Background
Asthma is a common obstructive airway disease, which currently affects around 300 million people worldwide and has a major debilitating impact on society [
1]. In most cases, asthma is associated with an allergic response towards inhaled aeroallergens. Patients with asthma suffer from inflammation of the airways, causing hyperresponsiveness to specific and non-specific stimuli. This inflammation is characterized by an increase in eosinophils, CD4
+ lymphocytes and T
H2 cytokines including IL-4, IL-5 and IL-13 [
2]. Moreover, remodeling of the bronchial tree is a significant pathology in severe asthma that contributes to airflow obstruction and loss of deep breath-induced bronchodilation [
3]. Remodeling of the airways is characterized by increased extracellular matrix deposition in the subepithelial airway compartment and marked thickening of the bronchial smooth muscle. All these changes are associated with airflow limitation in severe asthma [
4].
Current treatment for patients with asthma includes inhaled corticosteroids (ICS) and long-acting β
2-agonists (LABA). Moreover, the long-acting muscarinic antagonist (LAMA) tiotropium has recently been registered for the treatment of asthma. Clinical trials have shown beneficial effects on lung function by addition of tiotropium to standard treatment in moderate and severe asthma patients [
5‐
7]. In addition, treatment with tiotropium reduced the number of severe exacerbations [
5], suggesting that tiotropium might exert anti-inflammatory effects in these patients.
Anti-inflammatory effects of anticholinergics have indeed been observed in in vitro and in vivo studies using various experimental models [
8,
9]. In vitro, anticholinergics exert direct anti-inflammatory effects on inflammatory cells, including T cells [
10] and macrophages [
11], on epithelial cells [
12], and on airway smooth muscle cells [
13]. In addition, anticholinergics affect airway remodelling in vitro [
9]. Muscarinic receptors regulate proliferation of airway smooth muscle cells [
14] and fibroblasts [
15], fibroblast to myofibroblast transition [
16], and extracellular matrix deposition [
17,
18]. These findings have been confirmed in in vivo animal models, demonstrating inhibitory effects of tiotropium or muscarinic M
3 receptor knock-out on ovalbumin-induced inflammation and remodeling, including airway smooth muscle thickening, extracellular matrix deposition and mucus gland hypertrophy [
19‐
22]. Effects of tiotropium on ovalbumin-induced inflammation and remodeling were comparable to the effects of the corticosteroid budesonide [
22]. The effects of the combination of tiotropium and a corticosteroid on airway inflammation and remodeling are currently unknown. In vitro, it has been shown that the anticholinergic glycopyrrolate acts synergistically with budesonide in inhibiting TNF-α release from isolated monocytes [
23], suggesting that the combination of anticholinergics and corticosteroids might be more effective than the monotherapies in vivo.
In view of the above mentioned beneficial effects of tiotropium and corticosteroids on allergic airway inflammation and remodeling, combination therapy with anticholinergics and corticosteroids might have additive protective effects on airway inflammation and remodeling. Therefore, in the present study, the effects of pre-treatment with tiotropium and ciclesonide on airway inflammation and remodeling were investigated using guinea pig models of acute and chronic asthma. Guinea pig models are valuable for the evaluation of pathophysiological mechanisms and pharmacological interventions in asthma, since the mechanisms underlying the allergic asthmatic reaction in guinea pigs are more comparable to humans, and therefore more physiologically relevant compared to commonly used rodent models [
24]. In these guinea pig models, we demonstrate that tiotropium and ciclesonide do not inhibit
acute allergen-induced inflammation, but do inhibit
chronic allergen-induced airway inflammation and remodeling when applied in combination.
Discussion
The results of this study indicate that in vivo, tiotropium and ciclesonide do not protect against acute allergen-induced inflammation, but do protect against chronic allergen-induced airway inflammation and remodeling. Whereas there was only limited inhibition of airway eosinophilia and airway smooth muscle thickening after treatment with the monotherapies at threshold doses, allergen-induced alterations were significantly inhibited by pretreatment with the combination of both compounds. This suggests that combination therapy with tiotropium and ciclesonide might have beneficial effects on airway inflammation and remodeling. The potential for a steroid-sparing effect needs additional studies.
To our knowledge, this is the first study demonstrating functional interactions of anticholinergics and corticosteroids on inflammation and remodeling in vivo. Previously, it has been shown in mice and guinea pigs that monotherapy with higher doses of tiotropium or corticosteroids can inhibit allergen-induced inflammation and remodeling [
21,
22,
25]. Tiotropium and dexamethasone alone, both at a dose of 1 mg/kg, inhibit airway inflammation in response to ovalbumin in mice [
25]. Using the chronic guinea pig model as described in this study, we reported inhibitory effects of tiotropium and budesonide on inflammation and remodeling at 0.1 mM [
21,
22]. The effects observed on airway eosinophilia in the latter study were comparable to the effects observed with the highest dose in the current study (0.03 mM tiotropium and 0.1 mg/kg ciclesonide), suggesting that the inhibitory effects occur at much lower doses than previously thought, and that approximately 50 % inhibition of airway eosinophilia by these treatments is the maximal effect that can be achieved with anticholinergic or corticosteroid treatment in this model. In this study, we demonstrate that tiotropium and ciclesonide at even lower doses of 0.01 mM and 0.01 mg/kg respectively, significantly inhibit airway inflammation and remodeling when administered in combination, with no effect on inflammation or remodeling by the monotherapies at these doses.
Synergistic effects between anticholinergics and corticosteroids have also been observed in vitro. It has been shown that glycopyrrolate alone does not affect TNF-alpha release from monocytes, but synergistically enhances the inhibitory effects of budesonide on TNF-alpha release [
23]. This synergy between anticholinergics and corticosteroids on inflammatory processes is now confirmed by our in vivo findings. Randomized clinical trials investigating the effects of long-acting anticholinergics in asthma have only recently attracted attention, and there is no study that has investigated synergism between anticholinergics and corticosteroids in patients with asthma. However, beneficial effects of combining anticholinergics and corticosteroids have been reported. The addition of tiotropium to treatment for patients with uncontrolled asthma was shown to be more effective on improving asthma symptoms and lung function than doubling the dose of corticosteroids [
26]. Improvements in morning peak expiratory flow, the proportion of asthma-control days, forced expiratory volume in 1 s and daily symptom scores were reported in this crossover trial [
26]. Furthermore, randomized controlled clinical trials demonstrated that treatment with tiotropium, added on to ICS or ICS plus LABA, is an effective therapy for moderate and severe asthma patients as seen by improvements in lung function and reduction in the risk of severe asthma exacerbations [
5‐
7], indicating at least additive effects of tiotropium when added to ICS +/− LABA treatment.
The mechanistic basis for functional interactions between anticholinergics and corticosteroids is not yet clear, but it may well be that these drugs target specific and distinct pathophysiological processes. Ciclesonide, via glucocorticosteroid receptors, acts anti-inflammatory by repressing pro-inflammatory gene transcription [
27]. These mechanisms are most likely different from those targeted by tiotropium, which acts via G-protein coupled muscarinic receptors. In vitro evidence exists for anti-inflammatory and anti-remodeling effects of anticholinergics on airway cells via muscarinic receptors [
8]. Anticholinergics inhibit the release of neutrophil chemotactic mediators from a number of cells, including macrophages, fibroblasts, airway smooth muscle cells and epithelial cells [
11-
13]. Anticholinergics also inhibit parameters of remodeling, including enhanced MUC5AC expression, goblet cell metaplasia, and fibroblast to myofibroblast transition [
16,
28,
29]. In addition, bronchoconstriction is effectively targeted by tiotropium, and this may also have its effects on airway inflammation and remodeling [
30,
31]. This hypothesis is supported by recent data showing that repeated methacholine challenges induce remodeling in mild asthma patients [
32]. Moreover, we demonstrated that muscarinic M
3 receptor knock-out mice are protected from allergen-induced airway remodeling, even though there is still an inflammatory response in these animals [
20]. Because M
3 receptors mediate bronchoconstriction, which is abolished in M
3 receptor knock-out mice [
33], this may suggest that bronchoconstriction by itself might be an important driver of airway remodeling [
20,
31]. In support, we previously demonstrated that methacholine treatment promotes remodeling in guinea pig lung slices [
34]. Taken together, we propose that additive effects between anticholinergics and corticosteroids, as observed for tiotropium and ciclesonide in our study, are based on the different mechanisms they target.
Surprisingly, no anti-inflammatory effects of tiotropium and ciclesonide were observed in the acute asthma model in this study. Similar findings were observed for tiotropium and the long-acting β-agonist olodaterol in an acute guinea pig model [
35]. Apparently, chronic treatment is needed to unmask the anti-inflammatory effects in guinea pigs, as we do observe inhibition of inflammation in this study and previous studies after multiple allergen challenges [
22]. Because mast cell infiltration occurs already after the systemic sensitization against ovalbumin (i.e. prior to the first drug treatment), it may be that tiotropium cannot protect against the initial inflammatory response induced by the first allergen encounter, but requires prolonged treatment. In support, anticholinergics do not inhibit mast cell degranulation [
36]. Similarly, the effects of corticosteroids require prolonged treatment, as the same discrepancy between acute and chronic inflammation was seen for ciclesonide, which was substantially more effective, and at lower doses already, in inhibiting chronic allergen-induced inflammation compared with acute allergen-induced inflammation. Limitations in the delivery of the drugs do not explain the difference, as a single administration of tiotropium effectively prevents bronchoconstriction and the early and late asthmatic reaction [
35], and a single administration of corticosteroids inhibits the late asthmatic reaction in this model (unpublished observations). The mechanistic basis for the delay in the onset of action of tiotropium is unclear, however, it can be envisaged that the role of acetylcholine is further downstream in the pathophysiological process. This is supported by the fact that sensory nerves play an important role in the late asthmatic reaction, and not in the early asthmatic reaction [
37]. It may therefore be that an initial inflammatory response is required, which leads to epithelial damage, inflammatory mediator release and other mechanisms that enhance the cholinergic reflex, and thereby increase the role of acetylcholine later on.
The finding that tiotropium and ciclesonide protect against allergen-induced airway smooth muscle thickening is in agreement with our previous findings [
21]. The reduction in smooth muscle mass may be related to the reduction in airway inflammation, as many inflammatory mediators are reported to promote smooth muscle growth in allergic airway inflammation [
38]. Alternatively, increased cholinergic activity that results from airway inflammation promotes bronchoconstriction, which might drive airway remodeling as discussed above.
Competing interests
This study was supported by a grant from Boehringer Ingelheim (BI). RG and HMe have received funding for research from BI and RG has received lecture fees from BI.
Authors’ contributions
LK, HAM, HEM and RG designed the studies; LK, SB and MM performed the experiments; LK and SB analysed data; LK and RG interpreted the results of the experiments; LK, SB and RG prepared the figures; LK and RG drafted the manuscript and all authors read and approved the final version of the manuscript.