Introduction
Asthma is a major public health problem, affecting patients of all ages from infants to older adults. Morbidity and mortality are greater in older patients (> 65 yrs) despite a similar prevalence as that in younger asthmatics, and yet there is limited evidence on how age can influence the pathogenesis of asthma [
1,
2]. Older patients have a larger than predicted reduction in pulmonary function parameters even though physician-assessed severity, duration of diagnosed asthma, and smoking status were not different compared to younger adults. A significant increase in the comorbid diagnosis of chronic obstructive pulmonary disease is associated with asthma in older patients suggesting that long-standing asthma may lead to irreversible airflow obstruction [
3]. Asthma is characterized by airway hyperresponsiveness (AHR), an exaggerated narrowing of the airway in response to stimuli. AHR can reflect asthma severity, and has been associated with a variety of contributing factors [
4‐
7]. AHR has two distinct components. First, a transient component occurring after an allergen exposure that is linked to acute inflammation, second, a more persistent component, associated with the chronicity of the disease causing structural changes in the airways known as airway remodeling. Several features of airway remodeling can contribute to AHR, including: increased sensitivity, hypertrophy and hyperplasia of smooth muscle cells (SMCs), as well as increased subepithelial fibrosis characterized by increased collagen deposition [
8]. The development of structural changes persists during and after acute inflammation in allergic asthma subjects and in allergen challenge animal models with low inflammation [
9‐
11]. Some asthma patients develop AHR in association with airway remodeling [
12]. Furthermore, severe asthmatic patients under anti-inflammatory treatments will present loss in lung function despite inflammation being controlled [
13]. These facts illustrate that underlying non-inflammatory mechanisms regulate airway structure and function, which include epithelial thickening and subepithelial collagen deposition.
Caveolin 1 (Cav1) is the main structural and functional protein of caveolae. These are 10 to 100 nm wide invaginations of the plasma membrane found in many cell types. Cav1 acts as a scaffolding protein, as well as a regulatory protein in many signaling cascade protein complexes. Cav1 inhibits the activity of these signaling proteins by binding and releasing them in a controlled fashion [
14]. Cav1 has been shown to be involved in the regulation of both inflammation and fibrosis [
15‐
19]. One of the important pathways that Cav1 regulates is the Transforming Growth Factor (TGF)-β pathway. Cav1 binds to and inhibits the TGF-β type II receptor thus preventing downstream signaling including the phosphorylation of Smad2/3. Others and our group have demonstrated that
Cav1
-/-
mice have enhanced Smad2 phosphorylation and altered ECM deposition notably in the lungs [
18,
19]. We have shown that aged
Cav1
-/-
mice have decreased lung function (i.e. increased elastance and decreased compliance) mainly due to an increase in ECM deposition of collagen and elastin [
19]. The regulation of Cav1 has also been shown to be important in allergic airway disease [
20,
21]. We have previously shown that TGF-β signaling is enhanced in ovalbumin (OVA) challenged
Cav1
-/-
mice leading to enhanced airway remodeling [
21].
TGF-β1 is a pleiotrophic growth factor that participates in resolution of inflammation, as well as promotion of airway remodeling, especially promoting extracellular matrix (ECM) deposition. Anti-TGF-β1 antibody treatment prevents the progression of airway remodeling following allergen challenge in mice [
22]. More surprisingly, enhanced AHR has been reported in anti-TGF-β antibody treated OVA challenged mice associated with reduced Smad2 phosphorylation, marker of canonical TGF-β signaling pathway activation [
23]. The regulation of TGF-β signaling activity has been the focus of intensive studies as a potential therapeutic target but it has not yet been fully characterized.
This study was designed to evaluate the relationship between inflammation, structural changes, and lung function in aged and OVA-challenged mice. We hypothesized that structural alterations of the airways that develop with age in Cav1 deficient mice will predispose them to an increased response to allergen challenge. The effect of Cav1-deficiency on airway structure was investigated, and AHR was associated to change in airway structures.
Discussion
Murine models have been useful in studying the mechanisms that contribute to AHR [
22‐
25]. The relative contribution of airway remodeling to AHR is difficult to assess as most of these models involve an allergic response triggering both inflammation and airway remodeling. In this study, we explored the inflammatory responses before and after airway remodeling was established. Our main finding was that
Cav1
-/-
mice develop AHR as they age. At 6 months of age, the AHR was significantly increased compared to WT mice, but not at an earlier age. Furthermore, there was also a significantly increased thickening of the subepithelial matrix layer in
Cav1
-/-
mice as compared to WT mice at 6 months of age. In previous studies, we have demonstrated by trichrome and picrosirius red staining that subepithelial collagen deposition is increased in
Cav1
-/-
mice by the time they are 6 months of age [
17]. This suggests that AHR is related to collagen deposition in the bronchial subepithelial layer. Mathematical modeling has demonstrated that relatively minor increases in the thickness of the small airways can decrease the size of the airway caliber enough during muscle contraction to physiologically affect AHR, further supporting our findings [
21]. We have also previously demonstrated that subepithelial collagen deposition contributes to increased AHR in a chronic OVA allergic airways disease model and that reversal of subepithelial and total lung collagen by relaxin treatment can reduce AHR [
26].
The mechanisms for increased collagen deposition in the setting of Cav1 deficiency are incompletely understood. TGF-β promotes airway remodeling, especially promoting ECM deposition [
10]. In asthmatics, TGF-β regulated by leukotrienes, can activate lung fibroblasts and increase collagen deposition [
29,
30]. We have previously established that Cav1 was involved in the regulation of TGF-β signaling in murine lungs and that over time, the absence of Cav1 leads to increased TGF-β signaling and collagen deposition in the parenchyma [
17]. In this study we confirmed the increase in pSmad2 signaling in OVA challenged
Cav1
-/-
lungs. For the first time, we demonstrate the association of Cav1 deficiency with the development of increased AHR and thickening of the subepithelial layer. Cav1 interacts with and regulates a number of proteins in addition to TGF- β that are potentially important for regulation of AHR that we did not investigate in this study. For example, it has been well documented that Cav-1 participates in SMC contractility via Ca
2+-dependent mechanisms. Indeed, SMC expressed Cav-1 and the caveolae contain a number of proteins that participate in the regulation of Ca
2+[
31‐
34]. Importantly, Sathish and al. [
31] recently showed that Cav-1 regulates proteins (Orai1) that are important for calcium regulation. They also showed that an association between Cav1, calcium and inflammation.
After it was established that
Cav1
-/-
mice develop AHR and subepithelial thickness at 6 months of age, an OVA allergen sensitization and challenge was carried out at 2 months of age (before airway remodeling is established) and at 6 months of age (after airway remodeling is established) to explore differences in the inflammatory response. OVA challenge induced markedly elevated BAL cytokine levels in 2-month old WT and
Cav1
-/-
mice, but reduced cytokine levels in 6-month
Cav1
-/-
mice after the OVA allergen challenge. In a similar fashion, 6-month
Cav1
-/-
mice had a decreased number of inflammatory cells and percentage of eosinophils in the BAL after the OVA allergen challenge compared to 2-month
Cav1
-/-
mice. Increased number of mucous cells has been associated with increased IL-13, inflammation and eosinophilia [
35]. Importantly, both age and Cav1 expression influence these parameters of response to allergen challenge. In our study, eosinophilia and levels of IL-5 decreased in older mice and correlated with reduced mucous cell metaplasia. Six-month-old OVA challenged
Cav1
-/-
mice reduced IL-13 in the BAL corresponding to few mucous cells. Few reports investigating airway inflammation in aged sensitized and challenged mice are available and even less on airway remodeling. The decrease in eosinophilia and overall cytokine level in aged mice both WT and
Cav1
-/-
mice was consistent with 2 of 3 previous studies using similar allergen challenge protocols [
36‐
38]. Our findings suggest that the AHR observed in 6-month-old
Cav1
-/-
mice is independent of inflammation and more closely related to increased collagen around the airways. The work from Busse et al. also argues against a direct relationship between pulmonary inflammation or eosinophils and increased AHR, even though a different OVA sensitization and challenge protocol was used [
36].
In asthmatic patients, the persistence of AHR can be associated with airway remodeling after the resolution of inflammation [
6]. In addition, in mouse models, prolonged allergen challenge leads to persistent changes in the airways despite discontinuation of the allergen challenge [
7,
8]. These studies demonstrate a role of airway remodeling in AHR beyond the immediate inflammatory response. Other genetically engineered mouse models also point toward the importance of airway remodeling independent of inflammation [
7,
8]. For example, relaxin-deficient mice also show similar results, where an increase in airway remodeling leads to increased AHR [
7,
8,
26].
The timing of airway remodeling development in asthma is an area of increasing interest. Evidence suggests that remodeling of airways may develop long before symptoms of asthma and inflammation appear [
29]. In our study, older mice in both groups had less AHR and the BAL cytokines tended to be present at lower levels after the OVA allergen challenge. This suggests that some alterations in lung response to allergen challenge as the mice age were independent of the presence or absence of Cav1. Nevertheless, the reduction in inflammation was more prominent in the
Cav1
-/-
mice with greater reductions in eosinophils and IL-13. The decrease in inflammation in 6-month-old mice can account for the decrease in AHR seen in these mice compared to 2-month-old mice. Indeed, we demonstrated a decrease in the IL-4 levels in 6-month-old mice versus 2-month-old mice, and other studies have shown that treatment of sensitized mice with anti-IL-4 antibody prior to antigen stimulation reduces antigen-induced AHR, eosinophilia, and goblet cell metaplasia [
39]. In addition, IL-4 can induce AHR and goblet cell metaplasia independent of IL-13 [
31]. Despite the greater decrease in inflammation in the
Cav1
-/-
mice as they aged, the AHR of the
Cav1
-/-
remained greater than age-matched WT indication that remodeling rather than inflammation may play a role in the elevated AHR in 6-month-old OVA-challenged
Cav1
-/-
mice.
These findings have important implications, as they open avenues for possible therapeutic interventions to prevent the development of airway remodeling. Our data are supportive of the beneficial effects on airway remodeling after treatment with Cav1 scaffolding domain peptide. In addition, in other studies using relaxin-deficient mice, it has been demonstrated that treatment with relaxin is capable of reversing established airway remodeling and AHR [
27]. These studies suggest that the development of anti-fibrotic therapies that could potentially prevent lung function decline associated with airway remodeling is warranted. This approach may be especially relevant in severe asthma, where well-established treatments with inhaled corticosteroids have limited efficacy in the control of airway remodeling development and reversibility [
10,
29].
Even though subepithelial fibrosis is an established characteristic in airway remodeling, other studies have demonstrated that changes in myocytes and globet cells may also induce AHR. In this murine model, we identified an increase in subepithilial thickening as the main contributor of AHR in Cav1
-/-
mice, but because asthma is a heterogeneous disease, other cellular components may still play an important role in airway remodeling and AHR.
In conclusion, our findings demonstrate that Cav1 deficiency is associated with airway remodeling and the development of AHR. The airway remodeling progressed with age. Inflammation appears not to be required for the development of these age-related changes in Cav1 deficient mice. Cav1 may play a role, possibly through the TGF-β pathway, in the prevention of the development of subepithelial fibrosis and airway remodeling.
Competing interests
They authors declare that they have no competing interests.
Authors’ contributions
KEG: mouse work, lung functions, airway remodeling studies, interpretation of data, write up of the manuscript. SGR: morphometry analysis and write up of the manuscript. DJM: interpretation of data and write up of the manuscript. SKM: collagen analysis. MLKT: morphometry analysis and write up of the manuscript. CJLS: oversee of the entire project, interpretation of the results and write up of the manuscript. All authors read and approved the final manuscript.