy Immunol 2010;151:285–296 D OI :10.1159/000250437 T
oll-Like Receptor-9 Agonist Inhibits Airway Inflammation, Remodeling and Hyperreactivity in Mice Exposed to Chronic Environmental Tobacco Smoke and Allergen
D ae Jin Song a, b Myung Goo Min a Marina Miller a Jae Youn Cho a Hye Yung Yum a, c David H. Broide a
a
D epartment of Medicine, University of California San Diego, S an Diego, Calif. , USA; b D epartment of Pediatrics,College of Medicine, Korea University, and c
A topy Clinic,Seoul Medical Center,S eoul ,Korea
er, and AHR. The reduced airway remodeling in mice treated with the TLR-9 agonist was associated with significantly re-duced numbers of peribronchial MBP+ and peribronchial TGF- ? 1 + cells, and with significantly reduced levels of lung Th2 cytokines [interleukin-5 and interleukin-13] and TGF- ? 1.
C onclusion: These studies demonstrate that TLR-9-based therapies inhibit airway inflammation, remodeling and AHR in mice coexposed to ETS and allergen who exhibit en-hanced airway inflammation and remodeling.
C opyright ? 2009 S. Karger AG, Basel I ntroduc tion A
sthma is a disease characterized by airway inflam-mation and airway hyperreactivity (AHR)
[1] . A variety of environmental triggers can aggravate asthma includ-ing allergens, viruses, pollutants and tobacco smoke
[1].In terms of tobacco smoke exposure acting as a trigger for asthma, several studies have demonstrated that exposure to either high levels of tobacco smoke in active smokers [2–4]
or low levels of tobacco smoke exposure in non-smokers passively exposed to environmental tobacco
smoke (ETS)
[5–7] are associated with adverse asthma outcomes including increased prevalence of asthma, in-creased severity of asthma symptoms, increased frequen-K
ey Words T oll-like receptor-9 ? Airway hyperreactivity ?Airway inflammation ? Airway remodeling ?Eosinophils
A bstrac t
B ackground: As passive environmental tobacco smoke (ETS) exposure in nonsmokers can increase both asthma symp-toms and the frequency of asthma exacerbations, we uti-lized a mouse model, in which ovalbumin (OVA) + ETS induce significantly increased levels of eosinophilic airway inflam-mation and remodeling compared to either stimulus alone, to determine whether a Toll-like receptor-9 (TLR-9) agonist could reduce levels of airway inflammation, airway remodel-ing and airway hyperreactivity (AHR). M ethods: Mice treated with or without a TLR-9 agonist were sensitized to OVA and challenged with OVA + ETS for 1 month. AHR to methacho-line was assessed in intubated and ventilated mice. Lung Th2 cytokines and TGF- ? 1 were measured by ELISA. Lungs were processed for histology and immunohistology to quantify eosinophils, mucus, peribronchial fibrosis and smooth mus-cle changes using image analysis. R esults: Administration of a TLR-9 agonist to mice coexposed to chronic ETS and chron-ic OVA allergen significantly reduced levels of eosinophilic airway inflammation, mucus production, peribronchial fi-brosis, the thickness of the peribronchial smooth muscle lay-
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eceived: January 27, 2009 A ccepted after revision: July 2, 2009
P ublished online: October 22, 2009 6:31 A M
cy of asthma medication use and increased emergency room visits by asthmatic children. The importance of ETS exposure to asthma is further suggested from recent gene association studies demonstrating a link between a region on chromosome 17q21, combined with ETS, and asthma [8] . Experimental ETS challenge studies in hu-mans also indicate that such exposure has adverse effects on airflow and/or airway responsiveness in asthma [9, 10] . Thus, to reduce the adverse impact of ETS on asthma requires strategies that include reducing the number of smokers and, consequently, of ETS-exposed asthmatics. However, in the USA, although 45% of smokers each year make an attempt to quit, less than 5% of the general pop-ulation are successful [11] . As up to 68% of nonsmoking children with asthma in the inner cities of the USA are also exposed to ETS as assessed by salivary cotinine levels [7] , more immediate strategies are needed to reduce ad-verse asthma outcomes in ETS-exposed asthmatics in ad-dition to the long-term strategy of reducing the number of smokers.
S tudies in mouse models suggest a potential immuno-logic mechanism for the interaction of ETS and allergen resulting in adverse asthma outcomes. For example, stud-ies by our group [12] and others [13, 14] have demonstrat-ed that exposure of mice to the combination of ETS and ovalbumin (OVA) allergen induces significantly higher levels of Th2 cytokines, eosinophilic airway inflamma-tion, mucus expression, peribronchial fibrosis, thickness of the smooth muscle layer, and AHR compared to levels induced by exposure to either OVA alone or ETS alone. As recent studies suggest that inhaled and oral cortico-steroids, currently our most effective anti-inflammatory therapies in asthma, are not as effective in asthmatics who smoke [15–18] , there is a need to identify novel ther-apeutic interventions that inhibit Th2 immune responses both in asthmatics exposed and not exposed to ETS. We have previously demonstrated that administration of a Toll-like receptor-9 (TLR-9) ligand [i.e. immunostimula-tory sequences (ISS) of DNA containing a CpG motif] inhibits Th2 cytokine responses [19] , eosinophilic airway inflammation [19, 20] , airway remodeling [21–24]and AHR [19] in mouse models of asthma. In addition, con-jugation of the TLR-9 ligand to the major ragweed aller-gen A mb a 1 significantly reduces seasonal ragweed rhi-nitis symptoms in ragweed-allergic human subjects [25]. Based on the ability of TLR-9 ligands to inhibit Th2 cy-tokine responses [19] , as well as on our demonstration that mice coexposed to ETS and OVA allergen have en-hanced Th2 cytokine responses [12] , we have investigated in the present study whether a TLR-9 ligand would be ef-fective in reducing the enhanced airway inflammation, remodeling and AHR in mice induced by chronic coex-
posure to ETS and allergen.
M ethods
T herapeutic Intervention with TLR-9 Ligand in Chronic ETS
+ OVA-Exposed Mice
I n this study we have examined whether administration of a
TLR-9 ligand (i.e. ISS) inhibits airway inflammation, airway re-modeling and AHR in mice exposed to chronic ETS in combina-
tion with chronic OVA allergen for 1 month. Different groups of
8- to 10-week-old BALB/c mice (12 mice/group; The Jackson Lab-oratory, Bar Harbor, Me., USA) were chronically exposed to ETS
as well as to either no OVA, OVA or OVA + ISS. As controls we
also included mice not exposed to ETS (no OVA and OVA). The
results of an OVA + corticosteroid intervention in ETS + OVA-exposed mice are reported elsewhere [26] , and the present study focuses on the effect of ISS on airway inflammation, airway re-modeling, and AHR in ETS-exposed mice. Both intervention
studies (ISS or corticosteroid) used the same control groups to
limit the number of control mice required for these experiments.
The group of ETS-exposed mice that received the OVA + ISS were administered intraperitoneally endotoxin-free phosphorothioate
ISS-oligodeoxynucleotides (5 ?-TG ACTG TG AA C G T T C G A G A-
TGA-3 ?; Trilink, San Diego, Calif., USA; 100 ?g in 100 ?l of ster-
ile, endotoxin-free PBS), starting 1 day before the first intranasal
OVA challenge and then continued every other week for the dura-
tion of the 1-month period of combined ETS exposure and twice-
weekly OVA challenges. Previous studies in our laboratory have demonstrated that ISS inhibits OVA-induced eosinophilic in-flammation, airway remodeling and AHR when administered 1
day before OVA challenge [19, 21–24] , and that this inhibitory ef-
fect lasts at least 4 weeks [27] .
C hronic ETS + Chronic OVA Exposure
W e have previously demonstrated that chronic ETS exposure
alone does not increase airway inflammation, airway remodeling
or AHR in mice [12] . In contrast, coexposure of mice to chronic
ETS and chronic OVA allergen significantly increases levels of eosinophilic inflammation, airway remodeling and AHR as com-
pared to mice exposed to chronic OVA allergen with no ETS ex-
posure [12] .
C hronic ETS Exposure( f ig. 1 ). Three groups of mice (no OVA,
OVA and OVA + ISS) were exposed to chronic ETS (side-stream
smoke from 6 cigarettes/day each administered over approxi-
mately 5 min with a 15-min break between cigarettes, 5 days/
week) generated by burning 2R4F reference cigarettes (2.45 mg nicotine/cigarette; Tobacco Research Institute, University of Ken-
tucky, Lexington, Ky., USA) using a smoking machine (McChes-
ney-Jaeger CSM-SSM Single Cigarette Machine, CH Technolo-
gies USA, Inc., Westwood, N.J., USA) regulated by programma -
b le controls provided with JASPER Windows 9x/2000 software
over RS-232 communication ports (CH Technologies USA, Inc.)
as previously described in this laboratory [12].Each smoldering cigarette is puffed for approximately 2 s, once every 25 s, for a to-
tal of 12 puffs/cigarette, at a flow rate of 5 liters/min. The outflow
from the smoking machine was adjusted to mimic an exposure to
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ETS by producing a mixture of room air (98%) and mainstream smoke (2%). The mice were exposed to the ETS in a 12-port, nose-only, directed flow inhalation exposure system (Jaeger-NYU 12 port). Nose ports were monitored for total suspended particulates which we have previously reported to be 173 85.3 ?g/m3using a gravimetric method [12] . All animal experimental protocols were approved by the University of California, San Diego Animal Subjects Committee.
C hronic OVA Protocol(f ig.1).I n these studies,mice were im-munized subcutaneously on days 0, 7, 14 and 21 with 25 ?g of OVA (OVA, grade V; Sigma Chemicals, St. Louis, Mo., USA) adsorbed to 1 mg of alum (Aldrich) in 200 ?l normal saline as previously described [28] . OVA-challenged mice received intranasal OVA challenges on days 27, 29 and 31 under isoflurane (Vedco, Inc., St. Joseph, Mo., USA) anesthesia, which were then repeated twice a week for 1 month. The no-OVA age- and sex-matched control mice were sensitized but not challenged with OVA during the 1-month study. The groups of mice that were exposed to ETS had their first ETS exposure on day 33 after they had been sensitized with OVA subcutaneously, and received intranasal OVA challenges on days 27, 29 and 31 as previously described in this laboratory [28] .Chron-ic ETS was continued daily for the subsequent 1-month period of twice-weekly intranasal OVA challenges.
P rocessing of Lungs for Immunohistology
T he mice were sacrificed 24 h after the final chronic OVA and/ or chronic ETS challenge and bronchoalveolar lavage (BAL) fluid and lungs were analyzed as previously described [28] . The lungs in the different groups of mice were equivalently inflated with an in-tratracheal injection of a similar volume of 4% paraformaldehyde solution (Sigma Chemicals) to preserve the pulmonary architec-ture. These lungs were then processed as a batch for either histo-logic staining or immunostaining under identical conditions. Stained and immunostained slides were all quantified under iden-tical light microscope conditions, including magnification (!20), gain, camera position and background illumination. The quantita-tive histologic and image analysis of all coded slides was performed by research associates blinded to the coding of all the slides.
B AL and Peribronchial Eosinophils.Total BAL eosinophil counts and the number of peribronchial MBP+ cells were quanti-tated as previously described [28] . In brief, lung sections were pro-cessed for MBP immunohistochemistry using an anti-mouse MBP antibody (kindly provided by James Lee, PhD, Mayo Clinic, Scottsdale, Ariz., USA). The number of individual cells staining positive for MBP in the peribronchial space was counted using a light microscope. Results are expressed as the number of peri-bronchial cells staining positive for MBP/bronchiole with 150–200 ?m of internal diameter. At least 10 bronchioles were count-ed in each slide.
M ucus.The number of PAS-positive and PAS-negative airway epithelial cells in individual bronchioles were counted as previ-ously described in this laboratory [28] . At least 10 bronchioles were counted in each slide. Results are expressed as the percentage of PAS+ cells/bronchiole which is calculated from the number of PAS+ epithelial cells/bronchus divided by the total number of ep-ithelial cells of each bronchiole.
P eribronchial Fibrosis.The area of peribronchial trichrome staining in the paraffin-embedded lungs was outlined and quan-tified using a light microscope (Leica DMLS; Leica Microsystems, Inc., New York, N.Y., USA) attached to an image analysis system (Image-Pro Plus; Media Cybernetics, Bethesda, Md., USA) as pre-viously described [28] . Results are expressed as the area of tri-chrome staining/ ?m length of the basement membrane of bron-chioles 150–200 ?m of internal diameter.
P eribronchial TGF-?1+ Cells.The number of peribronchial cells expressing TGF- ?1were assessed in lung sections processed for immunohistochemistry using an anti-TGF- ?1primary anti-body (Santa Cruz, Calif., USA), the immunoperoxidase method and image analysis quantitation as previously described [28].Re-sults are expressed as the number of TG F- ?1+cells/bronchus [28] .
F ig. 1.Mice were immunized s.c. on days 0, 7, 14 and 21 with OVA (arrows pointing up). Intranasal OVA challenges were adminis-tered on days 27, 29 and 31, and then repeated twice a week for 1 month. Different groups of mice were either administered ETS alone or ETS in combination with OVA challenges. ETS was start-ed on day 33 (after mice had been sensitized and challenged on 3 occasions with intranasal OVA). The ETS was continued 5 days/
week for 1 month. ISS was administered on 3 occasions i.p. (ar-
rows pointing down) starting on day 26 with repeat doses on days
32 and 46. The mice were sacrificed 24 h after the final OVA chal-
lenge on day 60 and BAL fluid and lungs were analyzed.
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T hickness of the Peribronchial Smooth Muscle Layer.The thick-ness of the airway smooth muscle layer (the transverse diameter) was measured from the innermost aspect to the outermost aspect of the smooth muscle layer [28] . The smooth muscle layer thick-ness in at least 10 bronchioles of similar size (150–200 ?m) was calculated on each slide. Lung sections were also immunostained with an anti- ?-smooth muscle actin primary antibody (Sigma-Aldrich). The area of ?-smooth muscle actin staining was out-lined and quantified using a light microscope attached to an im-age analysis system as previously described [28] . Results are ex-pressed as the area of ?-smooth muscle actin staining/ ?m length of the basement membrane of bronchioles 150–200 ?m of inter-nal diameter.
A irway Hyperreactivity.AHR to methacholine (Mch) was as-sessed 24 h after the final chronic OVA and/or chronic ETS chal-lenge (after 1 month of repetitive OVA 8ETS challenges) in in-tubated and ventilated mice (flexiVent ventilator; Scireq, Mon-treal, Que., Canada) as previously described in this laboratory [29] . The frequency-independent airway resistance (R a w) was de-termined in mice exposed to nebulized PBS and Mch (3, 24 and 48 mg/ml) [29] . In addition to measuring R a w, the Scireq software also recorded tissue elastance (cm H 2O?s/ml) and compliance (ml/cm H 2O).
L ung Levels of Th2 Cytokines and TGF- ?1
L evels of Th2 cytokines (IL-5, IL-13) and TGF- ?1were mea-sured in BAL by ELISA (R&D Systems, Inc., Minneapolis, Minn., USA). The IL-5 assay has a sensitivity of 15 pg/ml while the IL-13 and TGF- ?1assays each have a sensitivity of 31 pg/ml.
P ercentage Reduction in Inflammation and Remodeling in
Response to ISS Therapy
T o calculate the percentage reduction in individual indices of airway inflammation and remodeling in response to ISS therapy, the absolute increase in each of these indices in response to OVA + ETS was calculated according to the formula: (OVA + ETS) – (no OVA + ETS). This value is the maximum increase induced by OVA + ETS above baseline values. The reduction of this value in-duced by ISS therapy was calculated as a percentage.
S tatistical Analysis
R esults in the different groups of mice were compared by ANOVA using the nonparametric Kruskal-Wallis test followed by posttesting using Dunn’s multiple comparison of means. All re-sults are presented as mean 8SEM. A statistical software pack-age (G raphPad Prism; G raphPad Software, San Diego, Calif., USA) was used for the analysis. p !0.05 was considered statisti-cally significant.
R esults
E ffect of ETS on OVA-Induced Airway Inflammation,
Airway Remodeling and AHR
E xposure of mice to chronic ETS alone did not induce an increase in BAL eosinophils ( f ig. 2 a), MBP+ peribron-chial eosinophils ( f ig. 2 b), TGF- ?1+cells ( f ig. 3 a), peri-bronchial fibrosis ( f ig. 3 b, 4), thickness of the peribron-chial smooth muscle layer ( f ig. 5 ), AHR ( f ig. 6 a) and mu-cus production ( f ig. 7 ) compared to non-ETS-exposed mice as previously reported in this laboratory [26].In contrast, chronic ETS in combination with chronic OVA allergen significantly increased all the indices of airway inflammation ( f ig. 2 ), airway remodeling ( f ig. 3 b, 4,5) and AHR ( f ig. 6 a) compared to chronic OVA allergen alone as previously demonstrated in this laboratory [26] .
F ig. 2.Eosinophils were quantitated by either Wright-
G iemsa staining in BAL fluid ( a) or by immunostaining lung sections with anti-MBP antibody ( b). In ETS-exposed mice challenged with OVA, ISS significantly reduced the number of BAL eosino-phils (p !0.0005; ETS + OVA + ISS vs. ETS + OVA) ( a) and peri-bronchial eosinophils (p !0.0005; ETS + OVA + ISS vs. ETS +
OVA) ( b).
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E ffect of TLR-9 Ligand on ETS + OVA-Induced
Eosinophilic Airway Inflammation
A dministration of ISS to mice exposed to chronic ETS + OVA allergen significantly reduced levels of BAL eo-sinophils by approximately 85% compared to chronic ETS + OVA allergen-challenged mice that did not receive ISS (ETS + OVA vs. ETS + OVA + ISS; p !0.0005) ( f ig. 2 a). Similarly, administration of ISS to mice exposed to chron-ic ETS + OVA allergen significantly reduced levels of peribronchial MBP+ eosinophils by approximately 78% compared to chronic ETS + OVA allergen-challenged mice that did not receive ISS (ETS + OVA vs. ETS + OVA + ISS; p !0.0005)(f ig.2b).
E ffect of TLR-9 Ligand on ETS + OVA-Induced
Peribronchial TGF- ?1+Cells
A dministration of ISS to mice exposed to chronic ETS + OVA allergen significantly reduced the number of peri-bronchial TGF- ?1+ cells by approximately 72% compared to chronic ETS + OVA allergen-challenged mice that did not receive ISS (ETS + OVA vs. ETS + OVA + ISS; p !
0.0001) ( f ig. 3 a).
E ffect of TLR-9 Ligand on ETS + OVA-Induced
Peribronchial Fibrosis
A dministration of ISS to mice exposed to chronic ETS + OVA allergen significantly reduced the area of peri-bronchial trichrome staining by approximately 44% com-pared to chronic ETS + OVA allergen-challenged mice that did not receive ISS (ETS + OVA vs. ETS + OVA + ISS; p !0.0001)(f ig.3b,4).
E ffect of TLR-9 Ligand on ETS + OVA-Induced
Thickness of Peribronchial Smooth Muscle Layer
A dministration of ISS to mice exposed to chronic ETS + OVA allergen significantly reduced the thickness of the peribronchial smooth muscle layer by approximately 53% compared to chronic ETS + OVA allergen-challenged mice that did not receive ISS (ETS + OVA vs. ETS + OVA + ISS; p !0.0001) ( f ig. 5 a).
I n addition to measuring the thickness of the smooth muscle layer, we also determined the area of peribron-chial ?-smooth muscle actin immunostaining. Adminis-tration of ISS to mice exposed to chronic ETS + OVA al-lergen significantly reduced the area of peribronchial ?-smooth muscle actin immunostaining by approximately 49% compared to chronic ETS + OVA allergen-chal-lenged mice that did not receive ISS (ETS + OVA vs. ETS + OVA + ISS; p !0.0001) ( f ig. 5 b).
E ffect of TLR-9 Ligand on ETS + OVA-Induced AHR
A dministration of ISS to mice exposed to chronic ETS + OVA allergen significantly reduced AHR compared to chronic ETS + OVA allergen-challenged mice that did not receive ISS (ETS + OVA vs. ETS + OVA + ISS; Mch 48 mg/ ml; p = 0.04) ( f ig. 6 a). Administration of ISS also reduced tissue elastance in OVA + ETS-challenged mice (p !0.05; Mch 48 mg/ml; ETS + OVA + ISS vs. ETS + OVA)
F ig. 3.a The number of peribronchial cells immunostaining pos-itive for TGF- ?1in mouse lungs was quantitated by image analy-sis. In ETS-exposed mice challenged with OVA, ISS significantly reduced the number of peribronchial cells immunostaining posi-tive for TGF- ?1(p !0.0001; ETS + OVA + ISS vs. ETS + OVA).b The area of peribronchial trichrome staining in mouse lungs
was quantitated in ?m2/?m length of bronchus by image analysis.
In ETS-exposed mice challenged with OVA, ISS significantly re-
duced the area of peribronchial trichrome staining (p !0.0001;
ETS + OVA + ISS vs. ETS + OVA).
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Trichrome PAS
No OVA + No ETS
No OVA + ETS
OVA + No ETS
OVA + ETS
OVA + ETS + ISS
F ig. 4. Lungs from the 5 groups of mice (no OVA, no OVA + ETS, OVA, OVA + ETS, OVA + ETS + ISS) were processed for tri-chrome staining to detect peribronchial fi-brosis (blue) and for PAS staining to detect epithelial mucus expression.
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F ig. 5.a The thickness of peribronchial smooth muscle layer was assessed by image analysis. b The area of the peribronchial region immunostaining positive with ?-smooth muscle actin antibody was quantitated by image analysis ( ?m2/?m length of the base-ment membrane of the bronchus). In ETS-exposed mice chal-lenged with OVA, ISS significantly reduced both the thickness of the peribronchial smooth muscle layer (p !0.0001; ETS + OVA + ISS vs. ETS + OVA) ( a) and the area of the peribronchial region immunostaining positive with ?-smooth muscle actin antibody (p !0.0001; ETS + OVA + ISS vs. ETS + OVA) ( b). SMA = Smooth muscle actin.
F ig. 6.a AHR to Mch was assessed 24 h after final chronic OVA
and/or chronic ETS challenge in intubated and ventilated mice.
Results are expressed as R a w in mice exposed to nebulized Mch (3,
24 and 48 mg/ml). In ETS-exposed mice challenged with OVA,
ISS significantly reduced AHR (p !0.04; Mch 48 mg/ml; ETS +
OVA + ISS vs. ETS + OVA). ISS also reduced tissue elastance in
OVA + ETS-challenged mice (p !0.05; Mch 48 mg/ml) ( b)and
improved compliance (p !0.001; Mch 48 mg/ml; ETS + OVA +
ISS vs. ETS + OVA) ( c).
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( f ig. 6 b ) and improved compliance (p ! 0.001; Mch 48 mg/ml; ETS + OVA + ISS vs. ETS + OVA) ( f ig. 6 c ). E ffect of TLR-9 Ligand on ETS + OVA-Induced Mucus Expression
A dministration of ISS to mice exposed to chronic ETS + OVA allergen significantly reduced levels of mucus ex-pression by approximately 58% compared to chronic ETS + OVA allergen-challenged mice that did not receive ISS (ETS + OVA vs. ETS + OVA + ISS; p ! 0.0001) ( f ig. 7 ).
E ffect of TLR-9 Ligand on ETS + OVA-Induced Lung Th2 Cytokines (IL-5, IL-13) and TGF-
? 1 A dministration of ISS to mice exposed to chronic ETS + OVA allergen significantly reduced levels of BAL IL-5 ( f ig. 8 a ), BAL IL-13 ( f ig. 8 b ) and TG F- ? 1( f ig. 8 c ) com-pared to chronic ETS + OVA allergen-challenged mice that did not receive ISS (ETS + OVA vs. ETS + OVA + ISS; p !0.05).
D isc ussion I n this study we demonstrated that a TLR-9 agonist (i.e. ISS) is very effective in reducing ETS-enhanced eo-sinophilic airway inflammation, airway remodeling and AHR in mice exposed to the combination of chronic ETS and chronic OVA allergen. In mice coexposed to ETS and OVA, the TLR-9 agonist inhibited the expression of cyto-kines that contribute to eosinophilic inflammation (i.e. IL-5), AHR (i.e. IL-13) and airway remodeling (i.e. TGF- ? 1 ). Previous studies from our [12, 26] and other labora-tories [13, 14] have demonstrated an enhanced Th2 re-sponse to the combination of ETS and allergen as opposed to either stimulus alone. However, the demon-stration that a TLR-9 agonist inhibits enhanced airway inflammation, remodeling and AHR in mice exposed to OVA + ETS is novel and most likely due to the ability of TLR-9-based therapies to inhibit Th2 responses to aller-gen
[19] , which are enhanced in mice coexposed to aller-gen and ETS
[12, 26] and reduced in those mice admin-istered the TLR-9 ligand. As corticosteroids exhibit a reduced anti-inflammatory effectiveness in smokers
[15–18] , it is important to identify therapeutic interven-tions that inhibit Th2 responses in the presence of ETS. In addition to having anti-inflammatory properties, ISS also inhibited airway remodeling in ETS-exposed mice. Studies have shown that smokers with asthma have a more rapid decline in lung function as compared to non-smokers with asthma
[2] . Thus, there is also a need to identify novel therapeutic interventions that inhibit air-way remodeling in smokers. In mice exposed to allergen + ETS, the increased numbers of cells expressing TGF- ? 1
may contribute to airway remodeling as several
[30–32],but not all [33] , studies in mice in which TGF- ? 1signal-ing is inhibited demonstrate reduced airway remodeling.
Thus, the ability of ISS to significantly reduce levels of lung TGF- ? 1 as well as the numbers of peribronchial cells
expressing TGF- ? 1
, such as eosinophils, in mice exposed to ETS + allergen may contribute to a reduction in levels of airway remodeling and airway responsiveness. How-ever, as TLR-9-based therapies influence several different cell types which express TLR-9, the resultant inhibit ef-fects on remodeling and airway responsiveness may, or may not, be linked.
T his study extends previous observations that ISS is able to inhibit inflammation, remodeling and AHR in
mice exposed to allergen alone
[19–24] to demonstrate that ISS can inhibit these asthma outcomes in mice ex-posed to the combination of ETS and allergen who have enhanced inflammation, remodeling and AHR. As we have previously demonstrated that ISS inhibits these air-way inflammation and remodeling outcomes in non-ETS-exposed OVA-challenged mice
[21] , we did not in-clude a group of ISS-treated, OVA-challenged, non-ETS-exposed mice in this study. Although the lack of a control ISS + OVA group is a limitation of our study, in compar-ing results of ISS therapy in this ETS + OVA exposure study to previous studies of ISS in non-ETS-exposed
F ig. 7. The percentage of bronchial epithelial cells staining posi-tive for PAS was quantitated by light microscopy. In ETS-exposed mice challenged with OVA, ISS significantly reduced the percent-age of PAS+ airway epithelial cells (p ! 0.0001; ETS + OVA + ISS vs. ETS + OVA).
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mice, we demonstrated that ISS inhibited airway inflam-mation and remodeling in mice exposed to ETS + OVA as effectively as in previous studies in which OVA-ex-posed mice were treated with ISS in the absence of ETS
exposure [21]
. For example, comparing the levels of inhi-bition of the indices of airway inflammation and airway remodeling in the current study to those of previous stud-ies by our group [21] demonstrates that ISS induced com-parable levels of inhibition of BAL eosinophilia (85 vs.
63%)
[21] , a reduction in smooth muscle thickness (53 vs. 45%) and a reduction in the number of PAS+ cells (58 vs. 64%). In addition, in the present study ISS reduced OVA + ETS-induced airway inflammation and remodeling to levels significantly below that of untreated mice exposed to OVA alone (OVA + no ETS + no ISS vs. OVA + ETS + ISS), suggesting that ISS was impacting both the OVA as well as the OVA + ETS effect on inflammation and re-modeling. These observations, demonstrating the poten-tial therapeutic utility of ISS in ETS-exposed mice, are
potentially important to the large number of asthmatic children and adults exposed to ETS, in whom ETS expo-sure is associated with adverse asthma outcomes
[6, 7, 34].The large number of asthmatics exposed to ETS has been
noted in pediatric and adult studies
[6, 7, 34] . For exam-ple, approximately 68% of nonsmoking urban children aged 8–14 years had evidence of ETS exposure as assessed by salivary cotinine levels [7] while approximately 29% of nonsmoking adult asthmatics aged 18–50 years in Cali-fornia reported some regular ETS exposure (defined as most days or nights) during an 18-month study period
[34] . The ETS-exposed asthmatics had increased asthma severity, increased health care utilization for asthma (emergency department visits, urgent physician visits and hospitalizations) and worse asthma-specific quality of life [34] . The asthmatics who reported cessation of ETS exposure over the 18-month study experienced a reduc-
F ig. 8. Levels of Th2 cytokines (IL-5, IL-13) and TGF- ? 1 in BAL were measured by ELISA. Administration of ISS to mice exposed to chronic ETS + OVA allergen significantly reduced levels of IL-5 ( a ), IL-13 ( b ) and TFG- ? 1 (
c ) compare
d to chronic ETS + OVA-challenged mic
e that did not receive ISS (p ! 0.05; ETS + OVA vs. ETS + OVA + ISS).
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tion in asthma severity and decreased health care utiliza-tion consistent with improved asthma [34] .Conversely, asthmatics with newly initiated significant exposure to ETS over the follow-up period had worsening of asthma severity and asthma-specific quality of life measures [34]. Overall, these studies suggest an important role of ETS in asthma severity and quality of life.
O ur study, using a mouse model, has the advantage of being able to accurately provide a well-defined level of exposure to ETS to determine whether ISS is effective in the presence of ETS. The mouse model also demonstrates that this level of ETS exposure has a biological effect in enhancing levels of airway inflammation, mucus, airway remodeling and AHR. The limitation of our study, as with all studies in mouse models, is that it is unknown how the results in the mouse model will translate to the effect of ISS in humans with asthma exposed to ETS. Fur-ther studies are also needed to determine whether ISS is effective in inhibiting asthmatic responses at higher dos-es of tobacco smoke exposure as noted in current smok-ers. As TLR agonists other than TLR-9 (TLR-2, TLR-4 and TLR-7/8) have inhibitory [35–37] or potentiating ef-fects [38, 39] on inflammation and AHR in OVA models depending upon the timing and route of administration of the TLR agonist, further study is needed to determine the effect of individual TLR agonists other than TLR-9 in mice exposed to OVA + ETS. Prior studies have also dem-onstrated increased levels of lipopolysaccharide (LPS) in IRF4 cigarettes (approximately 17,800 ng LPS/cigarette) as compared to mainstream smoke (approximately 120 ng LPS/cigarette), and considerably lower levels of LPS in ETS (approximately 18 ng LPS/cigarette) [40] . As the OVA + ETS as well as the OVA + ETS + ISS groups were both exposed to the same ETS (with potentially small amounts of LPS in the ETS), this is very unlikely to account for dif-ferences in asthma outcomes observed between these groups of mice.
A t present there are limited numbers of studies inves-tigating the therapeutic efficacy of ISS in humans with allergy and asthma. Studies of ISS conjugated to the ma-jor ragweed allergen Amb a 1 have demonstrated that in humans with allergic rhinitis the conjugate inhibits Th2 cytokine production by peripheral blood mononuclear cells in vitro [41–43] as well as by cells in the nasal mu-cosa in response to allergen challenge in vivo [44].The ISS-Amb a 1 conjugate also inhibits symptoms during the fall ragweed season in subjects with allergic rhinitis [25]. In limited studies in mild asthmatics utilizing an allergen challenge study design, nebulized ISS did not reduce the number of sputum eosinophils or the late-phase response to allergen challenge [45] . Thus, further studies are need-ed to determine whether ISS, which has demonstrated therapeutic efficacy in mouse [19–24] and primate mod-els of asthma [46] , is effective in humans with asthma.
I n summary, using a mouse model, our studies dem-onstrate that ISS significantly reduces levels of eosino-philic airway inflammation, mucus expression, airway remodeling and AHR in mice exposed to the combina-tion of ETS and allergen. However, further human stud-ies are needed to determine whether similar results would be observed in asthmatics treated with ISS who are ex-posed to ETS. Results from such human studies may be of particular importance to the large number of asthmat-ics exposed to ETS who have adverse asthma outcomes, as well as to children with the 17q21 gene variant who are at increased risk of developing asthma on exposure to ETS in early childhood [8] .
A cknowledgments
T his study was supported by a Tobacco-Related Disease Re-search Program (TRDRP) grant 12RT–0071 (D.H.B.) and Nation-al Institutes of Health (NIH) grants AI 38425, AI 70535 and AI 72115 (D.H.B.).
R eferenc es
Busse WW, Lemanske R Jr: Asthma. N Engl J Med 2001; 344:350–362.
Lange P, Parner J, Vestbo J, Schnohr P, Jensen G: A 15-year follow-up study of ventilatory function in adults with asthma. N Engl J Med 1998; 339: 1194–1200.
PG, Clark S, Camargo CA: Cigarette smok-ing among asthmatic adults presenting to 64 emergency departments. Chest 2003; 123: 1472–1479. Chaudhuri R, Livingston W, McMahon AD,
Lafferty J, Fraser I, Spears M, McSharry CP,
Thomson NC: Effects of smoking cessation
on lung function and airway inflammation
in smokers with asthma. Am J Respir Crit
Care Med 2006; 174: 127–133.
KN, Neveux LM, Palomaki GE, Knight GJ,
Pulkkinen AJ, Haddow JE: Association be-
tween exposure to environmental tobacco
smoke and exacerbations of asthma in chil-
dren. N Engl J Med 1993; 328: 1665–1669.
Eisner MD, Klein J, Hammond SK, Koren G,
Lactao G, Iribarren C: Directly measured
second hand smoke exposure and asthma
health outcomes. Thorax 2005; 60:814–821.
Kumar R, Curtis LM, Khiani S, Moy J, Sha-
lowitz MU, Sharp L, Durazo-Arvizu RA,
Shannon JJ, Weiss KB: A community-based
study of tobacco smoke exposure among in-
ner-city children with asthma in Chicago. J
Allergy Clin Immunol 2008; 122: 754–759.
6
:
3
1
A
M
MH, Boland A, Bousquet J, Chateigner N, Gormand F, Just J, le Moual N, Scheinmann P, Siroux V, Vervloet D, Zelenika D, Pin I, Kauffmann F, Lathrop M, Demenais F: Ef-fect of 17q21 variants and smoking exposure in early-onset asthma. N Engl J Med 2008;
359: 1985–1994.
Menon P, Rando RJ, Stankus RP, Salvaggio JE, Lehrer SB: Passive cigarette smoke-chal-lenge studies: increase in bronchial hyperre-activity. J Allergy Clin Immunol 1992; 89: 560–566.
0 Stankus RP, Menon PK, Rando RJ, G lind-
meyer H, Salvaggio JE, Lehrer SB: Cigarette smoke-sensitive asthma: challenge studies. J Allergy Clin Immunol 1988; 82:331–338.
1 Centers for Disease Control and Prevention
(CDC): Cigarette smoking among adults: United States, 2000. MMWR Morb Mortal Wkly Rep 2002; 51: 642–645.
2 Min MG, Song DJ, Miller M, McElwain S,
Ferguson P, Broide DH: Coexposure to envi-ronmental tobacco smoke increases levels of allergen-induced airway remodeling in mice.
J Immunol 2007; 178: 5321–5328.
3 Seymour BW, Pinkerton KE, Friebertshaus-
er KE, Coffman RL, G ershwin LJ: Second-hand smoke is an adjuvant for Th2 responses in a murine model of allergy. J Immunol 1997; 159: 6169–6175.
4 Rumold R, Jyrala M, Diaz-Sanchez DD: Sec-
ond-hand smoke induces allergic sensitiza-tion in mice. J Immunol 2001; 167: 4765–4770.
5 Chalmer GW, Macleod KJ, Little SA, Thom-
son LJ, McSharry CP, Thomson NC: Influ-ence of cigarette smoking on inhaled corti-costeroid treatment in mild asthma. Thorax 2002; 57: 226–230.
6 Lazarus SC, Chinchilli VM, Rollings NJ,
Boushey HA, Cherniack R, Craig TJ, Deykin A, DiMango E, Fish JE, Ford JG, Israel E, Ki-ley J, Kraft M, Lernanske RF, Leone FT, Mar-tin RJ, Pesola G R, Peters SP, Sorkness CA, Szefler SJ, Wechsler ME, Fahy JV, National Heart, Lung, and Blood Institute’s Asthma Clinical Research Network: Smoking affects response to inhaled corticosteroids or leu-kotriene receptor antagonists in asthma. Am J Respir Crit Care Med 2007; 175: 783–790.
7 Tomlinson JE, McMahon AD, Chaudhuri R,
Thompson JM, Wood SF, Thomson NC: Ef-ficacy of low and high dose inhaled cortico-steroid in smokers versus non-smokers with mild asthma. Thorax 2005; 60:282–287.
8 Chaudhuri R, Livingston E, McMahon AD,
Thomson L, Borland W, Thomson NC: Ciga-rette smoking impairs the therapeutic re-asthma. Am J Respir Crit Care Med 2003;
168: 1308–1311. 9 Broide D, Schwarze J, Tighe H, Gifford T,
Nguyen MD, Malek S, van Uden J, Martin-
Orozco E, Gelfand EW, Raz E: Immunostim-
ulatory DNA sequences inhibit IL-5, eosino-
philic inflammation, and airway hyperre -
s ponsiveness in mice. J Immunol 1998; 161:
7054–7062.
0 Ikeda RK, Miller M, Nayar J, Walker L, Cho
JY, McElwain K, McElwain S, Raz E, Broide
cells in a mouse model of ovalbumin allergen
induced chronic airway inflammation: mod-
ulation by immunostimulatory DNA se-
quences. J Immunol 2003; 171: 4860–4867.
1 Cho J, Miller M, Baek K, Han JW, Nayar J,
Rodriguez M, Lee SY, McElwain K, McEl-
wain S, Raz E, Broide DH: Immunostimula-
tory DNA inhibits transforming growth fac-
tor- ?expression and airway remodeling.
Am J Respir Cell Mol Biol 2004; 30:651–661.
2 Cho JY, Miller M, Baek KJ, Han JW, Nayar J,
Lee SY, McElwain K, McElwain S, Raz E,
Broide DH: Immunostimulatory DNA re-
verses established allergen-induced airway
remodeling. J Immunol 2004; 173: 7556–
7564.
3 Lee SY, Cho JY, Miller M, McElwain K,
McElwain S, Sriramarao P, Raz E, Broide
DH: Immunostimulatory DNA inhibits al-
lergen-induced peribronchial angiogenesis
in mice. J Allergy Clin Immunol 2006; 117:
597–603.
4 Cho JY, Miller M, McElwain K, McElwain S,
Shim JY, Raz E, Broide DH: Remodeling as-
sociated expression of matrix metallopro-
teinase 9 but not tissue inhibitor of metallo-
proteinase 1 in airway epithelium: modula -
t ion by immunostimulatory DNA. J Allergy
Clin Immunol 2006; 117: 618–625.
5 Creticos PS, Schroeder JT, Hamilton RG,
Balcer-Whaley SL, Khattignavong AP, Lind-
blad R, Li H, Coffman R, Seyfert V, Eiden JJ,
Broide D, Immune Tolerance Network: Im-
munotherapy with a ragweed-toll-like re-
ceptor 9 agonist vaccine for allergic rhinitis.
N Engl J Med 2006; 355: 1445–1455.
6 Song DJ, Min MG, Miller M, Cho JY, Broide
DH: Environmental tobacco smoke expo-
sure does not prevent corticosteroids reduc-
ing inflammation, remodeling, and airway
hyperreactivity in mice exposed to allergen.
Am J Physiol Lung Cell Mol Physiol 2009;
297:L380–L387.
7 Broide DH, Stachnick G, Castandeda D,
Nayar J, Miller M, Cho JY, Roman M, Zubel-
dia J, Hayashi T, Raz E: Systemic administra-
tion of immunostimulatory SNA sequences
mediates reversible inhibition of Th2 re-
sponses in a mouse model of asthma. J Clin
Immunol 2001; 21: 175–182.
8 Miller M, Cho JY, McElwain K, McElwain S,
Shim JY, Manni M, Baek JS, Broide DH: Cor-
ticosteroids prevent myofibroblast accumu-
lation and airway remodeling in mice. Am J
Physiol Lung Cell Mol Physiol 2006; 290:
L162–L169.
9 Lim DH, Cho JY, Song DJ, Lee SY, Miller M,
Broide DH: PI3K gamma-deficient mice
have reduced levels of allergen-induced eo-
sinophilic inflammation and airway remod-
eling. Am J Physiol Lung Cell Mol Physiol
2009; 296:L210–L219.
0 Le AV, Cho JY, Miller M, McElwain S, Gol-
gotiu K, Broide DH: Inhibition of allergen-
induced airway remodeling in Smad 3-defi-
cient mice. J Immunol 2007; 178: 7310–7316.
1 McMillan SJ, Xanthou G, Lloyd CM: Manip-
ulation of allergen-induced airway remodel-
ing by treatment with anti-TG F-beta anti-
body: effect on the Smad signaling pathway.
J Immunol 2005; 174: 5774–5780.
2 Alcorn J, Rinaldi L, Jaffe E, van Loon M,
Bates J, Janssen-Heininger Y, Irvin C: Trans-
forming growth factor- ?1suppresses airway
hyperresponsiveness in allergic airway dis-
ease. Am J Respir Crit Care Med 2007; 176:
974–982.
3 Fattouh R, Midence G, Arias K, Johnson J,
Walker T, Goncharova S, Souza K, Gregory
R, Lonning S, Gauldie J, Jordana M: Trans-
forming growth factor- ?regulates house
dust mite-induced allergic airway inflam-
mation but not airway remodeling. Am J
Respir Crit Care Med 2008; 177: 593–603.
4 Eisner MD, Yelin EH, Henke J, Shiboski SC,
Blanc PD: Environmental tobacco smoke
and adult asthma: the impact of changing ex-
posure status on health outcomes. Am J
Respir Crit Care Med 1998; 158: 170–175.
5 Akdis CA, Kussebi F, Pulendran B, Akdis M,
Lauener RP, Schmidt-Weber CB, Klunker S,
Isitmangil G, Hansjee N, Wynn TA, Dillon
S, Erb P, Baschang G, Blaser K, Alkan SS: In-
hibition of T helper 2-type responses, IgE
production and eosinophilia by synthetic li-
popeptides. Eur J Immunol 2003; 33: 2717–
2726.
6 Tuli? MK, Wale JL, Holt PG, Sly PD: Modifi-
cation of the inflammatory response to al-
lergen challenge after exposure to bacterial
lipopolysaccharide. Am J Respir Cell Mol
Biol 2000; 22:604–612.
7 Camateros P, Tamaoka M, Hassan M, Mari-
no R, Moisan J, Marion D, Guiot MC, Martin
JG, Radzioch D: Chronic asthma-induced
airway remodeling is prevented by toll-like
receptor-7/8 ligand S28463. Am J Respir Crit
Care Med 2007; 175: 1241–1249.
8 Lefort J, Singer M, Leduc D, Renesto P, Na-
hori MA, Huerre M, Créminon C, Chignard
M, Vargaftig BB: Systemic administration of
endotoxin induces bronchopulmonary hy-
perreactivity dissociated from TNF-alpha
formation and neutrophil sequestration into
the murine lungs. J Immunol 1998; 161: 474–
480.
9 Redecke V, H?cker H, Datta SK, Fermin A,
Pitha PM, Broide DH, Raz E: Cutting edge:
activation of toll-like receptor 2 induces a
Th2 immune response and promotes experi-
mental asthma. J Immunol 2004; 172: 2739–
2743.
6
:
3
1
A
M
0 Hasday JD, Bascom R, Costa JJ, Fitzgerald T, Dubin W: Bacterial endotoxin is an active component of cigarette smoke. Chest 1999; 115: 829–835.
1 Tighe H, Takabayashi K, Schwartz D, van Nest G, Tuck S, Eiden JJ, Kagey-Sobotka A, Creticos PS, Lichtenstein LM, Spiegelberg HL, Raz E: Conjugation of immunostimula-tory DNA to the short ragweed allergen Amb a 1 enhances its immunogenicity and reduc-es its allergenicity. J Allergy Clin Immunol 2000; 106: 124–134.
2 Marshall JD, Abtahi S, Eiden J, Tuck S, Mil-
ley R, Haycock F, Reid MJ, Kagey-Sobotka A,
Creticos PS, Lichtenstein LM, van Nest G:
Immunostimulatory sequence DNA linked
to the Amb a 1 allergen promotes T H1cyto-
kine expression while downregulating T H2
cytokine expression in PBMCs from human
patients with ragweed allergy. J Allergy Clin
Immunol 2001; 108: 191–197.
3 Simons FE, Shikishima Y, van Nest G, Eiden
JJ, HayGlass KT: Selective immune redirec-
tion in humans with ragweed allergy by in-
jecting Amb a 1 linked to immunostimula-
tory DNA. J Allergy Clin Immunol 2004; 113:
1144–1151.
4 Tulic MK, Fiset PO, Christodoulopoulos P,
Vaillancourt P, Desrosiers M, Lavigne F, Ei-
den J, Hamid Q: Amb a 1-immunostimula-
tory oligodeoxynucleotide conjugate immu-
notherapy decreases the nasal inflammatory
response. J Allergy Clin Immunol 2004; 113:
235–241.
5 Gauvreau GM, Hessel EM, Boulet LP, Coff-
man RL, O’Byrne PM: Immunostimulato -
r y sequences regulate interferon-inducible
genes but not allergic airway responses. Am
J Respir Crit Care Med 2006; 174: 15–20.
6 Fanucchi MV, Schelegle ES, Baker GL, Evans
MJ, McDonald RJ, Gershwin LJ, Raz E, Hyde
DM, Plopper CG, Miller LA: Immunostimu-
latory oligonucleotides attenuate airways re-
modeling in allergic monkeys. Am J Respir
Crit Care Med 2004; 170: 1153–1157.
6
:
3
1
A
M