Abstract
IL-9 overexpression protects against alveolar fibrosis induced by crystalline silica particles. This cytokine is also involved in allergic asthma. In the present study, we examined the effect of IL-9 overexpression on the subepithelial fibrotic response, a feature of asthmatic remodeling, induced by chronic exposure to Alternaria alternata extract. IL-9–overexpressing mice (Tg5) and their wild-type counterparts (FVB) were intranasally exposed to A. alternata extract or PBS (controls) twice a week during 3 mo. At the end of the allergic challenge, enhanced pause (Penh) measured in response to methacholine and fibrotic parameters, such as collagen and fibronectin lung content, were significantly higher in Tg5 compared with FVB. Staining of lung sections with Masson's Trichrome also showed more collagen fibers in peribronchial areas of treated Tg5 mice. A similar recruitment of inflammatory cells was observed in challenged FVB and Tg5 mice, except for eosinophils, which were significantly more abundant in the lung of Tg5. High serum levels of IgE and IgG1 in both strains indicated that FVB and Tg5 developed a strong type 2 immune response. The concentration of the eosinophil chemoattractant RANTES and the profibrotic mediator connective tissue growth factor (CTGF) was higher in the BAL of challenged Tg5 than FVB. These results demonstrate a profibrotic role of IL-9 in an airway remodeling model, possibly involving eosinophils and CTGF. These data also highlight a dual role of IL-9 in lung fibrosis, being anti- or profibrotic depending on the alveolar or airway localization of the process, respectively.
Our group has recently shown that Tg5 mice, which overexpress the type 2 cytokine IL-9, are partly protected against exaggerated collagen accumulation in silica-induced pulmonary fibrosis (5, 6). IL-9 has also been proposed as a therapeutic target for asthma (7). Indeed, IL-9 and its receptor IL-9R are expressed in bronchial biopsies of patients with asthma but not in healthy subjects (8). In experimental models, treatment of ovalbulmin (OVA)-challenged mice with anti–IL-9 antibodies decreases airway hyperreactivity (AHR), accumulation of eosinophils, and airway epithelium damage (9, 10). In addition, it has been reported that the lungs of mice overexpressing IL-9 display characteristic features of asthma as well as airway remodeling. These mice have a stronger AHR to metacholine than wild-type mice and accumulate more eosinophils and lymphocytes in the lung (11–13). Moreover, they develop hypertrophic airway epithelium and smooth muscle cells, and a subepithelial fibrosis. Altogether, these data raise the issue of a possible promoting role of IL-9 in airway fibrosis. Therefore, we examined the effect of IL-9 on ECM accumulation in an experimental model of bronchial asthma.
Most experimental models of asthma and airway remodeling use OVA as a surrogate allergen (14, 15). Contrary to OVA, spores of the mold Alternaria alternata are an important cause of allergic rhinitis and asthma in humans (16). We have recently shown that the spores of this mold induce lung allergy in a murine model (17). Here, we show that the overexpression of IL-9 exacerbates airway subepithelial fibrosis induced by chronic intranasal instillation of A. alternata extract. This effect is associated with an increased growth factor production and recruitment of eosinophils.
Tg5 mice constitutively express high levels of IL-9 in all organs and were described previously (18). Eight- to 10-wk-old female Tg5 mice and their background congeners, FVB, were obtained from the Ludwig Institute (Brussels, Belgium) and maintained under standard laboratory conditions. Mice were intranasally instilled twice a week during a total of 13 wk with 10 μg Alternaria extract (prepared as described in Ref. 15) resuspended in 100 μl PBS. Control mice were instilled with PBS only. Each experimental group comprised 4–7 mice. Mice were killed with an overdose of sodium pentobarbital (12 mg/animal, intraperitoneally) 24 h after the last intranasal instillation. Animals were bled by cardiac puncture and their bronchoalveolar lavage (BAL) and lungs were collected. Data shown in this report are representative of two independent experiments.
Airway obstruction in response to methacholine inhalation was measured in naive mice (time 0) and 4, 7, 10, and 13 wk after the onset of the challenge period using a whole-body plethysmography system (EMKA, Paris, France) as previously described (17). Enhanced pause (Penh) was recorded upon exposure to increasing concentrations of methacholine as a reflection of airway obstruction (19). In naive mice, concentrations of 12.5, 25, and 50 mg methacholine/ml PBS were nebulized in the plethysmograph to measure Penh. Because immunized Tg5 mice were more susceptible to metacholine, lower doses (1.25, 2.5, and 5 mg/ml) were used at Weeks 4, 7, 10, and 13.
BALs were performed by cannulating the trachea and lavaging the lungs four times with 1 ml NaCl 0.9%. This first lavage was centrifuged (200 × g, 10 min, 4°C) and the cell-free supernatant (BALF) was used for biochemical measurements. Three additional BALs were performed with 1 ml NaCl 0,9% on the same mouse. Cell pellet from the first and subsequent lavages were pooled and centrifuged as previously. Total cell number for each animal was determined, and differentials were assessed by examining at least 200 cells on cytospin slides stained with Diff-Quick (Dade Berhing, Brussels, Belgium).
Lavaged lungs were perfused with NaCl 0,9%, excised, and placed in 3 ml ice-cold PBS. Lungs were homogenized on ice with an Ultra-Turrax T25 homogenizer (Janke and Kunkel, Brussels, Belgium) and stored at −80°C. Total collagen deposition was estimated by measuring OH-proline, a specific amino acid of collagen. Part of the lung homogenate was hydrolyzed in HCl 6 N at 108°C during 24 h and OH-proline was quantified by high-performance liquid chromatography (20). The rest of the lung homogenate was centrifuged (2,600 × g, 10 min, 4°C) and the supernatant was used for the quantification of soluble collagen and fibronectin. Soluble collagen was measured by the Sircol collagen assay kit (Biocolor, Belfast, Ireland) according to manufacturer's instructions. Fibronectin was determined by enzyme-linked immunosorbent assay (ELISA) as previously detailed (21).
Whole lungs were collected and inflated with 3.6% buffered formaldehyde (Sigma, St. Louis, MO). After overnight fixation, lungs were embedded in paraffin. Five-micrometer-thick sections were stained with Masson's Trichrome.
Antibodies against A. alternata were analyzed in the sera by ELISA. Specific IgE, IgG1, and IgG2a were determined using an indirect ELISA. Plates were coated with mold extracts and saturated with skimmed milk proteins. Serial 2-fold dilutions of sera were applied for 16 h. Peroxidase-labeled rat anti-mouse IgE (LO-ME-3, IMEX; UCL, Brussels, Belgium), anti-mouse IgG1 (LO-MG1–13, IMEX), or anti-mouse IgG2a (LO-MG2a-9, IMEX) were then added during 3 h. Finally, the plates were washed and developed by addition of 100 μl TMB (Immunopure TMB substrate kit; Pierce, Rockford, IL). Optical density (O.D.) was measured at 450 nm.
The following analyses were all performed on the BALF. Total proteins were assayed as described previously (5). Cytokines such as IL-4, IL-13, IL-5, eotaxin, RANTES (regulated upon activation normal T cell expressed and presumably secreted), granulocyte macrophage colony-stimulating factor (GM-CSF), and active transforming growth factor (TGF)-β1 were quantified by ELISA using mouse Quantikine kits according to manufacturer's instructions (R&D Systems, Minneapolis, MN). IFN-γ and prostaglandin (PG) E2 levels were also determined by ELISA with the OptEIA Set mouse IFN-γ (BD Biosciences, San Diego, CA) and PGE2 Biotrak Enzymeimmunoassay (EIA) System (Amersham Biosciences, Freiburg, Germany), respectively. For connective tissue growth factor (CTGF), 20 μl of BALF were separated on 10% sodium dodecyl sulfate-polyacrylamide gel. After blotting proteins on a nitrocellulose membrane, blots were probed with 1:1,000 rabbit anti-CTGF antibody (Abcam, Cambridge, UK), which was detected with 1:10,000 anti-rabbit IgG-HRP (Cell Signaling, Boston, MA). Blots were developed using the Supersignal West Pico Maximum Sensitivity Substrate (Pierce). Band intensity was quantified using Pixel Quantification v1.0 in Adobe Photoshop 7.0. Pixel intensities were normalized to the BAL total protein concentrations.
Data are presented as means ± SEM. Penh differences were assessed using a two-way ANOVA (SAS/STAT version 8 software; SAS Institute, Cary, NC) (see details in figure legends). Other differences were evaluated using t tests with GraphPad InStat version 3.05 for Windows 95/NT (GraphPad Software, San Diego, CA). Statistical significance was considered at P < 0.05.
FVB and Tg5 mice were chronically instilled intranasally with an A. alternata extract during 13 wk. Whole-body plethysmography was used to assess airway obstruction of mice challenged with Alternaria. Before challenge, Tg5 mice were already more sensitive to methacholine at doses above 12.5 mg/ml (Figure 1A). After the onset of the challenge period, FVB and Tg5 mice displayed similarly increased Penh values after 4 wk (Figure 1B). However, from the seventh week, while the Penh increase was transient in FVB mice, strong Penh values were maintained in challenged Tg5 mice until the end of the challenge period (Figures 1C and 1D). Low concentrations of methacholine (maximum 5 mg/ml) were used to assess the airway obstruction of challenged mice because immunized Tg5 mice did not survive to higher doses. The use of these low doses explains why we did not observe any significant increase of Penh values in FVB mice 7 wk after the start of the challenge and later. Indeed, we can speculate that challenged FVB mice would have had higher Penh values with higher doses of methacholine, since markers of a chronic allergic reaction such as serum IgE levels and BAL eosinophils were still very much increased in FVB after 13 wk of treatment (see below).
Figure 1. Tg5 mice chronically instilled with A. alternata have higher Penh values than FVB mice in response to inhaled metacholine. FVB and Tg5 mice were intranasally instilled with PBS (Ctl) or A. alternata extract (Aa) during 13 wk. Experiments were performed at time 0 (A) and after 4 (B), 7 (C), and 13 (D) wk, 24 h after the last challenge of the time period. We performed a two-way ANOVA considering as factors methacholine doses and strain/treatment for each time point (P < 0.0001) followed by a multiple comparison analysis (least squares means) for strain/treatment factor. P < 0.05 between FVB and Tg5 at time 0; P > 0.05, P < 0.0001, P < 0.0001 for differences between FVB Aa and Tg5 Aa respectively at 4 wk, 7 wk, and 13 wk.
[More] [Minimize]Chronic administration of allergens can lead to the development of airway subepithelial fibrosis, one of the main features of airway remodeling (14, 22). In this work, we studied the ECM deposition occurring in association with chronic inflammation induced by A. alternata and the effect of IL-9 overexpression on this phenomenon. The accumulation of collagen was monitored in lung homogenates and histologic sections. Contrary to previous studies (6, 11), collagen and fibronectin lung contents were not significantly different between naive FVB and Tg5. After 3 mo of A. alternata challenges, FVB lungs contained higher levels of OH-proline and soluble collagen than control mice (Figures 2A and 2B). In response to the mold, Tg5 lungs also accumulated collagen, but to a significantly larger extent than FVB mice. Indeed, OH-proline lung contents were ∼ 1.4-fold greater in challenged Tg5 mice compared with challenged FVB mice, and 1.7-fold more soluble collagen was found in Tg5 lungs. Fibronectin, another protein of the ECM, was also quantified in lung homogenates. While fibronectin levels were already increased in challenged FVB mice compared with naive FVB, challenged Tg5 mice accumulated nearly twice more fibronectin than FVB mice, pointing again to a stronger fibrotic response in Tg5 mice (Figure 2C).
Figure 2. Levels of fibrosis markers are higher in Tg5 mice than in FVB in response to A. alternata. FVB and Tg5 mice were intranasally instilled with PBS (Ctl) or A. alternata extract (Aa) during 13 wk. Lungs were collected after this period. (A) OH-proline lung contents were measured in lung homogenates (OH-proline gives an estimate of the total lung collagen content), (B) soluble collagen, and (C) fibronectin in the supernatant of lung homogenates. ***P < 0.001 between PBS- and A. alternata–challenged mice. §§P < 0.01 and §§§P < 0.001 between A. alternata–challenged FVB and Tg5 mice.
[More] [Minimize]These results were confirmed histologically on lung sections stained with Masson's Trichrome. Normal lung architecture was observed in naive FVB and Tg5 mice (Figures 3A and 3B). Sections from challenged FVB and Tg5 mice showed intense inflammation in the peribronchial and alveolar areas, with numerous inflammatory cells (Figures 3C and 3D). While thickening of the airway epithelium was visible in challenged FVB mice, a much stronger collagen deposition was observed underneath the airway epithelium of challenged Tg5 mice. No alveolar fibrosis was observed in any strain after instillation of A. alternata extract.
Figure 3. Tg5 mice chronically instilled with A. alternata accumulate more collagen in peribronchial areas than FVB. FVB (A, C) and Tg5 (B, D) mice were intranasally instilled with PBS (Ctl) or A. alternata extract (Aa) during 13 wk. Lungs were collected after this period. Lung sections were stained with Masson's Trichrome. Collagen fibers are visualized in blue. (A, B) Mice instilled with PBS; (C, D) mice instilled with A. alternata extract.
[More] [Minimize]Asthmatic reactions are characterized by a type 2–polarized immune response associated with the overproduction of IgE and IgG1 (23, 24). We therefore compared the effect of IL-9 overexpression on the levels of specific IgE and IgG1 after chronic intranasal instillation of A. alternata extracts. As expected, a strong overproduction of these specific antibodies was observed in the serum of challenged FVB mice after 13 wk of treatment (Figures 4A and 4B), showing that the immune response to chronic instillation of A. alternata extract is consistent with this characteristic feature of allergic reactions. Similar specific IgE and IgG1 antibody titers were found in challenged FVB and Tg5 mice (Figures 4A and 4B), indicating that both strains developed a type 2 immune response to a similar extent. A weak IgG2a increase (associated with a type 1 immune response) was also found in both strains (Figure 4C). This response was slightly stronger in the Tg5 strain.
Figure 4. A. alternata induces a strong specific IgE and IgG1 production both in FVB and Tg5 mice. FVB and Tg5 mice were intranasally instilled with PBS (Ctl) or A. alternata extract (Aa) during 13 wk. Specific IgE (A), IgG1 (B), and IgG2a (C) were determined in sera collected after this period.
[More] [Minimize]The pulmonary inflammatory status was assessed in the BAL fluid (BALF) of mice 13 wk after the first allergen challenge. The total protein content in the BALF, a marker of permeability of the capillary–bronchoalveolar barrier, was significantly and similarly increased in challenged FVB and Tg5 mice (Figure 5A). Cellular inflammation was monitored by counting total leukocytes in BAL. No inflammation was observed in naive FVB and Tg5 mice, as shown by low levels of inflammatory cells found in both strains. A similarly increased recruitment of inflammatory cells was observed in challenged wild-type and transgenic mice (Figure 5B), except for eosinophils which were significantly more abundant in treated Tg5 BAL (Table 1).
Figure 5. Total protein contents and cell number in BALs of A. alternata–treated FVB and Tg5 mice. FVB and Tg5 mice were intranasally challenged with PBS (Ctl) or A. alternata extract (Aa) during 13 wk. (A) Total protein contents were quantified in BALFs collected after this period. (B) Total leukocytes were counted in BALs collected after this period. ***P < 0.001 between PBS and A. alternata–challenged mice.
[More] [Minimize]Macrophages | Neutrophils | Eosinophils | Lymphocytes | ||||
|---|---|---|---|---|---|---|---|
| 104 cells (mean ± SEM) | |||||||
| FVB Ctl | 1.28 ± 0.28 | 0.03 ± 0.01 | 0.02 ± 0.01 | 0 | |||
| FVB Aa | 12.02 ± 1.51* | 4.93 ± 0.65* | 4.24 ± 0.71* | 1.95 ± 0.31* | |||
| Tg5 Ctl | 1.33 ± 0.44 | 0.04 ± 0.02 | 0.04 ± 0.02 | 0 | |||
| Tg5 Aa | 13.00 ± 2.76* | 3.86 ± 0.77* | 13.51 ± 3.43*† | 2.31 ± 0.63* | |||
To determine the cytokine profile induced by the overexpression of IL-9 in presence of the allergenic mold extract, several factors known for their capacity to recruit eosinophils (eotaxin, IL-5, GM-CSF, and RANTES), for their fibrotic activities (TGF-β1, IL-13, and IL-4) and for their antifibrotic activities (PGE2 and IFN-γ) were measured by ELISA in the BALF of naive and challenged mice after 13 wk of treatment. Both eosinophil chemoattractants eotaxin and RANTES were found to be strongly induced by Alternaria (Figures 6A and 6B). While eotaxin was similarly increased by the treatment in FVB and Tg5, five times more RANTES was measured in Tg5. To assess whether IL-9 reduced the production of antifibrotic mediators, the levels of the prototypic type 1 cytokine, IFN-γ, and of the arachidonic metabolite PGE2 were also measured in BALF. IFN-γ was not modified by the chronic instillation of Alternaria either in FVB or Tg5 mice (Figure 6D) and PGE2 was increased similarly by the challenge in both strains (Figure 6C). Levels of IL-13 and TGF-β1 in BALF were already higher in naive Tg5 than in FVB: 0.71 ± 0.28 pg/ml in FVB versus 7.4 ± 2.01 pg/ml in Tg5 for IL-13, and 37.84 ± 21.48 pg/ml in FVB versus 74.36 ± 3.39 pg/ml in Tg5 for active TGF-β1. After Alternaria challenge, a similar increase of these mediators was found in both strains (Figures 6 E and F). CTGF was also assessed by western blot in identical volumes of BALF. Interestingly, although CTGF was not or barely detectable in non-treated FVB and Tg5 mice and Alternaria-treated FVB, a predominant and strong band at around 70 kD, corresponding to the CTGF dimer (25), was observed in the BALF of Alternaria-challenged Tg5 animals (Figure 7). IL-4, IL-5, and GM-CSF were not detected in any of the samples (data not shown).
Figure 6. Levels of inflammatory and fibrotic mediators in A. alternata–treated FVB and Tg5 mice. FVB and Tg5 mice were intranasally instilled with PBS (Ctl) or A. alternata extract (Aa) during 13 wk. (A) Eotaxin, (B) RANTES, (C) PGE2, (D) IFN-γ, (E) active TGF-β1, (F) IL-13 were quantified by ELISA in BALFs collected after this period. *P < 0.05, **P < 0.01, ***P < 0.001 between PBS and A. alternata–challenged mice. §P < 0.05 between control FVB and Tg5 mice. $$$P < 0.0001 between Aa-treated FVB and Tg5 mice.
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Figure 7. Higher level of CTGF in BAL of challenged Tg5 mice compared with FVB. Detection of CTGF by Western blot analysis of equal amounts of BALF from individual control and Aa-treated FVB and Tg5 mice. CTGF 70 kD band intensity was quantified using Pixel Quantification v1.0 in Adobe Photoshop 7.0. Pixel intensities were normalized to the BAL total protein concentrations shown in Figure 5. Results are presented in arbitrary units.
[More] [Minimize]IL-9 has been clearly implicated in asthmatic reactions both in humans and in experimental models. However, little is known on the influence of this cytokine on the development of fibrosis associated with airway remodeling. The main purpose of this study was to investigate the effect of the IL-9 overexpression on the development of airway fibrosis induced by the chronic administration of an allergen. We show that overexpression of IL-9 exacerbates part of the allergic reaction (eosinophils) and also the fibrotic process accompanying the asthmatic response induced by the chronic intranasal instillation of Alternaria extract. This profibrotic effect of IL-9 is in contrast with its antifibrotic action in a model of interstitial fibrosis (5).
It has previously been reported that IL-9–overexpressing mice display subepithelial fibrosis (6, 11). However, in the present study, we observed no significant difference in collagen accumulation, or in inflammation between naive FVB and Tg5 mice. Working for several years with Tg5 mice (5, 6, 26), we have observed that the lung inflammatory and fibrotic status in these mice seems to depend on the conditions in the animal care facility. Indeed, when Tg5 mice are bred in SPF conditions, we do not observe any difference in BAL eosinophils or collagen accumulation compared with FVB. Our results suggest that overexpression of IL-9 alone, accompanied by an induction of IL-13, is not sufficient to induce an increased collagen deposition. We therefore believe that most of the pulmonary effects resulting from IL-9 overexpression require an additional external stimulus. In the present case, the effect of IL-9 was revealed by the allergenic mold Alternaria alternata.
Although overexpression of IL-9 did increase the intensity of subepithelial fibrosis in our model of chronic asthma, the deficiency for its receptor (IL-9R knockout mice) did not affect this process (data not shown). Similarly, the implication of IL-9 in the inflammatory asthmatic reaction to OVA could not be shown in IL-9–deficient mice (27), whereas treatment of OVA challenged mice with anti–IL-9 antibodies reduced the asthmatic reaction (9, 10), raising the hypothesis that animals genetically lacking the IL-9 signaling develop an alternative pathway, which is not induced by a short-term anti–IL-9 treatment. Overall, these data suggest that IL-9 can exacerbate airway fibrosis but is probably dispensable for its development, at least in response to chronic instillation of Alternaria (11, 27–30).
Our observations are in accordance and in apparent discrepancy with previous data on IL-9: while IL-9 overexpression or its pulmonary administration induces airway subepithelial collagen deposition (11, 29), this same overexpression protects mice against alveolar fibrosis induced by silica (5). It therefore appears that both fibrotic processes differ at certain points, probably because of their different localization in the lung and/or of varying effector cells involved. It also suggests a dual role of IL-9 in fibrotic processes depending on the localization of fibrosis and/or the source of IL-9.
PGE2 is a potent antifibrotic mediator that reduces fibroblast proliferation and collagen production and contributes to the control of pulmonary fibrosis in vivo (31). We previously observed higher PGE2 contents in the BALFs of Tg5 mice and an increased capacity of IL-9–overexpressing macrophages to produce PGE2 in response to silica (6). As in patients with asthma (32), we observed increased levels of PGE2 after challenge. We did, however, not find any difference in PGE2 levels between both strains, neither in naive or challenged animals, providing a possible explanation for the fact that IL-9–overexpressing mice were not protected against collagen accumulation in this model of asthma.
Another important difference between experimental models of airway remodeling and alveolar fibrosis is the recruitment of eosinophils. Indeed, the number of these cells is increased after chronic OVA challenge. Moreover, contrary to wild-type mice, IL-5 receptor knockout mice (deficient in eosinophil recruitment) are protected against peribronchial collagen accumulation induced by chronic exposure to OVA (33). In contrast, in experimental models of parenchymal lung fibrosis, eosinophils are only weakly recruited during the fibrotic stage (34, 35) and seem not to be necessary for the development of the disease (36, 37). In this work, we observed that eosinophils accumulate in the lung in response to the chronic instillation of A. alternata extract and that IL-9 overexpression potentiates this increase (38). Since several authors have linked the recruitment of eosinophils to AHR and fibrosis intensity (3, 39) and since this cell type was the only one modulated by IL-9 overexpression among those investigated here, we suggest that eosinophils might be one of the mediators responsible of the profibrotic effect of IL-9 in the airways.
To explain the increased recruitment of eosinophils in treated Tg5 mice, we assessed the expression of several chemoattractants for these cells. Whereas IL-5, GM-CSF, and eotaxin were not detected or similarly induced by Alternaria in both strains, the overexpression of RANTES induced by the mold was strongly exacerbated in Tg5. RANTES is produced by various cell types, including eosinophils, T lymphocytes, epithelial cells, and fibroblasts (40–43). When stimulated with recombinant IL-9, secretions from human primary bronchial epithelial cells were shown to increase T cell migration. Part of this chemoattractant activity was attributable to RANTES (44). Thus, our results suggest that IL-9 can also induce the overproduction of RANTES in the lung but only under specific conditions (e.g., chronic instillation of Alternaria), possibly explaining the increased recruitment of eosinophils in treated Tg5 mice.
Eosinophils are thought to contribute to the establishment of subepithelial fibrosis in asthma via their production of TGF-β. Indeed, eosinophils were found to be the main source of this profibrotic factor in patients with asthma (3, 45). TGF-β induces the proliferation and differentiation of fibroblasts in myofibroblasts and increases their production of ECM proteins, making of this molecule the profibrotic factor par excellence (46, 47). Although we observed more eosinophils and fibrosis in challenged Tg5 mice compared with FVB, we did not note any difference in active TGF-β protein level. TGF-β receptor expression on fibroblasts was, however, not assessed. Cells producing TGF-β could also be different in challenged FVB and Tg5 mice. Moreover, it is possible that the expression/activation of TGF-β occurs at other time points not measured in this study.
TGF-β can induce the production of another profibrotic factor, CTGF, by smooth muscle cells and fibroblasts (48, 49). CTGF stimulates fibroblasts proliferation and ECM production and is involved in several fibrotic processes (50). CTGF is also thought to be implicated in airway remodeling since it is more expressed in airway smooth muscle cells from individuals with asthma than from healthy subjects (51). In this study, CTGF protein expression was more strongly induced in challenged Tg5 BAL compared with FVB. Our data suggest that CTGF overproduction in challenged Tg5 mice is responsible for increased airway fibrosis in this strain and that the strong CTGF expression in Tg5 mice could be due to an early or local increase of TGF-β production/signaling or to another unidentified factor (52, 53).
To conclude, we have shown that IL-9 overexpression exacerbates airway fibrosis induced by the chronic instillation of an allergenic mold, probably via an enhanced eosinophil recruitment and/or CTGF production. We also have demonstrated that IL-9 can have opposite effects in similar processes of pulmonary ECM accumulation when induced at different tissular locations.
The authors thank Yousof Yakoub, Francine Uwambayineme, and Christelle Rochard for their excellent technical assistance, and Jean-François Heilier for his help with statistics.
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