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Am J Physiol Lung Cell Mol Physiol 296: L185-L197, 2009. First published November 21, 2008; doi:10.1152/ajplung.90367.2008
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New asthma biomarkers: lessons from murine models of acute and chronic asthma

Emmanuel Di Valentin,1,* Céline Crahay,2,* Nancy Garbacki,3 Benoit Hennuy,4 Maud Guéders,2 Agnès Noël,2 Jean-Michel Foidart,2 Johan Grooten,5 Alain Colige,3 Jacques Piette,1 and Didier Cataldo2

1Laboratory of Fundamental Virology and Immunology, 2Laboratory of Biology of Tumours and Development, 3Laboratory of Connective Tissues Biology, and 4Transcriptomic Unit, GIGA-Research, University of Liège, Liège; and 5Department for Molecular Biomedical Research (DMBR), VIB-Ghent University, Ghent (Zwijnaarde), Belgium

Submitted 30 June 2008 ; accepted in final form 17 November 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Many patients suffering from asthma are not fully controlled by currently available treatments, and some of them display an airway remodeling leading to exaggerated lung function decline. The aim of the present study was to unveil new mediators in asthma to better understand pathophysiology and propose or validate new potential therapeutic targets. A mouse model of asthma mimicking acute or chronic asthma disease was used to select genes undergoing a modulation in both acute and chronic conditions. Mice were exposed to ovalbumin or PBS for 1, 5, and 10 wk [short-, intermediate-, and long-term model (ST, IT, and LT)], and gene expression in the lung was studied using an Affymetrix 430 2.0 genome-wide microarray and further confirmed by RT-PCR and immunohistochemistry for selected targets. We report that 598, 1,406, and 117 genes were upregulated and 490, 153, 321 downregulated at ST, IT, and LT, respectively. Genes related to mucous secretion displayed a progressively amplified expression during the allergen exposure protocol, whereas genes corresponding to growth and differentiation factors, matrix metalloproteinases, and collagens were mainly upregulated at IT. By contrast, genes related to cell division were upregulated at ST and IT and were downregulated at LT. In this study, besides confirming that Arg1, Slc26a4, Ear11, and Mmp12 genes are highly modulated throughout the asthma pathology, we show for the first time that Agr2, Scin, and Cd209e genes are overexpressed throughout the allergen exposure and might therefore be considered as suitable new potential targets for the treatment of asthma.

microarray; ovalbumin-induced asthma; airway inflammation; remodeling

ASTHMA IS A COMPLEX CHRONIC inflammatory disease characterized by airway inflammation and hyperresponsiveness obstruction, which is at least partially reversible. Eosinophilic inflammation is a hallmark of asthma and correlates to bronchial hyperresponsiveness and disease severity (21). The activation of a network of cytokines/chemokines including the production of various soluble mediators leads to eosinophil accumulation and airway hyperresponsiveness. Among those mediators, IL-13, which is produced by CD4+ T helper type 2 lymphocytes (Th2 cells), induces the bronchi to become hyperreactive and promotes IgE production and airway eosinophilia (40).

During the past 2–3 decades, epidemiology of asthma has been found to increase dramatically with prevalence ranging from 1% to 18%, reflecting wide variations between different regions and populations (23). During their lifetime, asthmatics display higher rates of lung function decrease than normal individuals, related to a remodeling of the airway walls (18, 28). Major features of asthma-induced airway remodeling consist of epithelial damages, smooth muscle cell hyperplasia, glandular hyperplasia, and airway wall fibrosis including a thin collagen layer deposition in the lamina reticularis of airway epithelium (13, 15, 28).

Current treatments for asthma are not effective in controlling most of airway remodeling features and do not allow all patients to reach control of the disease from a clinical point of view. For these reasons, new therapeutic targets in asthma are eagerly needed to allow getting control of acute inflammation, hyperresponsiveness, and airway remodeling.

Mouse models of asthma following allergen exposure have been developed and thoroughly characterized, reproducing airway hyperresponsiveness, inflammation, and remodeling (30).

In the present study, a mouse model of asthma allowing the development of both acute and chronic responses to allergen was used to identify, by a transcriptomic analysis, new potential therapeutic targets relevant to the disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Protocol of sensitization and allergen exposure. BALB/c mice were used following "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research, and the experimental protocols were approved by the local animal ethical committee (University of Liège) under the no. 03/158. Two different protocols were used. For the "short-term" (ST) exposure protocol, 6- to 8-wk-old BALB/c males mice were sensitized on days 1 and 8 by intraperitoneal injection of 10 µg of ovalbumin (OVA Grade III; Sigma-Aldrich, Schnelldorf, Germany) emulsified in aluminum hydroxide (AlumInject; Perbio, Erembodegem, Belgium). Mice were subsequently divided into 2 groups: 1 group of mice was only exposed to PBS aerosol (sham challenge), and the other group was subjected to ovalbumin (OVA) 1% aerosol for 30 min. Aerosols were generated daily by ultrasonic nebulizer (DeVilbiss 2000) from days 21 to 27. After determination of airway reactivity, death of mice was performed on day 28 as previously reported (6). Three different experiments have been made, and measurements have been performed on cohorts of 12 mice per experimental condition (4 PBS and 8 OVA-treated mice). An "intermediate-term" (IT) and "long-term" (LT) exposure protocol was designed and adapted from the ST exposure protocol. Amendments were made to the ST exposure protocol as follows: mice were sensitized by an intraperitoneal injection on days 1 and 11, and groups of mice were exposed to PBS or OVA aerosols 5 days/wk (odd weeks) from days 22 to 90. The protocols are illustrated in Fig. 1A.


Figure 1
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  		Fig. 1. A: experimental procedure. Short-term (ST), intermediate-term (IT), and long-term (LT) PBS/ovalbumin (OVA) sensitization and exposure protocols. BALB/c males mice were sensitized on days 1 and 8 by intraperitoneal injection of 10 µg of OVA. Mice were subsequently exposed to daily PBS aerosol or OVA 1% aerosol for 30 min for ST, IT, or LT duration (see MATERIALS AND METHODS for the detailed protocol). B: methacholine responsiveness. Mice were exposed to increasing doses of methacholine (2.5–12.5 g/l). To take into account every individual dose-response curve, data are expressed as area under the curve of dose-response graphs (arbitrary units; A.U.). C: quantification of peribronchial inflammation. Airway inflammation quantified from hematoxylin and eosin-stained slides is expressed as a bronchial inflammation score as reported previously (6). D: eosinophilic inflammation score. The number of eosinophils was determined using a digitalized picture and was reported to the length of the epithelial basement membrane. Mean scores were measured as described in MATERIALS AND METHODS. Results are expressed as means ± SE, and the comparison between groups was performed using Mann-Whitney U test. P < 0.05({star}). NS = not significant (P ≥ 0.05).

 
Assessment of airway remodeling in histology. Mice were anesthetized by intraperitoneal injection (200 µl) of a mixture of ketamine (10 mg/ml; Merial, Brussels, Belgium) and xylazine (1 mg/ml; VMD, Arendonk, Belgium). A tracheotomy was performed by insertion of a 20-gauge polyethylene catheter into the trachea. A ligature was made around the catheter to avoid leaks and disconnections. Mice were ventilated with a flexiVent small animal ventilator (SCIREQ, Montréal, Québec, Canada) at a frequency of 250 breaths/min and a tidal volume of 10 ml/kg. A positive end-expiratory pressure was set at 2 hPa. Measurement started after 2 min of mechanical ventilation. A sinusoidal 1-Hz oscillation was then applied to the tracheal tube and allowed a calculation of dynamic resistance, elastance, and compliance of the airway by multiple linear regressions. A second maneuver consisting in an 8-s forced oscillatory signal ranging frequencies between 0.5 and 19.6 Hz allowed measurement of impedance to evaluate tissue damping, tissue elastance, and tissue hysteresivity (14). Following measurement of baseline lung function, mice were exposed to a saline aerosol (PBS) followed by increasing doses of methacholine aerosols (3, 6, 9 and 12 g/l; ICN, Asse-Relegem, Belgium), and a dose-response curve was recorded for each animal. Aerosols were generated by an ultrasonic nebulizer (SCIREQ) and delivered to the inspiratory line of the flexiVent using a bias flow of medical air following the manufacturer's instructions. Each aerosol was delivered for 10 s, and periods of measurement as described above were made at 1-min intervals following each aerosol.

Remodeling assessment in histology. The thorax was opened, and the left main bronchus was clamped. The right lung was infused with 4% paraformaldehyde, embedded in paraffin, and processed for histology and immunohistochemistry. Sections of 5-µm thickness were cut off from paraffin and stained with hematoxylin and eosin. The extent of peribronchial inflammation was quantified as a score related to the thickness of inflammatory cells around the bronchi, as previously described (6). The number of eosinophils was quantified after specific Congo red staining by manual counting in randomly selected bronchi and normalized to the perimeter of corresponding epithelial basement membrane.

Glandular hyperplasia was quantified as percentage of Alcian blue-stained goblet cells per total epithelial cells in randomly selected bronchi.

Masson's trichrome staining was used to detect peribronchial collagen deposition. A score from 0 to 3 was applied to each observed bronchi: 0, no collagen around bronchi; 1, a thin layer of collagen; 2, a cluster of collagen; and 3, a thick layer of collagen.

Results of methacholine hyperresponsiveness, inflammation score, and eosinophil numbers are expressed as means ± SE, and comparison between groups was performed using Mann-Whitney U test. Mann-Whitney U test was performed using GraphPad InStat (GraphPad; http://www.graphpad.com). Values of P <0.05 were considered as significant.

Immunohistochemistry. Mice lung sections of 5-µm thickness were treated successively by 0.1% trypsin (Sigma-Aldrich), 3% H2O2 (Merck, Darmstadt, Germany), 1% Triton X-100 (Merck), and 10% BSA (Sigma-Aldrich) and then incubated for 2 h at room temperature with primary antibody. These antibodies were specific for, respectively, type III (Col3, anti-type III collagen), type V (Col5, anti-type V collagen, 20541; Novotec), and type VI (Col6, anti-type VI collagen) collagens; {alpha}-smooth muscle actin ({alpha}-Sma/Acta2, anti-{alpha}-smooth muscle actin/FITC, F3777; Sigma-Aldrich); anterior gradient 2 (Agr2, anti-Agr2 homolog, SP7216P; Acris Antibodies); Cd209e antigen (anti-mouse Signr4, BAF1528; R&D Systems); Fc receptor, IgG, low affinity II (Fcgr2, anti-mouse CD32/CD16, AF1460; R&D Systems); complement component 1, q subcomponent, {alpha}-polypeptide (anti-C1qa, SC-27661; Santa Cruz Biotechnology); chemokine (C-C motif) ligand 8 (Ccl8, anti-CCL8/MCP2, MAB790; R&D Systems); chitinase 3-like 3 [Chi3l3 or Ym1, rabbit anti-Ym1 provided by Dr. S. Kimura (39)] and arginase 1 (Arg1, anti-arginase-1, 610708; BD Transduction Laboratories). Slides were then washed with PBS and incubated with horseradish peroxidase-conjugated secondary antibodies (DAKO, Heverlee, Belgium).

Measurement of anti-OVA-specific IgE. After mice death, blood was taken from the heart for OVA-specific IgE measurement. Plates (Immuno BreakApart Maxisorp C8; NUNC) were coated with OVA. Serum was added. Plates were then incubated with a biotinylated polyclonal rabbit anti-mouse IgE (401100; Calbiochem). A serum pool from OVA-sensitized animals was used as internal laboratory standard. One unit was arbitrarily defined as 1:100 dilution of this pool (6).

Lung RNA extraction and cDNA preparation. For each time-point experiment (ST, IT, or LT), PBS-treated (n = 4) and OVA-treated (n = 8) mice were killed. The right lobe of the lung was directly incubated in RNAlater RNA Stabilization Reagent (76104; Qiagen) for 1 night at 4°C and then stored at –80°C. Tissue disruption was performed with a Mikro-Dismembrator (Sartorius Stedim Biotech, Vilvoorde, Belgium), and homogenization was done with QIAshredder kit (79654; Qiagen). Total lung RNA was extracted and purified using QIAprep Spin Miniprep Kit and RNase-Free DNase I (27106 and 79254; Qiagen) according the manufacturer's instructions. For real-time RT-PCR and microarray studies, RNA pools were used. The control pool (PBS) contained equal amount of RNA from the lung of four control mice. For OVA-treated mice, two independent pools (pool 1 and pool 2; each containing RNA from 4 different mice) were processed separately.

Transcriptome analysis. The RNA quality was assessed by the Experion automated electrophoresis system using the RNA StdSens Analysis Kit (Bio-Rad). Four micrograms of total RNA were labeled using the GeneChip Expression 3'-Amplification One-Cycle Target Labeling Kit (Affymetrix, Santa Clara, CA) following the manufacturer's protocol. The cRNA was hybridized to GeneChip Mouse Genome 430 2.0 (Affymetrix) according to the manufacturer's protocol. Briefly, double-stranded cDNA was synthesized from 4 µg of total RNA primed with a poly-(dT)-T7 oligonucleotide. The cDNA was used in an in vitro transcription reaction in the presence of T7 RNA polymerase and biotin-labeled modified nucleotides for 16 h at 37°C. Biotinylated cRNA was purified and then fragmented (35–200 nucleotides) together with hybridization controls and hybridized to the microarrays for 16 h at 45°C. Using Fluidics Station (Affymetrix), the hybridized biotin-labeled cRNA was revealed by successive reactions with streptavidin R-phycoerythrin conjugate, biotinylated anti-streptavidin antibody, and streptavidin R-phycoerythrin conjugate. The arrays were finally scanned with an Affymetrix/Hewlett-Packard GeneChip Scanner 3000 7G.

The data were generated with the MAS 5.0 algorithm included in GeneChip Operating Software (GCOS). Values obtained for PBS treatment condition have been considered as baseline for pairwise comparison with OVA-induced experimental conditions.

The probe sets have been filtered on signal log ratio (greater than 0.6 for upregulated and less than –0.6 for downregulated transcripts) and on P value associated with the change status (<0.001 for upregulated probe sets, >0.999 for downregulated probe sets). Lists of differentially expressed genes obtained from two different OVA-treated pools [OVA 1 and OVA 2] vs. PBS pools and corresponding to each experimental time point W1 (ST), W5 (IT) and W10 (LT) are shown in Supplemental Tables 1

Formula

3 (available in the data supplement online at the AJP-Lung Cellular and Molecular Physiology web site). Pools were made with RNA obtained from a minimum of three mice.

Real-time PCR. A 1-µg portion of RNA was reverse-transcribed in 15-µl reaction mixture containing 2.5 mM random primers (Amersham Pharmacia Biotech, Diegem, Belgium), 16 mM DTT, 1.6 mM 2-deoxynucleotide 5'-triphosphate (dNTP; Roche, Vilvoorde, Belgium), 0.35 µl of RNA guard (Amersham Pharmacia Biotech), 250 units of Moloney murine leukemia virus RT (Life Technologies, Merelbeke, Belgium), and first-strand buffer (Life Technologies). Resulting cDNA was subjected to quantitative real-time PCR using the SYBRgreen mix method (RT-SN2x-005; Eurogentec, Liège, Belgium). Primers were designed using Primers Express software and were obtained from Eurogentec (Table 1). Real-time PCR experiments were run on an ABI 7700 instrument, and data were analyzed using Sequence Detection System software (Applied Biosystems). Results were normalized using β2-microglobulin (β2m) and hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) transcripts. Experiments were done at least in triplicate. Differences (n-fold) between samples were calculated using the standard curve method and the 2(–{Delta}{Delta}CT) method (20). P values were calculated using GraphPad QuickCalcs software (t-test).


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  		Table 1. Primers used for qRT-PCR

 
Functional analysis. To determine which pathways were significantly regulated, lists of differentially expressed (up- and downregulated) genes (Supplemental Tables 1–3) were uploaded in Ingenuity Pathway Analysis software (IPA 6.0; Ingenuity Systems; http://www.ingenuity.com). Results are shown in Supplemental Tables 4–6.

GOstat tool (http://gostat.wehi.edu.au/cgi-bin/goStat.pl; Ref. 2) was used to find overrepresented gene ontology (GO) biological processes (BP). Lists of differentially expressed (up- and/or downregulated) genes [OVA pools 1 and 2 vs. PBS for W1 (ST), W5 (IT), and W10 (LT); Supplemental Tables 1–3] were used as input into the analysis software. Each gene list was loaded in the Group IDs box, and the Affymetrix Mouse Genome 430 2.0 Array database was selected. The minimal length of GO pathways was 4; the selected GO super category was "Biological Process," with a prefilter of P < 0.1 for the output. The outputs are shown in Supplemental Tables 7–9.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Assessment of airway inflammation, sensitization, and hyperresponsiveness. Mice exposed to PBS or OVA for 1 (ST), 5 (IT), and 10 (LT) wk were characterized by assessment of airway responsiveness, airway inflammation, and anti-OVA-specific IgE production in serum and airway remodeling (Fig. 1A).

Airway resistance was measured in all mice by using the flexiVent system. At the end of each protocol of allergen exposure, mice showed increased airway resistance compared with corresponding control groups (sham exposed to PBS). Dose-response curves measuring airway resistance (expressed as area under the curve, AUC) after increasing doses of methacholine were recorded and displayed a significant higher reactivity in OVA-exposed mice in ST and IT exposure models compared with corresponding PBS-exposed mice (Fig. 1B).

ST and IT allergen-exposed mice displayed significant higher peribronchial inflammation compared with control mice (Fig. 1C; P < 0.05). Eosinophilic infiltration of the bronchial walls was significantly increased in each experimental group compared with control but decreased over time, with lower levels being observed in LT compared with ST and IT groups (Fig. 1D).

Anti-OVA-specific IgE levels were significantly increased in mice after OVA exposure (ST, 343.25 ± 96.24 vs. 4.80 ± 1.35; IT, 363.50 ± 67.58 vs. 7.6 ± 2.82; LT, 240.00 ± 53.17 vs. 2.66 ± 0.66; P < 0.005).

Assessment of airway remodeling in histology. Obvious mucous hyperplasia can be evidenced in our experimental model of asthma. Percentages of goblet cell in the bronchial epithelium were progressively increased over time in allergen-exposed mice and were significantly higher than controls at every time point studied (P < 0.05; Fig. 2, top).


Figure 2
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  		Fig. 2. Remodeling of lung after PBS or OVA exposures. Mice exposed to PBS or OVA were killed at various time points, and their lungs were examined by immunohistochemistry (magnification, x40). Graph results are expressed as means ± SE, and the comparison between groups was performed using Mann-Whitney U test. P < 0.05({star}); P >= 0.05 (NS, not significant). Only 1 representative example of PBS-exposed animals is provided in this figure, but every immunohistochemistry or staining was compared with the appropriate control matched for time of exposure to PBS. There were no differences among the PBS-exposed mice for any duration. Top: goblet cell staining with Alcian blue (bronchial walls in blue) and goblet cells percentage in bronchi. Middle: collagen deposition in lung tissue (Masson's trichrome staining) and calculated collagen score. Bottom: collagen type III (Col3), collagen type V (Col5), and {alpha}-smooth muscle cell actin ({alpha}-Sma/Acta2) detection by immunohistochemistry in lung tissue.

 
When evaluated by Masson's trichrome staining, an increased collagen deposition was observed in allergen-exposed lung of the IT and LT groups but not in the ST group (Fig. 2, middle). This was further confirmed by immunohistochemistry showing subepithelial accumulation of collagen types III (Col3) and V (Col5) at IT and LT but not at ST. By contrast, {alpha}-smooth muscle actin staining was increased throughout allergen exposure (Fig. 2, bottom).

Transcriptome analysis. Transcriptome analysis was carried out using Affymetrix microarrays. For each experimental condition, total RNA pools from lungs of four animals were used. Allergen-exposed cohorts were studied in duplicate (2 independent pools containing total RNA from 4 different mice) for all time points (ST, IT, and LT). Comparisons were made between allergen- and PBS-exposed mice (OVA pools vs. PBS pool for the corresponding time point) taking into account, for each, only genes similarly regulated in the two cohorts of allergen-exposed mice.

Figure 3 shows scatter plots of gene expression in lungs in response to OVA or sham challenge for each time point (ST, IT, LT; raw data of modulated genes are shown in Supplemental Tables 1–3). After ST exposure to OVA, roughly equivalent numbers of genes were found to be up- or downregulated, 598 and 490 genes, respectively. On the contrary, modulated genes were mainly upregulated after IT exposure (1,406 up- vs. 153 downregulated) and mainly downregulated after LT exposure (117 up- vs. 321 downregulated; Fig. 3). Top 15 up- (Fig. 3, white boxes) and downregulated (Fig. 3, black boxes) genes are provided for each condition. In this top 15, arginase 1 gene (Arg1) appears to be constantly and strongly upregulated.


Figure 3
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  		Fig. 3. A: scatter plots of mouse lung gene expression in response to OVA exposure corresponding to each time point. For each time-point experiment (ST, IT, or LT), 4 PBS-treated (pool PBS) and 8 OVA-treated mice (divided in 2 groups, pool 1 OVA and pool 2 OVA) were killed. Lung RNA was extracted, and the RNA pools (PBS pool, pool 1 OVA, and pool 2 OVA) were then submitted to a transcriptomic analysis using Affymetrix microarrays. Log2 fold change between OVA pool 1 vs. PBS pool or OVA pool 2 vs. PBS pool were calculated (Supplemental Tables 1–3). The scatter plots show the comparison of log2 fold change between the 2 biological replicates for each time point of the OVA exposure: pool 1 OVA (vs. PBS) compared with pool 2 OVA (vs. PBS). B: listing of top 15 up- and downregulated genes and their respective fold induction (FI) corresponding to each time-point experiment (extracted from Supplemental Tables 1–3).

 
Different groups of up- or downregulated genes were selected according to their known or putative functions (Fig. 4). Expression of genes related to mucous secretion displayed a progressive increase during allergen exposure and were mostly upregulated in the LT group. Growth factors, matrix metalloproteinases (MMPs), and collagen genes were mainly overexpressed at IT. Among those groups, however, Timp1 and Col6a2 displayed a constant upregulation over time, whereas Igf1 and Mmp12 were more specifically upregulated after a short-time allergen exposure (Fig. 4). Genes implicated in cell division were generally overexpressed in ST group and downregulated in the LT group, suggesting that cell cycle is activated at early time points. In the group of genes related to innate immunity, cytokines, interleukins, and chemokines, we did not find any common trend of preferential gene modulation over time, although several individual genes (Ccl11, Ccl8, Ccl9, and Ccr5) appeared to be significantly overexpressed in the three different experimental protocols (ST, IT, and LT).


Figure 4
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  		Fig. 4. Selected genes displaying modulation of expression (at least at 1 time point) in the microarray analysis. TLR, Toll-like receptor.

 
As one of the main focuses of our study was to determine which genes could represent suitable therapeutic targets during the whole course of asthma, genes regulated in all three experimental conditions were considered of special interest. Some of them were already known to take part to asthma pathology (Fig. 5A), whereas others were not previously reported to be implicated (Fig. 5B, "Potential new targets"). Interestingly, in this potential new targets category, most genes are upregulated at ST and IT and downregulated at LT.


Figure 5
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  		Fig. 5. Shown are genes undergoing a significant modulation (and corresponding proteins) for each time point determined by microarray analysis. A: modulated genes previously described as modulated in asthma. B: potential new targets for asthma treatment. NC, no change.

 
RT-PCR validations. As validation for microarray measurements, quantitative RT-PCR (qRT-PCR) assays were performed using the same RNA samples as in our transcriptomic analysis, and RNA were samples obtained with a second experimental design. Eighteen gene products from the lists presented in Figs. 4 and 5 were arbitrarily selected and quantified including genes undergoing different patterns of regulation or previously reported to be strongly up- or downregulated during asthma. qRT-PCR measurements were always tightly correlated with microarray results (Fig. 6).


Figure 6
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  		Fig. 6. Validation of microarray analysis results by quantitative RT-PCR experiments (qRT-PCR). RT-PCR assays were performed in duplicate: by using RNA samples in our transcriptomic analysis and by using RNA samples from a 2nd data set obtained by performing a separate experiment. The graphs show a compilation of these 2 RT-PCR assays (error bars represent standard deviations). mRNA levels were determined by quantitative real-time PCR and were normalized by using the β2-microglobulin and hypoxanthine guanine phosphoribosyl transferase (Hprt) transcripts. Differences (n-fold) between samples (OVA vs. PBS) were calculated using the standard curve method and the 2(–{Delta}{Delta}CT) method (20). PBS fold induction was arbitrarily set as 1. P values (qRT-PCR OVA experiments vs. PBS) were calculated using t-test (GraphPad QuickCalcs software; http://www.graphpad.com). Microarray error bars correspond to the standard deviation between the 2 microarray experiments.

 
Immunohistochemistry validations. Since genes upregulated at each time point were considered to be promising new therapeutic targets in asthma, their protein products were analyzed by immunohistochemistry (Agr2, Cd209e, Col6, and C1qa). Other genes previously described as upregulated in asthma have also been studied for validation [Fcgr2 (16), Ccl8 (11), Chi3l3 (44), and Arg1 (48)]. Immunohistochemical analysis were carried out on lung sections from ST, IT, and LT groups and have shown that Cd209e, Ccl8, and Fcgr2 were mostly produced by immune cells, whereas Arg1 and Chi3l3 were produced by epithelial and immune cells. Agr2 production was mainly detectable in goblet cells. As expected, Col6 and C1qa productions were detectable in the extracellular matrix from the peribronchial area (Fig. 7).


Figure 7
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  		Fig. 7. Immunohistochemical analysis of PBS or OVA-exposed mice lung sections using antibodies directed against Agr2, Cd209e, Col6, C1qa, Fcgr2, Ccl8, Chi3l3, and Arg1 (magnification, x40). Only 1 representative example of PBS-exposed animals is provided in this figure, but every immunohistochemistry was compared with the appropriate control matched for time of exposure to PBS. There were no differences among the PBS-exposed mice for any duration.

 
Modulated pathways and biological processes. Finally, an analysis of modulated pathways and GO BP was performed using Ingenuity Pathway Analysis and GOstat software, respectively (entire results are shown in Supplemental Tables 4–6 and 7–8). Based on these data, several pathways and biological processes more specifically regulated at a defined time point (Fig. 8A) or modulated throughout allergen exposure (Fig. 8B) were selected.


Figure 8
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  		Fig. 8. Summary of selected gene ontology (GO) biological processes (BP) and pathways that were modulated during OVA exposure. GO BP was determined using GOstat software, and pathways were obtained with Ingenuity Pathway Analysis software (Supplemental Tables 4–9). A: pathways and BP regulated at specific time points. {uparrow} Or {downarrow} means up- or downmodulated BP/pathway. B: pathways and BP modulated throughout allergen exposure; the 3 arrows correspond to each time points. MHCII, major histocompatibility complex II; GM-CSF, granulocyte/macrophage colony-stimulating factor; TGFβ, transforming growth factor-β; PI3K, phosphatidylinositol 3-kinase.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
New therapeutics controlling different aspects of asthma, especially LT airway remodeling, are urgently needed as the prevalence and the severity of the disease are both dramatically increasing. In this context, identification of genes regulated during the whole course of allergen exposure is of potential interest.

In the present study, a transcriptomic analysis was performed to identify genes differentially regulated at the 3 time points in OVA-exposed mice compared with controls. Among genes systematically up- or downregulated after allergen exposure, ~20 were previously described to be associated with asthma, further validating our experimental model. More specifically, Arg1 (48), Mmp12 (7), and Ear11 (Rnase3) (8) were previously shown to be upregulated in ST model of asthma induction. Our data confirm this strong induction at ST but further demonstrate their constant overexpression as the allergic stimulation persists. This work resulted also in the identification of 36 other genes regulated throughout the disease but not previously known to be associated with asthma. Among them, only 14 were upregulated at each time point, including Cd209e, Agr2, and Scin.

Cd209e (DC-Signr4), identified in cell of the immune system, displays a 70% homology with human DC-SIGN expressed by human dendritic cells (26, 34, 46) and, therefore, may play key roles in dendritic cells-T lymphocytes interaction participating in the exaggerated activation of T lymphocytes during asthma.

The Agr2 gene (also referred to as hAG-2 or Gob-4) appears of particular interest as it encodes a secreted protein expressed in mucous-secreting cells and endocrine organs (35). Using RT-PCR and immunohistochemistry, we have demonstrated that Agr2 protein is continuously overexpressed in our mice model of asthma and is mainly localized in goblet cells. It has been reported to be also highly expressed in adenocarcinomas of the esophagus, pancreas, breast, and prostate (17, 19, 38, 43) and to promote tumor growth, cell migration, and cellular transformation (38). It has been also suggested to be involved in the inflammatory bowel disease and in epithelial barrier function (45). Transcription of Agr2 gene can be induced by transcription factors typical for epithelial goblet cells such as Foxa1 and Foxa2 (36, 45). However, none of these two genes were modulated in our microarray experiments, suggesting another pathway for Agr2 activation in asthma.

Interestingly, two other genes also overexpressed throughout allergen exposure and involved in secretion processes were identified: Scin and Slc26a4.

Scin (adseverin) is a Ca2+-activated actin filament severing and capping protein (31). This protein is able to rearrange the apical actin cap, present in airway goblet cell, that acts as a physical barrier to mucin secretion (9). Therefore, an overexpression of Scin in goblet cells might allow secretory granules to reach airway lumen. Scin protein might also have other roles in asthma pathogenesis since it is involved in cell differentiation by inducing rearrangements of the actin cytoskeleton, and its expression is positively regulated by PKC and MEK signaling (25).

The product of Slc26a4 gene (pendrin, pds), an anion transporter present at apical cell surface, allows airway epithelial cells secreting thiocyanates, which are implicated in mucosal surface innate host defenses. Interestingly, Slc26a4 expression was reported to be increased by IL-4 in bronchial epithelial cells (29). Very recently it was also reported to be induced by IL-13 in tracheal epithelial cells and to be overexpressed in a murine model of asthma leading to mucous overproduction (24). In that specific model, Slc26a4 was overexpressed in the lungs of sensitized mice exposed to allergens for limited durations, whereas in the present study, upregulation was observed in the ST exposure model but also after IT (6-fold induction) and LT (11-fold induction) treatment, pointing to Slc26a4 as a potential therapeutic target of choice in asthma.

These three genes involved in mucous production (Agr2, Scin, and Slc26a4) are expressed throughout the allergen exposure and before expression of other well-known mucous gene such as Clca3, Muc5ac, and Muc5b. These data suggest an early and pivotal role of Arg2, Scin, and Slc26a4 for mucous secretion by goblet cells. We have also observed that the expression of all these "mucous genes" was constantly upregulated in microarray experiments (Fig. 4) showing a good correlation with the remodeling assessment performed in histology (Fig. 2).

Of interest is also the first demonstration that Pon1 is the only gene to be downregulated in all experimental conditions in our model. Pon1 gene encodes for paraoxonase, which has been suggested to have a protective role against oxidation induced by reactive oxygen species in asthma (10). Pon1 decrease could therefore participate in asthma development.

Some rather classical mediators of asthma, such as IL-13 or MMP-9, have actually not been found to be increased in our genome-wide transcriptomic analysis. Previous analysis by microarrays in asthma also failed to report any modulation of those two genes (27, 48). Various hypotheses could explain this apparent discrepancy. Regarding IL-13, we hypothesize that this very potent cytokine is produced by a very limited amount of cells (mainly Th2 lymphocytes), and therefore its overexpression could be missed by a microarray studying a mixture of RNA purified from the whole lung. This could be a potential limitation of microarray studies in diseases such as asthma. For MMP-9, on the contrary, the number of producing cells (mostly granulocytes and macrophages) is quite high in the lung parenchyma and the absence of an upregulation of MMP-9 mRNA in our microarray experiments confirm previous studies showing that MMP-9 protein is mainly preformed in the bone marrow during the granulocytes maturation process and secreted when those cells are activated. MMP-9 production in lungs is therefore formally underestimated when assessing gene expression (5). However, in the present study, we have detected MMP-12 overexpression after acute allergen exposure persisting over time. This could seem contradictory with the above discussion about MMP-9. Indeed, MMP-12, unlike MMP-9, is produced in loco by macrophages and dendritic cells (4). It is therefore expected that MMP-12 expression is higher when the inflammation is maximal (ST exposure model).

Comparison between genes thoroughly overexpressed in our microarray experiments (35 genes listed in Fig. 5) and those modulated in 2 other studies reporting gene expression profiling in mouse asthma models (27, 48) reveals 14 common genes: Arg2, Arg1, Ccl8, Ccl11, Chi3l3, C1qa, C1qb, C1qc(g), Fcgr2b, Igf1, Scin, Serpina3g, Serpina3n, and Timp1. This correlation both validates the reproducibility of such data and reinforces the potential importance of these genes as therapeutic targets for asthma pathology as they are found to be overexpressed in different conditions such as different allergens and exposure protocols used. When comparing our transcriptomic analysis and a very recent proteome analysis (41) of lungs in a mouse chronic asthma model, some genes modulated in this analysis can also be detected at the protein level in the mouse chronic asthma model: e.g., Chi3l4 (Ym2), Retnla (Fizz1), Chia, Col6a1, or Cox5a.

These correlations both validate the reproducibility of such data and reinforce the potential importance of these genes as therapeutic targets for asthma pathology as they are found to be overexpressed in different conditions such as different allergens and exposure protocols used.

Study of the expression of defined genes has provided the identification of new potential target genes in asthma pathology but does not always give a general picture of all the involved regulating networks. To address this problem, pathways and GO BP analyses were carried out for each time point of our model (full results are shown in Supplemental Tables 4–9). Several selected pathways and biological processes of relevance were modulated at specific time points (Fig. 8A), whereas others were constantly modulated throughout the allergen exposure (Fig. 8B).

Genes associated with inflammatory response are acutely upregulated at ST. Interestingly, upregulation of genes encoding for proteins involved in actin cytoskeleton signaling was observed at ST and IT. This observation was also made very recently by Wong and Zhao (41) using a proteomic approach. In the IT, however, activated biological processes correspond to organ development, morphogenesis, angiogenesis, and so forth, suggesting that active remodeling of lung or bronchi is occurring at this stage. β-Catenin/Wnt pathway has been suggested to be a reliable biomarker of asthma and proposed to be a therapeutic target (33, 37). However, in our microarray experiments, this pathway is upregulated only at IT. Similar conclusions can be drawn for IL-6 signaling pathway, which has also been reported to be involved in asthma pathology (1). Indeed, in the present study, IL-6, gp130 (IL-6st), and IL-6r were found to be upregulated only in IT conditions.

In the LT exposure model, downregulation predominates among biological processes, and cell-to-cell interactions (e.g., T cell receptor signaling and antigen receptor-mediated pathway) could be lowered compared with previous states (Fig. 8A).

Although some biological processes or pathways are constantly upregulated, some others can be both up- and downregulated according to allergen-exposure duration (Fig. 8B). For instance, cell cycle is activated in the ST and IT conditions but downregulated at the LT time point, suggesting that cell proliferation predominates in ST and IT rather than in LT conditions. In this setting, new therapeutic targets have to be searched in relevant pathways that are constantly activated at the three time points studied. For instance, arginine metabolism (49), IGF-1 signaling (42), and chemokines signaling (3, 22, 47), previously suggested as being important in asthma, are constantly upregulated in our study. Complement cascade is also activated in the three time points of our model. Previous authors have shown that C3 and C5 play a role in asthma pathogenesis, and our observation of a continuous upregulation of complement cascade pathway (C3 and C1q genes) further validates the idea that complement could be a suitable therapeutic target (12, 32). Furthermore, an upregulation of all subunits of C1q complex (C1qa, C1qb, and C1qc) was observed in our model throughout the allergen exposure, suggesting that a therapeutic intervention could take place at an earlier stage of the complement cascade, targeting C1q.

This work, representing the first complete analysis concerning genes involved in the progressive development of asthma pathology, has permitted us to establish a list of genes and pathways continuously overexpressed throughout the disease. In addition, several potential new target genes have been proposed and validated by immunohistochemistry. In conclusion, this study provides new insights in understanding the mechanisms prevailing in the establishment of asthma phenotype and unveils new potential therapeutic targets in asthma.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by the "Région Wallonne" (Ministère de la Région wallonne Direction générale des Technologies, de la Recherche et de l'Énergie, Division de la Recherche et de la Coopération scientifique, no. 415907), the FNRS (Brussels, Belgium), and the IAP 6/18 and IAP 6/35 networks (funded by the Interuniversity Attraction Poles Programme, initiated by the Belgian State, Science Policy Office).


   ACKNOWLEDGMENTS
 
We thank Dr. Shioko Kimura for providing the antibody directed anti-Chi3l3/4 (Ym1/Ym2), Nathalie Renotte, Christine Fink, and Fabienne Perrin for technical assistance.

A. Colige, J. Piette, and D. Cataldo are senior research associate, research director, and research associate from the Fonds National de la Recherche Scientifique (FNRS; Brussels, Belgium).


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Di Valentin, GIGA-Research Laboratory of Fundamental Virology and Immunology, Univ. of Liège, B-4000 Liège, Belgium (e-mail: edivalentin{at}ulg.ac.be)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* E. Di Valentin and C. Crahay contributed equally to this work. Back


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