Coexposure to environmental tobacco smoke increases levels of allergen-induced airway remodeling in mice

Environmental tobacco smoke (ETS) can increase asthma symptoms and the frequency of asthma attacks. However, the contribution of ETS to airway remodeling in asthma is at present unknown. In this study, we have used a mouse model of allergen-induced airway remodeling to determine whether the combination of chronic exposure to ETS and chronic exposure to OVA allergen induces greater levels of airway remodeling than exposure to either chronic ETS or chronic OVA allergen alone. Mice exposed to chronic ETS alone did not develop significant eosinophilic airway inflammation, airway remodeling, or increased airway hyperreactivity to methacholine. In contrast, mice exposed to chronic OVA allergen had significantly increased levels of peribronchial fibrosis, increased thickening of the smooth muscle layer, increased mucus, and increased airway hyperreactivity which was significantly enhanced by coexposure to the combination of chronic ETS and chronic OVA allergen. Mice coexposed to chronic ETS and chronic OVA allergen had significantly increased levels of eotaxin-1 expression in airway epithelium which was associated with increased numbers of peribronchial eosinophils, as well as increased numbers of peribronchial cells expressing TGF-beta1. These studies suggest that chronic coexposure to ETS significantly increases levels of allergen-induced airway remodeling (in particular smooth muscle thickness) and airway responsiveness by up-regulating expression of chemokines such as eotaxin-1 in airway epithelium with resultant recruitment of cells expressing TGF-beta1 to the airway and enhanced airway remodeling.     

Neutralisation of Interleukin-13 in Mice Prevents Airway Pathology Caused by Chronic Exposure to House Dust Mite

Background

Repeated exposure to inhaled allergen can cause airway inflammation, remodeling and dysfunction that manifests as the symptoms of allergic asthma. We have investigated the role of the cytokine interleukin-13 (IL-13) in the generation and persistence of airway cellular inflammation, bronchial remodeling and deterioration in airway function in a model of allergic asthma caused by chronic exposure to the aeroallergen House Dust Mite (HDM).

Methodology/Principal Findings

Mice were exposed to HDM via the intranasal route for 4 consecutive days per week for up to 8 consecutive weeks. Mice were treated either prophylactically or therapeutically with a potent neutralising anti-IL-13 monoclonal antibody (mAb) administered subcutaneously (s.c.). Airway cellular inflammation was assessed by flow cytometry, peribronchial collagen deposition by histocytochemistry and airway hyperreactivity (AHR) by invasive measurement of lung resistance (R_L) and dynamic compliance (C_dyn). Both prophylactic and therapeutic treatment with an anti-IL-13 mAb significantly inhibited (P<0.05) the generation and maintenance of chronic HDM-induced airway cellular inflammation, peribronchial collagen deposition, epithelial goblet cell upregulation. AHR to inhaled methacholine was reversed by prophylactic but not therapeutic treatment with anti-IL-13 mAb. Both prophylactic and therapeutic treatment with anti-IL-13 mAb significantly reversed (P<0.05) the increase in baseline R_L and the decrease in baseline C_dyn caused by chronic exposure to inhaled HDM.

Conclusions/Significance

These data demonstrate that in a model of allergic lung disease driven by chronic exposure to a clinically relevant aeroallergen, IL-13 plays a significant role in the generation and persistence of airway inflammation, remodeling and dysfunction.     

Inflammatory and mechanical factors of allergen-induced bronchoconstriction in mild asthma and rhinitis.

We studied whether different bronchial responses to allergen in asthma and rhinitis are associated with different bronchial inflammation and remodeling or airway mechanics. Nine subjects with mild asthma and eight with rhinitis alone underwent methacholine and allergen inhalation challenges. The latter was preceded and followed by bronchoalveolar lavage and bronchial biopsy. The response to methacholine was positive in all asthmatic but in only two rhinitic subjects. The response to allergen was positive in all asthmatic and most, i.e., five, rhinitic subjects. No significant differences between groups were found in airway inflammatory cells or basement membrane thickness either at baseline or after allergen. The ability of deep inhalation to dilate methacholine-constricted airways was greater in rhinitis than in asthma, but it was progressively reduced in rhinitis during allergen challenge. We conclude that 1) rhinitic subjects may develop similar airway inflammation and remodeling as the asthmatic subjects do and 2) the difference in bronchial response to allergen between asthma and rhinitis is associated with different airway mechanics.     

Divergent immune responses to house dust mite lead to distinct structural-functional phenotypes

Asthma is a chronic airway inflammatory disease that encompasses three cardinal processes: T helper (Th) cell type 2 (Th2)-polarized inflammation, bronchial hyperreactivity, and airway wall remodeling. However, the link between the immune-inflammatory phenotype and the structural-functional phenotype remains to be fully defined. The objective of these studies was to evaluate the relationship between the immunologic nature of chronic airway inflammation and the development of abnormal airway structure and function in a mouse model of chronic asthma. Using IL-4-competent and IL-4-deficient mice, we created divergent immune-inflammatory responses to chronic aeroallergen challenge. Immune-inflammatory, structural, and physiological parameters of chronic allergic airway disease were evaluated in both strains of mice. Although both strains developed airway inflammation, the profiles of the immune-inflammatory responses were markedly different: IL-4-competent mice elicited a Th2-polarized response and IL-4-deficient mice developed a Th1-polarized response. Importantly, this chronic Th1-polarized immune response was not associated with airway remodeling or bronchial hyperresponsiveness. Transient reconstitution of IL-4 in IL-4-deficient mice via an airway gene transfer approach led to partial Th2 repolarization and increased bronchial hyperresponsiveness, along with full reconstitution of airway remodeling. These data show that distinct structural-functional phenotypes associated with chronic airway inflammation are strictly dependent on the nature of the immune-inflammatory response.     

Respiratory tolerance is inhibited by the administration of corticosteroids

Corticosteroids constitute the most effective current anti-inflammatory therapy for acute and chronic forms of allergic diseases and asthma. Corticosteroids are highly effective in inhibiting the effector function of Th2 cells, eosinophils, and epithelial cells. However, treatment with corticosteroids may also limit beneficial T cell responses, including respiratory tolerance and the development of regulatory T cells (T(Reg)), which actively suppress inflammation in allergic diseases. To examine this possibility, we investigated the effects of corticosteroid administration on the development of respiratory tolerance. Respiratory exposure to Ag-induced T cell tolerance and prevented the subsequent development of allergen-induced airway hyperreactivity. However, treatment with dexamethasone during the delivery of respiratory Ag prevented tolerance, such that allergen sensitization and severe airway hyperreactivity subsequently occurred. Treatment with dexamethasone during respiratory exposure to allergen eliminated the development of IL-10-secreting dendritic cells, which was required for the induction of IL-10-producing allergen-specific T(Reg) cells. Therefore, because allergen-specific T(Reg) cells normally develop to prevent allergic disease and asthma, our results suggest that treatment with corticosteroids, which limit the development of T(Reg) cells and tolerance to allergens, could enhance subsequent Th2 responses and aggravate the long-term course of allergic diseases and asthma.     

Treatment of established asthma in a murine model using CpG oligodeoxynucleotides

Allergen immunotherapy is an effective but underutilized treatment for atopic asthma. We have previously demonstrated that CpG oligodeoxynucleotides (CpG ODN) can prevent the development of a murine model of asthma. In the current study, we evaluated the role of CpG ODN in the treatment of established eosinophilic airway inflammation and bronchial hyperreactivity in a murine model of asthma. In this model, mice with established ovalbumin (OVA)-induced airway disease were given a course of immunotherapy (using low doses of OVA) in the presence or absence of CpG ODN. All mice then were rechallenged with experimental allergen. Untreated mice developed marked airway eosinophilia and bronchial hyperresponsiveness, which were significantly reduced by treatment with OVA and CpG. CpG ODN leads to induction of antigen-induced Th1 cytokine responses; successful therapy was associated with induction of the chemokines interferon-gamma-inducible protein-10 and RANTES and suppression of eotaxin. Unlike previous studies, these data demonstrate that the combination of CpG ODN and allergen can effectively reverse established atopic eosinophilic airway disease, at least partially through redirecting a Th2 to a Th1 response.     

IMD-4690, a Novel Specific Inhibitor for Plasminogen Activator Inhibitor-1, Reduces Allergic Airway Remodeling in a Mouse Model of Chronic Asthma via Regulating Angiogenesis and Remodeling-Related Mediators

Plasminogen activator inhibitor (PAI)-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp). IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.     

Compartmental and Temporal Dynamics of Chronic Inflammation and Airway Remodelling in a Chronic Asthma Mouse Model

Background

Allergic asthma is associated with chronic airway inflammation and progressive airway remodelling. However, the dynamics of the development of these features and their spontaneous and pharmacological reversibility are still poorly understood. We have therefore investigated the dynamics of airway remodelling and repair in an experimental asthma model and studied how pharmacological intervention affects these processes.

Methods

Using BALB/c mice, the kinetics of chronic asthma progression and resolution were characterised in absence and presence of inhaled corticosteroid (ICS) treatment. Airway inflammation and remodelling was assessed by the analysis of bronchoalveolar and peribronichal inflammatory cell infiltrate, goblet cell hyperplasia, collagen deposition and smooth muscle thickening.

Results

Chronic allergen exposure resulted in early (goblet cell hyperplasia) and late remodelling (collagen deposition and smooth muscle thickening). After four weeks of allergen cessation eosinophilic inflammation, goblet cell hyperplasia and collagen deposition were resolved, full resolution of lymphocyte inflammation and smooth muscle thickening was only observed after eight weeks. ICS therapy when started before the full establishment of chronic asthma reduced the development of lung inflammation, decreased goblet cell hyperplasia and collagen deposition, but did not affect smooth muscle thickening. These effects of ICS on airway remodelling were maintained for a further four weeks even when therapy was discontinued.

Conclusions

Utilising a chronic model of experimental asthma we have shown that repeated allergen exposure induces reversible airway remodelling and inflammation in mice. Therapeutic intervention with ICS was partially effective in inhibiting the transition from acute to chronic asthma by reducing airway inflammation and remodelling but was ineffective in preventing smooth muscle hypertrophy.     

Potential therapeutic target discovery by 2D-DIGE proteomic analysis in mouse models of asthma

As asthma physiopathology is complex and not fully understood to date; it is expected that new key mediators are still to be unveiled in this disease. The main objective of this study was to discover potential new target proteins with a molecular weight >20 kDa by using two-dimensional differential in-gel electrophoresis (2D-DIGE) on lung parenchyma extracts from control or allergen-exposed mice (ovalbumin). Two different mouse models leading to the development of acute airway inflammation (5 days allergen exposure) and airway remodeling (10 weeks allergen exposure) were used. This experimental setting allowed the discrimination of 33 protein spots in the acute inflammation model and 31 spots in the remodeling model displaying a differential expression. Several proteins were then identified by MALDI-TOF/TOF MS. Among those differentially expressed proteins, PDIA6, GRP78, Annexin A6, hnRPA3, and Enolase display an increased expression in lung parenchyma from mice exposed to allergen for 5 days. Conversely, Apolipoprotein A1 was shown to be decreased after allergen exposure in the same model. Analysis on lung parenchyma of mice exposed to allergens for 10 weeks showed decreased calreticulin levels. Changes in the levels of those different mediators were confirmed by Western blot and immunohistochemical analysis. Interestingly, alveolar macrophages isolated from lungs in the acute inflammation model displayed enhanced levels of GRP78. Moreover, intratracheal instillation of anti-GRP78 siRNA in allergen-exposed animals led to a decrease in eosinophilic inflammation and bronchial hyperresponsiveness. This study unveils new mediators of potential importance that are up- and down-regulated in asthma. Among up-regulated mediators, GRP-78 appears as a potential new therapeutic target worthy of further investigations.     

Recombinant human platelet-activating factor-acetylhydrolase inhibits airway inflammation and hyperreactivity in mouse asthma model

Numerous in vitro and in vivo studies in both animal models and human asthmatics have implicated platelet-activating factor (PAF) as an important inflammatory mediator in asthma. In a murine asthma model, we examined the anti-inflammatory activities of recombinant human PAF-acetylhydrolase (rPAF-AH), which converts PAF to biologically inactive lyso-PAF. In this model, mice sensitized to OVA by i.p. and intranasal (i.n.) routes are challenged with the allergen by i.n. administration. The OVA challenge elicits an eosinophil infiltration into the lungs with widespread mucus occlusion of the airways and results in bronchial hyperreactivity. The administration of rPAF-AH had a marked effect on late-phase pulmonary inflammation, which included a significant reduction in airway eosinophil infiltration, mucus hypersecretion, and airway hyperreactivity in response to methacholine challenge. These studies demonstrate that elevating plasma levels of PAF-AH through the administration of rPAF-AH is effective in blocking the late-phase pulmonary inflammation that occurs in this murine allergen-challenge asthma model. These results suggest that rPAF-AH may have therapeutic effects in patients with allergic airway inflammation.     

T-cell-mediated inflammation does not contribute to the maintenance of airway dysfunction in mice.

T-cell-mediated airway inflammation is considered to be critical in the pathogenesis of airway hyperresponsiveness (AHR). We have described a mouse model in which chronic allergen exposure results in sustained AHR and aspects of airway remodeling and here sought to determine whether eliminating CD4(+) and CD8(+) cells, at a time when airway remodeling had occurred, would attenuate this sustained AHR. Sensitized BALB/c mice were subjected to either brief or chronic periods of allergen exposure and studied 24 h after brief or 4 wk after chronic allergen exposure. In both models, mice received three treatments with anti-CD4 and -CD8 monoclonal antibodies during the 10 days before outcome measurements. Outcomes included in vivo airway responsiveness to intravenous methacholine, CD4(+) and CD8(+) cell counts of lung and spleen using flow cytometric analysis, and airway morphometry using a computer-based image analysis system. Compared with saline control mice, brief allergen challenge resulted in AHR, which was eliminated by antibody treatment. Chronic allergen challenge resulted in sustained AHR and indexes of airway remodeling. This sustained AHR was not reversed by antibody treatment, even though CD4(+) and CD8(+) cells were absent in lung and spleen. These results indicate that T-cell-mediated inflammation is critical for development of AHR associated with brief allergen exposure, but is not necessary to maintain sustained AHR.     

Manipulation of allergen-induced airway remodeling by treatment with anti-TGF-beta antibody: effect on the Smad signaling pathway

Airway inflammation and remodeling are important pathophysiologic features of chronic asthma. Previously, we have developed a mouse model of prolonged allergen challenge which exhibits many characteristics of chronic asthma such as goblet cell hyperplasia and subepithelial collagen deposition, in association with an increase in lung expression of the profibrotic mediator, TGF-beta. The aim of this study was to determine the effects of blockade of TGF-beta on the development of airway inflammation and remodeling using our murine model of prolonged allergen challenge. Importantly anti-TGF-beta Ab was administered therapeutically, with dosing starting after the onset of established eosinophilic airway inflammation. Therapeutic treatment of mice with anti-TGF-beta Ab significantly reduced peribronchiolar extracellular matrix deposition, airway smooth muscle cell proliferation, and mucus production in the lung without affecting established airway inflammation and Th2 cytokine production. Thus, our data suggest that it might be possible to uncouple airway inflammation and remodeling during prolonged allergen challenge. In addition, anti-TGF-beta Ab treatment was shown to regulate active TGF-beta signaling in situ with a reduction in the expression of phospho-Smad 2 and the concomitant up-regulation of Smad 7 in lung sections. Therefore, this is the first report to suggest that anti-TGF-beta Ab treatment prevents the progression of airway remodeling following allergen challenge even when given in a therapeutic mode. Moreover, the molecular mechanism behind this effect may involve regulation of active TGF-beta signaling.     

Influence of sensitization and allergen provocation procedures on the development of allergen-induced bronchial hyperreactivity in conscious, unrestrained guinea-pigs

The effects of different sensitization and allergen provocation regimens on the development of allergen-induced bronchial hyperreactivity (BHR) to histamine were investigated in conscious, unrestrained guinea-pigs. Similar early and late phase asthmatic reactions, BHR for inhaled histamine after the early (6 h) as well as after the late reaction (24 h), and airway inflammation were observed after a single allergen provocation in animals sensitized to produce mainly IgG or IgE antibodies, respectively. Repeating the allergen provocation in the IgE-sensitized animals after 7 days, using identical provocation conditions, resulted in a similar development of BHR to histamine inhalation. Repetition of the allergen provocation during 4 subsequent days resulted in a decreased development of BHR after each provocation, despite a significant increase in the allergen provocation dose necessary to obtain similar airway obstruction. The number of inflammatory cells in the bronchoalveolar lavage was not significantly changed after repeated provocation, when compared with a single allergen provocation. Finally, we investigated allergen-induced bronchial hyperreactivity by repetition of the sensitization procedure at day 7 and 14 (booster), followed by repeated allergen provocation twice a week for 5 weeks. Surprisingly, no BHR to histamine could be observed after either provocation, while the number of inflammatory cells in the bronchoalveolar lavage fluid after 5 weeks was enhanced compared with controls. These data indicate that both IgE and IgG sensitized guinea-pigs may develop bronchial hyperreactivity after a single allergen provocation. Repeated allergen exposure of IgE sensitized animals causes a gradual fading of the induced hyperreactivity despite the on-going presence of inflammatory cells in the airways, indicating a mechanism of reduced cellular activation.     

The airway epithelium in asthma

Asthma is a T lymphocyte-controlled disease of the airway wall caused by inflammation, overproduction of mucus and airway wall remodeling leading to bronchial hyperreactivity and airway obstruction. The airway epithelium is considered an essential controller of inflammatory, immune and regenerative responses to allergens, viruses and environmental pollutants that contribute to asthma pathogenesis. Epithelial cells express pattern recognition receptors that detect environmental stimuli and secrete endogenous danger signals, thereby activating dendritic cells and bridging innate and adaptive immunity. Improved understanding of the epithelium's function in maintaining the integrity of the airways and its dysfunction in asthma has provided important mechanistic insight into how asthma is initiated and perpetuated and could provide a framework by which to select new therapeutic strategies that prevent exacerbations and alter the natural course of the disease.     

Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity

Asthma is caused by T-helper cell 2 (Th2)-driven immune responses, but the immunological mechanisms that protect against asthma development are poorly understood. T-cell tolerance, induced by respiratory exposure to allergen, can inhibit the development of airway hyperreactivity (AHR), a cardinal feature of asthma, and we show here that regulatory T (T(R)) cells can mediate this protective effect. Mature pulmonary dendritic cells in the bronchial lymph nodes of mice exposed to respiratory allergen induced the development of T(R) cells, in a process that required T-cell costimulation via the inducible costimulator (ICOS-ICOS-ligand pathway. The T(R) cells produced IL-10, and had potent inhibitory activity; when adoptively transferred into sensitized mice, T(R) cells blocked the development of AHR. Both the development and the inhibitory function of regulatory cells were dependent on the presence of IL-10 and on ICOS-ICOS-ligand interactions. These studies demonstrate that T(R) cells and the ICOS-ICOS-ligand signaling pathway are critically involved in respiratory tolerance and in downregulating pulmonary inflammation in asthma.     

Inflammatory cell distribution in guinea pig airways and its relationship to airway reactivity.

Although airway inflammation and airway hyperreactivity are observed after allergen inhalation both in allergic humans and animals, little is known about the mechanisms by which inflammatory cells can contribute to allergen-induced airway hyperreactivity. To understand how inflammatory cell infiltration can contribute to airway hyperreactivity, the location of these cells within the airways may be crucial Using a guinea pig model of acute allergic asthma, we investigated the inflammatory cell infiltration in different airway compartments at 6 and 24 h (i.e. after the early and the late asthmatic reaction, respectively) after allergen or saline challenge in relation to changes in airway reactivity (AR) to histamine. At 6 h after allergen challenge, a threefold (p < 0.01) increase in the AR to histamine was observed. At 24 h after challenge, the AR to histamine was lower, but still significantly enhanced (1.6-fold, p < 0.05). Adventitial eosinophil and neutrophil numbers in both bronchi and bronchioli were significantly increased at 6 h post-allergen provocation as compared with saline (p < 0.01 for all), while there was a strong tendency to enhanced eosinophils in the bronchial submucosa at this time point (p = 0.08). At 24h after allergen challenge, the eosinophilic and neutrophilic cell infiltration was reduced. CD3+ T lymphocytes were increased in the adventitial compartment of the large airways (p < 0.05) and in the parenchyma (p < 0.05) at 24h post-allergen, while numbers of CD8+ cells did not differ from saline treatment at any time point post-provocation. The results indicate that, after allergen provocation, inflammatory cell numbers in the airways are mainly elevated in the adventitial compartment. The adventitial inflammation could be important for the development of allergen-induced airway hyperreactivity.     

Endotoxin responsiveness and subchronic grain dust-induced airway disease

Endotoxin is one of the principal components of grain dust that causes acute reversible airflow obstruction and airway inflammation. To determine whether endotoxin responsiveness influences the development of chronic grain dust-induced airway disease, physiological and airway inflammation remodeling parameters were evaluated after an 8-wk exposure to corn dust extract (CDE) and again after a 4-wk recovery period in a strain of mice sensitive to (C3H/HeBFeJ) and one resistant to (C3H/HeJ) endotoxin. After the CDE exposure, both strains of mice had equal airway hyperreactivity to a methacholine challenge; however, airway hyperreactivity persisted only in the C3H/HeBFeJ mice after the recovery period. Only the C3H/HeBFeJ mice showed significant inflammation of the lower airway after the 8-wk exposure to CDE. After the recovery period, this inflammatory response completely resolved. Lung stereological measurements indicate that an 8-wk exposure to CDE resulted in persistent expansion of the airway submucosal cross-sectional area only in the C3H/HeBFeJ mice. Collagen type III and an influx of cells into the subepithelial area participated in the expansion of the submucosa. Our findings demonstrate that subchronic inhalation of grain dust extract results in the development of chronic airway disease only in mice sensitive to endotoxin but not in mice that are genetically hyporesponsive to endotoxin, suggesting that endotoxin is important in the development of chronic airway disease.     

Overall asthma control achieved with budesonide/formoterol maintenance and reliever therapy for patients on different treatment steps

Background

Adjusting medication for uncontrolled asthma involves selecting one of several options from the same or a higher treatment step outlined in asthma guidelines. We examined the relative benefit of introducing budesonide/formoterol (BUD/FORM) maintenance and reliever therapy (Symbicort SMART^® Turbuhaler^®) in patients previously prescribed treatments from Global Initiative for Asthma (GINA) Steps 2, 3 or 4.

Methods

This is a post hoc analysis of the results of five large clinical trials (>12000 patients) comparing BUD/FORM maintenance and reliever therapy with other treatments categorised by treatment step at study entry. Both current clinical asthma control during the last week of treatment and exacerbations during the study were examined.

Results

At each GINA treatment step, the proportion of patients achieving target levels of current clinical control were similar or higher with BUD/FORM maintenance and reliever therapy compared with the same or a higher fixed maintenance dose of inhaled corticosteroid/long-acting β₂-agonist (ICS/LABA) (plus short-acting β₂-agonist [SABA] as reliever), and rates of exacerbations were lower at all treatment steps in BUD/FORM maintenance and reliever therapy versus same maintenance dose ICS/LABA (P < 0.01) and at treatment Step 4 versus higher maintenance dose ICS/LABA (P < 0.001). BUD/FORM maintenance and reliever therapy also achieved significantly higher rates of current clinical control and significantly lower exacerbation rates at most treatment steps compared with a higher maintenance dose ICS + SABA (Steps 2-4 for control and Steps 3 and 4 for exacerbations). With all treatments, the proportion of patients achieving current clinical control was lower with increasing treatment steps.

Conclusions

BUD/FORM maintenance and reliever therapy may be a preferable option for patients on Steps 2 to 4 of asthma guidelines requiring a more effective treatment and, compared with other fixed dose alternatives, is most effective in the higher treatment steps.