Novel approaches to the management of noneosinophilic asthma

Abstract

Noneosinophilic airway inflammation occurs in approximately 50% of patients with asthma. It is subdivided into neutrophilic or paucigranulocytic inflammation, although the proportion of each subtype is uncertain because of variable cut-off points used to define neutrophilia. This article reviews the evidence for noneosinophilic inflammation being a target for therapy in asthma and assesses clinical trials of licensed drugs, novel small molecules and biologics agents in noneosinophilic inflammation. Current symptoms, rate of exacerbations and decline in lung function are generally less in noneosinophilic asthma than eosinophilic asthma. Noneosinophilic inflammation is associated with corticosteroid insensitivity. Neutrophil activation in the airways and systemic inflammation is reported in neutrophilic asthma. Neutrophilia in asthma may be due to corticosteroids, associated chronic pulmonary infection, altered airway microbiome or delayed neutrophil apoptosis. The cause of poorly controlled noneosinophilic asthma may differ between patients and involve several mechanism including neutrophilic inflammation, T helper 2 (Th2)-low or other subtypes of airway inflammation or corticosteroid insensitivity as well as noninflammatory pathways such as airway hyperreactivity and remodelling. Smoking cessation in asthmatic smokers and removal from exposure to some occupational agents reduces neutrophilic inflammation. Preliminary studies of ‘off-label’ use of licensed drugs suggest that macrolides show efficacy in nonsmokers with noneosinophilic severe asthma and statins, low-dose theophylline and peroxisome proliferator-activated receptor gamma (PPARγ) agonists may benefit asthmatic smokers with noneosinophilic inflammation. Novel small molecules targeting neutrophilic inflammation, such as chemokine (CXC) receptor 2 (CXCR2) antagonists reduce neutrophils, but do not improve clinical outcomes in studies to date. Inhaled phosphodiesterase (PDE)₄ inhibitors, dual PDE₃ and PDE₄ inhibitors, p38MAPK (mitogen-activated protein kinase) inhibitors, tyrosine kinase inhibitors and PI (phosphoinositide) 3kinase inhibitors are under development and these compounds may be of benefit in noneosinophilic inflammation. The results of clinical trials of biological agents targeting mediators associated with noneosinophilic inflammation, such as interleukin (IL)-17 and tumor necrosis factor (TNF)-α are disappointing. Greater understanding of the mechanisms of noneosinophilic inflammation in asthma should lead to improved therapies.

Introduction

Personalized medicine in asthma aims to individualise treatment using noninvasive biomarkers that predict a beneficial response or that identify individuals who are at risk of adverse effects [Agustí et al. 2015]. Several airway inflammatory phenotypes are recognized that help identify a therapeutic response to specific treatments in asthma. For example, eosinophilic airway inflammation, which is usually identified on the basis of sputum or blood eosinophilia, predicts patients with asthma that are likely to obtain a favourable therapeutic response to corticosteroids [Pavord et al. 1999; Little et al. 2000; Green et al. 2002a; Bacci et al. 2006; Berry et al. 2007] and to monoclonal antibodies targeting interleukin (IL)-5 [Pavord et al. 2012; Katz et al. 2014; Thomson, 2014]. Type 2 helper T-cell (Th2)-high subtype of asthma is associated with increased epithelial expression of IL-4, IL-5 and IL-13 [Woodruff et al. 2009; Arron et al. 2013] and is considered to overlap with eosinophilic airway inflammation [Arron et al. 2013]. Evidence from clinical trials suggests that the presence of type-2 eosinophilic inflammation predicts a therapeutic response not only to corticosteroids [Woodruff et al. 2009], but to monoclonal antibodies targeting specific cytokines such as IL-5 [Bel et al. 2014; Ortega et al. 2014] and IL-13 [Corren et al. 2011]. Many patients with asthma have noneosinophilic asthma, sometimes associated with neutrophilic inflammation or have a Th2-low type of inflammation. Compared with type-2 eosinophilic inflammation there are relatively few interventions available for non-type 2 inflammatory subgroups. This article aims to discuss the evidence that noneosinophilic airway inflammation, with or without neutrophilic inflammation, is an appropriate target for therapy in asthma and also aims to assess the results of recent clinical trials of licensed drugs, novel small molecules and biologics agents in the treatment of noneosinophilic asthma.

Is noneosinophilic airway inflammation an appropriate target for therapy in asthma?

A number of factors need to be considered when attempting to answer the question of whether noneosinophilic inflammation is an appropriate target for treatment in asthma including the criteria used to define neutrophilic and eosinophilic inflammation, the stability of noneosinophilic inflammation over time, the prevalence of noneosinophilic inflammation, the strength of evidence for the involvement of noneosinophilic inflammation in clinical features of asthma and the cause(s) of noneosinophilic airway inflammation.

Definition of eosinophilic and neutrophilic airway inflammation

Noneosinophilic airway inflammation is a term used to describe a subtype of asthma associated with normal numbers of sputum eosinophils. The noneosinophilic phenotype is subdivided into neutrophilic inflammation, when neutrophil numbers are raised above a defined cut-off level or paucigranulocytic inflammation, when both eosinophil and neutrophil numbers are normal. In addition, some individuals have a mixed type of inflammation, when there is sputum neutrophilia and eosinophilia. Cut-off levels used to define sputum eosinophilia most commonly used are ⩾2% [Mcgrath et al. 2012; Hastie et al. 2013], >2% [Peters et al. 2014] or ⩾3% [Schleich et al. 2013; Zhang et al. 2014; Wagener et al. 2015]. A ⩾3% cut-off is reported to be the most precise value to identify eosinophilic airway inflammation [Simpson et al. 2010]. Sputum eosinophil counts are associated with bronchial tissue eosinophil numbers suggesting that they provide a good indicator of airway eosinophilic pathology [Arron et al. 2014]. The cut-off for a raised sputum neutrophil count is not clearly established with a wide range of values reported in the literature: >40% [Nair et al. 2012; Moore et al. 2014), ⩾50% [Chaudhuri et al. 2014], >61% [Simpson et al. 2009], >65% [Nair et al. 2015] and ⩾76% [Schleich et al. 2013]. The most appropriate cut-off value that identifies individuals in whom neutrophils are activated and contributing to the pathogenic processes in asthma is not certain. In addition, sputum neutrophils do not correlate with bronchial tissue numbers bringing into doubt their predictive value for identifying neutrophil-induced airway pathology [Arron et al. 2014]. In addition to the presence of noneosinophilic inflammation, Haldar and Pavord proposed that the criteria for a diagnosis of noneosinophilic asthma should include objective evidence of airflow obstruction or airway hyperreactivity, a raised asthma control questionnaire (ACQ) score (>1.5) and the absence of a significant smoking history, fixed airflow obstruction or associated bronchiectasis [Haldar and Pavord, 2007]. In the current article, the criteria for noneosinophilic asthma include the presence of noneosinophilic inflammation as defined above plus objective evidence of asthma, but the review also includes data from patients with both normal and raised ACQ scores, who have a significant smoking history or who have fixed airflow obstruction.

Stability of sputum cell counts

Published data on the long term stability of sputum neutrophil and eosinophil counts is conflicting. Some studies report stable sputum cell counts in patients with mild to severe asthma follow-up over 6 months [Berry et al. 2007], 12 months [Green et al. 2002a], 2 years [Jayaram et al. 2006] and 5 years [Simpson et al. 2006; Van Veen et al. 2009]. In contrast, sputum inflammatory cell phenotype changed in 48.6% of patients with severe asthma over 1 year among patients recruited to the BIOmarkers in Severe Chronic AIRway Disease (BIOAIR) study [Kupczyk et al. 2014a]. Similar variability in sputum cell counts has been reported by others [Hancox et al. 2012] and in one study a stable inflammatory phenotype was found in only one-third of patients [Al-Samri et al. 2010]. Transient sputum eosinophilia is reported in up to 40% patients with noneosinophilic inflammation [Bacci et al. 2012; McGrath et al. 2012]. The potential for the lack of stability in noneosinophilic inflammation over time needs to be accounted for in intervention studies targeting sputum inflammatory cell biomarkers.

Prevalence of noneosinophilic airway inflammation

The different cut-off values used to define elevated sputum cell counts, particularly sputum neutrophils, may explain the variation in prevalence figures for noneosinophilic inflammation between studies. Nevertheless, overall up to 50% of adults and adolescents with stable mild to severe asthma, and in some studies higher proportions, have noneosinophilic inflammation [Gibson et al. 2001; Green et al. 2002b; Simpson et al. 2006; Wang et al. 2011; McGrath et al. 2012; Schleich et al. 2013; Moore et al. 2014; Brooks et al. 2016 ]. For example, a review of sputum cytology data from 995 patients with mild to moderate asthma enrolled in clinical trials undertaken by the Asthma Clinical Research Network (ACRN) reported that noneosinophilic inflammation (sputum eosinophils <2%) was present in 64% of patients not taking inhaled corticosteroid and 83% of patients taking inhaled corticosteroids. In a subgroup of patients followed up for 6 months, 47% of the inhaled corticosteroid-free patients and 72% of those taking inhaled corticosteroids had persistent noneosinophilic inflammation [McGrath et al. 2012]. In a cluster analysis performed on 423 patients recruited to the Severe Asthma Research Program (SARP) cohort, four asthma inflammatory subphenotypes were identified (cut-off values used to define sputum eosinophilia ⩾2% and sputum neutrophilia >40%) [Moore et al. 2014]. Two groups had mild-to-moderate allergic asthma with minimal or eosinophil-predominant sputum inflammation whereas the other two subphenotypes had moderate-to-severe asthma with neutrophil-predominant or mixed granulocytic inflammation [Moore et al. 2014]. A study in a small group of adults with stable asthma found 51.7% of subjects had a paucigranulocytic phenotype, 27.6% neutrophilic inflammation and 17.2% eosinophilic inflammation [Wang et al. 2011].

Involvement of neutrophilic and noneosinophilic airway inflammation in asthma

Evidence for the involvement of noneosinophilic inflammation in asthma is based mainly on studies examining the association between sputum inflammatory phenotypes and clinical outcomes in asthma, including current symptom control, exacerbations, airflow obstruction and therapeutic response to corticosteroids. Further evidence is provided by reports of local activation of neutrophils and systemic inflammation in neutrophilic asthma.

Current symptom control

The severity of current symptoms is in general similar or slightly lower in noneosinophilic or neutrophilic subgroups of asthma compared to eosinophilic subgroups [Cowan et al. 2010; Hastie et al. 2010; Wood et al. 2012; Schleich et al. 2013, 2014; Baines et al. 2014; Newby et al. 2014].

Exacerbations

Sputum neutrophilia is found in up to 80% of exacerbations in adults with asthma [Turner et al. 1995; Fahy et al. 1995; Lamblin et al. 1998; Green et al. 2002a; Jayaram et al. 2006; Maneechotesuwan et al. 2007; Wang et al. 2011], although the predominant sputum cell type can alter during successive exacerbations [D’Silva et al. 2007]. Sputum eosinophilia is a better predictor of future exacerbations than sputum neutrophilia [Jatakanon et al. 2000; Leuppi et al. 2001; Kupczyk et al. 2014b; Schleich et al. 2014]. For example, a cluster analysis performed on patients recruited to the BIOAIR study identified two clusters with raised sputum eosinophil counts that accounted for 83% of patients who had two or more severe exacerbations during follow-up for 1 year [Kupczyk et al. 2014b]. One of these clusters had a mixed inflammatory profile with raised sputum neutrophils (43% percent of patients). A further cluster had a raised neutrophil count and a normal (cut-off values used to define sputum eosinophilia ⩾2% and sputum neutrophilia >40%) eosinophil count (11% of patients) and a noneosinophilic paucigranulocytic inflammation was found in only 6% of cases. Patients with severe asthma associated with eosinophil inflammation have more intubations than noneosinophilic patients [Wenzel et al. 1999].

Airflow obstruction

Sputum neutrophilia is associated with reduced lung function and based on this finding it has been speculated that airway neutrophils may contribute to the development of persistent airflow obstruction in asthma [Little et al. 2002; Shaw et al. 2007]. Against this hypothesis, a recent cluster analysis of lung function decline and sputum eosinophil count performed in 97 patients with severe asthma identified a noneosinophilic group in whom the decline in forced expiratory volume in 1 second (FEV₁) was −14 ml per year compared to an eosinophilic group with highly variable eosinophil counts that had a greater rate of decline in FEV₁ of −41 ml per year [Newby et al. 2014]. These findings suggest that eosinophilic inflammation, particularly when there is high variability in eosinophil count, is a greater risk factor for the development of persistent airflow obstruction than noneosinophilic inflammation. Bronchodilator reversibility and airway hyperresponsiveness are similar in eosinophilic and noneosinophilic asthma [Berry et al. 2007; McGrath et al. 2012], although one study noted greater airway hyperresponsiveness in persistent or intermittent eosinophilic groups [McGrath et al. 2012].

Impaired response to inhaled corticosteroids

Noneosinophilic inflammation is associated with an impaired therapeutic response to inhaled corticosteroids [Pavord et al. 1999; Green et al. 2002a; Bacci et al. 2006; Berry et al. 2007; Thomson et al. 2009; McGrath et al. 2012], although the lack of efficacy may not be complete. Several clinical studies performed in small numbers of patients with noneosinophilic asthma suggest that this group may obtain some benefit from inhaled corticosteroids although less than that found in eosinophilic patients [Godon et al. 2002; Cowan et al. 2010; Lemière et al. 2011]. Intermittent eosinophilia might be a factor accounting for corticosteroid sensitivity in some of these patients [Bacci et al. 2012; McGrath et al. 2012; Suárez-Cuartín et al. 2015].

Evidence for neutrophil activation in asthma

There is evidence to suggest that the innate immune system is activated in chronic asthma. Firstly, sputum IL-8 and neutrophil elastase concentrations and innate immune receptors –Toll-like receptors (TLR)2, TLR4 and CD14 – as well as pro-inflammatory IL-8 and IL-1β gene expression levels are increased in neutrophilic asthma compared to non-neutrophilic asthma [Simpson et al. 2007; Wood et al. 2012]. Secondly, neutrophil activation, as measured by sputum myeloperoxidase (MPO) levels, is positively associated with sputum neutrophil numbers in asthma [Little et al. 2002]. Thirdly, specific sputum gene expression signatures are reported to discriminate eosinophilic asthma from noneosinophilic asthma as well as to predict a beneficial response to inhaled corticosteroids [Baines et al. 2014]. In this study, noneosinophilic asthma was identified by increased sputum cell expression of IL-1β, alkaline phosphatase, tissuenonspecific isozyme (ALPL) and CXCR2, whereas eosinophilic asthma was characterized by increased expression of Charcot–Leyden crystal protein or galectin-10 (CLC), carboxypeptidase A3 (CPA3) and deoxyribonuclease I-like 3 (DNASE1L3). The NLRP3 inflammasome is upregulated in neutrophilic asthma and may increase the production of IL-1β [Simpson et al. 2014b; Kim et al. 2015a]. Anti-inflammatory responses may be impaired in noneosinophilic asthma based on reduced sputum galectin-3 concentrations, which increases uptake of apoptotic neutrophils and reduced IL-1RA/IL-1β ratio, and which might increase pro-inflammatory actions of IL-1β [Gao et al. 2015]. In addition, soluble receptor for advanced glycation end-products (RAGE), which is a pattern-recognition receptor is deficient in bronchoalveolar lavage (BAL) samples in neutrophilic asthma [Sukkar et al. 2012]. The T-cell granzyme B pathway, which is thought to mediate apoptosis of epithelial cells, might be defective in noneosinophilic asthma, based on the finding of a higher ratio of the expression of granzyme B to its inhibitor in T cells in this group compared to eosinophilic asthma [Simpson et al. 2014a].

Systemic inflammation is increased in patients with neutrophilic airway inflammation. The proportion of patients with elevated CRP, IL-6 and neutrophil elastase concentrations is higher in neutrophilic asthma compared to a non-neutrophilic group [Baines et al. 2011; Wood et al. 2012]. Neutrophilic inflammation is associated with increased α-defensin and neutrophil protease gene expression in blood [Baines et al. 2011]. In noneosinophilic asthma, blood neutrophils released significantly higher levels of IL-8 at rest [Baines et al. 2010]. In a small study, gene expression markers of systemic inflammation were associated with higher basal metabolic index (BMI), greater history of cigarette smoking, lower forced vital capacity (FVC)% predicted, and increased sputum neutrophils [Fu et al. 2013].

Potential inflammatory processes leading to noneosinophilic airway inflammation

Several inflammatory processes could lead to noneosinophilic inflammation and airway damage in asthma although the exact immunological mechanisms are unclear (Figure 1) [Trejo Bittar et al. 2015]. Uncertainty in the clinical relevance of experimental animal models of noneosinophilic inflammation has hampered progress in understanding the involvement of neutrophils and noneosinophilic inflammation in the pathogenesis of asthma. Stimuli such as viruses, cigarette smoke and pollutants could induce the release of chemoattractants including IL-8 to recruit neutrophils to the airways. In experimental asthma models, the release of IL-17A and IL-17F from activated Th17 cells stimulates the synthesis of neutrophil chemoattractants including CXCL1 and IL-8 from the airway epithelium. [Newcomb and Peebles, 2013]. Interferon gamma (IFN-γ) may also be involved in the pathogenesis of severe asthma associated with neutrophilic and eosinophilic inflammation, possibly in part through the release of IFN-γ from Th1 cells [Raundhal et al. 2015]. Data from patients with severe asthma and an experimental murine asthma model implicate high IFN-γ immune responses and low secretory leukocyte protease inhibitor expression (SLPI) in airway epithelial cells with airway hyperresponsiveness [Raundhal et al. 2015]. Neutrophils are a potential source of oxygen free radicals and enzymes and their ability to activate other airway cell types [Futosi et al. 2013]. Neutrophils in asthma are implicated in causing mucus gland hyperplasia and hypersecretion, airway hyperreactivity and remodelling as well as corticosteroid insensitivity. Interestingly, Th1 and Th17 cells may induce airway hyperreactivity or remodelling independently of neutrophil activation.

Figure 1.

Schematic diagram of potential pathways leading to noneosinophilic inflammation and airway damage in severe asthma.

Several inflammatory pathways could potentially lead to noneosinophilic inflammation and airway damage in asthma although the exact mechanisms are unclear. Possible pathways are briefly summarized in the schematic diagram. Stimuli such as viruses, cigarette smoke and pollutants could induce the release of chemoattractants including IL-8 to recruit neutrophils to the airways. The release of IL-17A and IL-17F from activated Th17 cells could stimulate the synthesis of neutrophil chemoattractants, such as CXCL1 and IL-8 from the airway epithelium. IFN-γ may also be involved in noneosinophilic asthma, possibly in part through its release from Th1 cells. Inflammatory mediators released by neutrophils are implicated in causing mucus gland hyperplasia and hypersecretion, airway hyperreactivity and remodelling as well as corticosteroid insensitivity in asthma. Th1 and Th17 cells may induce airway hyperreactivity or remodelling independently of neutrophil activation.

Abbreviations: CXCL1, chemokine (C-X-C motif) ligand; IFN, interferon; IL, interleukin; LT, leukotriene; MMP, matrix metalloproteinase; MPO, myeloperoxidase; ROC, reactive oxygen species; TNF-α, tumor necrosis factor α.

Limited information has been published on the immunopathological characteristics of noneosinophilic inflammation in asthma compared with other inflammatory airway phenotypes including Th2-low inflammation, Th17-high inflammation or a combination of Th2/Th17 profiles. Bronchial biopsy studies of patients with noneosinophilic asthma or with Th2-low inflammation, report reduced submucosal eosinophil numbers and normal subepithelial basement membrane thickness in both groups [Wenzel et al. 1999; Berry et al. 2007; Woodruff et al. 2009]. In contrast, bronchial eosinophil numbers and subepithelial basement membrane thickness are both increased in eosinophilic asthma and in Th2-high asthma [Wenzel et al. 1999; Berry et al. 2007; Woodruff et al. 2009]. Mast cell numbers are increased in eosinophilic asthma [Wenzel et al. 1999] and Th2-high asthma [Dougherty et al. 2010], whereas mast cell numbers are normal in the submucosa of patients with severe noneosinophilic asthma [Wenzel et al. 1999] and in the epithelium of nonsmokers with Th2-low asthma [Dougherty et al. 2010]. Mast cell numbers in airway smooth muscle are increased in both noneosinophilic and eosinophilic asthma [Berry et al. 2007]. Bronchial biopsy neutrophil numbers are increased by a similar degree in noneosinophilic severe asthma and eosinophilic severe asthma [Wenzel et al. 1999]. Neutrophil numbers in Th2-low asthma have not been reported. A lower proportion of subjects with noneosinophilic asthma are atopic compared to eosinophilic asthma (18% versus 66%) [Berry et al. 2007] and (58% versus 83%) [Gibson et al. 2001]. Severe asthma associated with neutrophilia has significantly higher sputum levels of Th17-related cytokines (CXCL1, CXCL10, Chemokine (C-C motif) ligand (CCL)2, IL-6 and IL-8) compared with severe asthmatics with other inflammatory phenotypes [Manni et al. 2014]. The proportion of Th17 lymphocytes and the ratio of Th17 to regulatory T cells (Treg) in the peripheral blood is greater in patients with noneosinophilic asthma taking inhaled corticosteroids compared to an eosinophilic asthma group [Furukawa et al. 2015]. Approximately one-third of patients with severe eosinophilic asthma have a Th17-high signature that is associated with a Th2-low gene expression profile [Choy et al. 2015]. The number of patients with noneosinophilic severe asthma in this study was not sufficient to determine their Th17 profile [Choy et al. 2015]. Taken together, these findings suggest that noneosinophilic inflammation and Th2-low inflammation in nonsmokers with asthma share some similar immunopathological features, including normal eosinophil numbers, submucosal mast cell numbers and subepithelial basement membrane thickness. There is a need for further studies to establish the similarities and differences in endotypes of noneosinophilic, Th2-low and Th17-high inflammation to help identify subgroups of patients for targeted therapies.

Factors accounting for neutrophilic airway inflammation in asthma

Several factors either alone or in combination could explain raised sputum neutrophil counts in asthma (Table 1). Corticosteroids inhibit apoptosis of neutrophils [Cox, 1995] and their use in asthma may contribute to sputum neutrophilia [Saffar et al. 2011]. In addition, Th2-targeted therapies, including oral corticosteroids may contribute to the development of Th17-high neutrophilic inflammation [Choy et al. 2015; Shum, 2015]. In support of corticosteroids causing neutrophilia in asthma, inhaled corticosteroid withdrawal from patients with moderate asthma resulted in only one patient with neutrophilic inflammation although the reintroduction of inhaled fluticasone for 4 weeks resulted in a raised neutrophil count in only 5% of patients [Cowan et al. 2010]. In one study of patients with severe oral corticosteroid-dependent asthma associated with increased sputum neutrophil number, markers of neutrophil activation including oxidative burst and surface granular receptor expression were similar to patients with mild asthma [Nair et al. 2015]. In contrast, corticosteroid use was not associated with sputum neutrophilia in the SARP cohort, suggesting that continuous corticosteroid exposure may not be the only influence on sputum neutrophil numbers in severe asthma [Moore et al. 2014]. Comorbid conditions such bronchiectasis or severe airflow obstructions occurring in association with asthma may result in neutrophilic inflammation. Delayed human neutrophil apoptosis has been reported in severe asthma [Uddin et al. 2010], possibly due to epithelial growth factor-induced release of mediators with neutrophil chemotactic and anti-apoptotic actions from bronchial epithelial cells [Uddin et al. 2010; Uddin et al. 2013]. Macrophage efferocytosis is impaired in noneosinophilic asthma, which may cause airway neutrophilia [Simpson et al. 2013]. Lastly, an altered airway microbiome has been implicated in airway neutrophilia. Airway colonization determined by terminal restriction fragment length polymorphism (T-RFLP) analysis is associated with more severe airway obstruction and longer duration of disease as well as neutrophilic airway inflammation and raised sputum IL-8 levels [Simpson et al. 2013; Green et al. 2014]. Taken together, these findings suggest that the cause of airway neutrophilia in asthma is likely to be complex, possibly due to corticosteroid treatment inducing impaired apoptosis of neutrophils and Th17 mediated neutrophilic inflammation, delayed apoptosis of neutrophils due to epithelial growth factor release and ineffective macrophage efferocytosis of neutrophils, as well as an altered airway microbiome.

Table 1.

Possible factors accounting for neutrophilic airway inflammation in asthma.

• Corticosteroid treatment causing reduced apoptosis of neutrophils and contributing to Th17-mediated neutrophilic inflammation

• Neutrophilia associated with chronic sinopulmonary infection or bronchiectasis

• Delayed human neutrophil apoptosis in severe asthma due to epithelial growth factor-induced release of mediators with neutrophil chemotactic and anti-apoptotic actions from bronchial epithelial cells

• Impaired macrophage phagocytosis of neutrophils

• Neutrophilia associated with an altered airway microbiome

Clinical phenotypes associated with noneosinophilic inflammation

Noneosinophilic inflammation, either paucigranulocytic or neutrophilic, occurs in a range of clinical phenotypes that account for approximately 50% of never smokers or exsmokers with mild to severe asthma or that have controlled or uncontrolled asthma (Table 2). Noneosinophilic inflammation, with or without neutrophilic inflammation, is commonly found in smokers with asthma [Chalmers et al. 2002; Boulet et al. 2006; Thomson et al. 2013]. A high BMI is associated with noneosinophilic asthma in some people [Haldar et al. 2008], although others have submucosal eosinophilia [Desai et al. 2013]. Approximately two-thirds of cases of occupational asthma due to low molecular weight agents have noneosinophilic inflammation [Anees et al. 2002], which is associated with a poor asthma prognosis [Lemiere et al. 2014]. Nonoccupational-induced asthma that is exacerbated by work exposures is associated with the noneosinophilic phenotype [Lemière et al. 2013]. Additional factors associated with higher neutrophil counts include older age [Brooks et al. 2013], exposure to environmental pollution through living close to car pollution [Wallace et al. 2011], exposure to occupational particulate matter [Simpson et al. 2015] and respiratory infections.

Table 2.

Clinical phenotypes and factors associated with noneosinophilic airway inflammation in asthma.

Mild to severe asthma in nonsmokers or exsmokers (both controlled and uncontrolled)

Smokers with asthma

High basal metabolic index (subgroup)

Occupational asthma (subgroup)

Factors associated with higher neutrophil counts

- Older age

- Exposure to environmental pollution

- Respiratory infections

Biomarkers that can identify noneosinophilic airway inflammation

Are there biomarkers that can identify patients with noneosinophilic airway inflammation? Blood eosinophil numbers are moderately associated with sputum eosinophils [Schleich et al. 2013; Zhang et al. 2014; Wagener et al. 2015]. Using a cut-off for a blood eosinophil count of >0.22 × 10⁹/l [Schleich et al. 2013], >0.26 × 10⁹/l [Zhang et al. 2014] or ⩾0.27 × 10⁹/l [Wagener et al. 2015] accurately predicts sputum eosinophilia. In contrast, another study reported that blood eosinophils had a poor predictive value of 47% for sputum eosinophilia (⩾3% cut-off) although this was better in severe asthma (71%) [Hastie et al. 2013]. In patients with mild to severe asthma, blood eosinophils were reported to be better than serum periostin and exhaled nitric oxide in identifying sputum eosinophilia [Wagener et al. 2015]. Blood neutrophil number has a weak relationship with sputum neutrophil count [Schleich et al. 2013; Zhang et al. 2014] and has a poor predictive value for sputum neutrophilia (64% or 38% for a cut-off of ⩾40% or ⩾61% respectively) [Hastie et al. 2013]. In one study, exhaled nitric oxide predicted inhaled corticosteroid response for airway hyperreactivity in noneosinophilic asthma (area under the curve 0.81), with an optimum cut-off point of 33 ppb [Cowan et al. 2010].

Which inflammatory phenotype to target?

In summary, noneosinophilic airway inflammation is found in approximately 50% of patients with mild to severe asthma. The proportion of this group with neutrophilic inflammation is less certain because of variable cut-off points used in different studies to define neutrophilia. Current symptoms, rate of exacerbations and rate of decline in lung function are generally less severe in noneosinophilic asthma compared to eosinophilic asthma. Noneosinophilic inflammation is associated with an impaired response to inhaled corticosteroids. There is some evidence that neutrophils are activated in the airways of patients with neutrophilic asthma and that biomarkers of systemic inflammation is increased in this group. Neutrophilia in asthma may be due to corticosteroids, associated chronic pulmonary infection, an altered airway microbiome or delayed neutrophil apoptosis, particularly in severe disease. Noneosinophilic asthma and Th2-low asthma may share some common immunopathological features, but further investigation is required. Due to the lack of effective specific therapies targeting noneosinophilic inflammation including neutrophilic inflammation there is currently no definitive evidence for the involvement of these inflammatory phenotypes in chronic asthma. Additional pathways may account for poor asthma control in patients with noneosinophilic asthma including Th1 inflammation, Th17 inflammation, or a combination of Th2 and Th17 inflammation, as well as corticosteroid insensitivity (Figure 1). Recent work suggests a reciprocal relationship between Th2 and Th17 pathways in severe disease and that corticosteroid treatment may contribute to the emergence of a Th17-high profile [Choy et al. 2015]. Noninflammatory mechanisms may also be important in some individuals including airway hyperreactivity and airway remodelling.

Treatments targeting noneosinophilic airway inflammation

Many patients with asthma continue to have poorly controlled disease despite treatment with currently available therapies. There is an unmet need for novel treatments that will impact favourably on clinical outcomes in patients with noneosinophilic inflammation. Nonpharmacological interventions, ‘off-label’ use of licensed drugs, novel small molecules and biologics agents are being investigated as possible treatments of noneosinophilic inflammation in asthma (Table 3 and Figure 2).

Table 3.

Treatments targeting noneosinophilic airway inflammation in asthma.

Nonpharmacological interventions

Avoidance from exposure to environmental and occupational pollutants

Smoking cessation

Dietary supplementation with vitamin D3

‘Off-label’ use of licensed drugs

Macrolides

Statins

Low-dose theophylline

Peroxisome proliferator-activated receptor-γ (PPARγ) agonists

Novel small molecule drugs

Drugs targeting neutrophilic inflammation

C-X-C chemokine receptor (CXCR)2 antagonists

5-lipoxygenase-activating protein (FLAP) inhibitors

Phosphodiesterase (PDE) inhibitors

PDE4 inhibitors

Dual PDE3 and PDE4 inhibitors

Protein kinase inhibitors

p38 mitogen-activated protein kinase (MAPK) inhibitors

Narrow spectrum kinase inhibitors

Tyrosine kinase inhibitors

Phosphoinositide 3 (PI3)-kinase inhibitors

PI3K-δ inhibitors

Dual PI3Kδ/γ inhibitors

Biological agents

Interleukin (IL)-17A receptor blockers

IL-17A blockers

Tumor necrosis factor (TNF)-α receptor blockers

IL-1β monoclonal antibody blockers

Soluble IL-1 receptor monoclonal antibody blockers

IL-6 monoclonal antibody blockers

Figure 2.

Targets and potential therapies for treating noneosinophilic airway inflammation in asthma.

Noneosinophilic airway inflammation is found in approximately 50% of patients with asthma. The proportion of this group with neutrophilic inflammation is less certain because of variable cut-off points used to define neutrophilia. The higher the cut-off value used to define sputum neutrophilia the greater the proportion of patients that are classified as having paucigranulocytic inflammation. Pathways that may account for poor asthma control in patients with noneosinophilic asthma including neutrophilic inflammation, associated inflammatory phenotypes (Th1-high inflammation, Th17-high inflammation, a combination of Th2 and Th17 inflammation, mast cell-induced inflammation, and other inflammatory mechanisms) as well as corticosteroid insensitivity. Noninflammatory mechanisms such as airway hyperreactivity and airway remodelling may be important in causing symptoms in some individuals. Potential treatments targeting specific pathways are listed in the diagram. Novel small molecules targeting neutrophilic inflammation, such as CXCR2 antagonists reduce neutrophils, but do not improve clinical outcomes. Smoking cessation in asthmatic smokers and removal from exposure to occupational agents reduces neutrophilic inflammation. The results of clinical trials of biological agents targeting mediators associated with noneosinophilic inflammation, such as IL-17 and TNF-α are disappointing. Preliminary studies of ‘off-label’ use of licensed drugs suggest that macrolides show efficacy in nonsmokers with noneosinophilic severe asthma and statins, low-dose theophylline and PPARγ agonists may benefit asthmatic smokers with noneosinophilic inflammation and associated corticosteroid insensitivity. Inhaled PDE₄ inhibitors, dual PDE₃ and PDE₄ inhibitors, p38MAPK inhibitors, tyrosine kinase inhibitors and PI3kinase inhibitors are under development and these compounds may be of benefit in treating noneosinophilic inflammation and corticosteroid insensitivity. Long-acting bronchodilators or bronchial thermoplasty are possible treatment options for symptomatic patients with paucigranulocytic inflammation in whom there is no evidence of activated inflammatory pathways or corticosteroid insensitivity that could be targeted by specific therapies.

Abbreviations: CXCR, C-X-C chemokine receptor; FLAP, 5-lipoxygenase-activating protein; IL, interleukin; PDE, phosphodiesterase; PI3K, phosphoinositide 3-kinase; PPARγ,: peroxisome proliferator-activated receptor-γ.

Nonpharmacological interventions

Avoidance from exposure to environmental and occupational pollutants may reduce neutrophilic inflammation in asthma. In a study of smokers with asthma, of which a subgroup quit smoking for 6 weeks, the proportion of sputum neutrophils reduced, corticosteroid sensitivity improved and the FEV₁ increased compared to those who continued to smoke [Chaudhuri et al. 2006]. After cessation of exposure to occupational agents, neutrophilic inflammation reduced in people where their asthma was cured or improved compared to those where there was no improvement [Maghni et al. 2004].

Several clinical trials have examined the effect of dietary supplements of vitamin D in asthma, based on the anti-inflammatory and corticosteroid-enhancing actions of vitamin D [Nanzer et al. 2013; Zhang et al. 2014; Xystrakis et al. 2006]. Two large randomized clinical trials of vitamin D3 supplementation in patients with asthma and vitamin D insufficiency [VIDA and ViDiAs trials), although not selected for specific airway inflammatory cell profiles or corticosteroid insensitivity, reported no improvement in clinical outcomes [Castro et al. 2014; Martineau et al. 2015; Denlinger et al. 2015 ]. Interestingly, vitamin D supplementation reduces eosinophilic inflammation in patients with nonatopic asthma, suggesting that certain inflammatory phenotypes might benefit from vitamin D3 supplementation [De Groot et al. 2015].

‘Off-label’ use of licensed drugs

Several drugs licensed for the treatment of medical conditions other than asthma have been investigated for their efficacy in asthma, including patients with noneosinophilic inflammation. Candidate drugs have been chosen usually because of preclinical evidence of anti-inflammatory effects that might be relevant to treatment of asthma. Some examples are reviewed below.

Macrolides

Macrolides may be of benefit in the treatment of chronic asthma [Reiter et al. 2013], including noneosinophilic asthma [Simpson et al. 2008], although prescribing macrolides as a long-term treatment increases the risk of adverse drug effects and the development of microbial resistance [Cameron et al. 2012]. The mechanism of action of macrolides in the treatment of airway diseases is not known, but could be due to antibacterial or anti-inflammatory actions, which include inhibition of nuclear factor (NF)-κB and other transcription factors as well as reduction in neutrophil migration or function [Culic et al. 2001; Fujitani and Trifilieff, 2003; Simpson et al. 2008; Cameron et al. 2012; Kobayashi et al. 2013]. Macrolides have additional potentially beneficial properties including antiviral actions [Gielen et al. 2010; Schögler et al. 2015] and an ability to restore corticosteroid sensitivity by inhibiting the phosphoinositide 3-kinase (PI3K) pathway and restoring histone deacetylase (HDAC)2 activity [Spahn et al. 2001; Kobayashi et al. 2013; Hao et al. 2015] and by attenuating TNFα and IL-17 immune responses [Essilfie et al. 2015]. Two recent exploratory clinical trials have investigated the effects of macrolides in noneosinophilic asthma. In one trial, smokers with mild to moderate asthma associated with noneosinophilic inflammation were randomized to azithromycin 250 mg per day or placebo [Cameron et al. 2013]. After 12 weeks, treatment with azithromycin was not associated with improvements in morning peak expiratory flow (PEF), ACQ score, asthma quality of life questionnaire (AQLQ) score and methacholine PC₂₀ compared to placebo and did not alter induced sputum differential counts, bacterial load, C. pneumonia, M. pneumoniae seropositivity or upper airway respiratory virus prevalence. In another randomized controlled trial patients with exacerbation-prone severe asthma received low-dose azithromycin or placebo as an add-on treatment to combination therapy of inhaled corticosteroids and long-acting β₂ agonists for 6 months [Brusselle et al. 2013]. The rate of severe exacerbations and lower respiratory tract infections requiring treatment with antibiotics was not reduced by azithromycin. In a predefined subgroup with noneosinophilic severe asthma (blood eosinophilia ⩽200/µl) there was a reduction in the rate of primary endpoints in azithromycin-treated patients [Brusselle et al. 2013]. Azithromycin improved AQLQ scores, but did change ACQ scores or lung function. Based on these findings, further clinical trials of macrolides in noneosinophilic severe asthma are indicated. Novel analogues of macrolides have been developed that have enhanced anti-inflammatory properties than current macrolides, such as solithromycin (CEM-101) [Kobayashi et al. 2013a; Kobayashi et al. 2013b] or that lack antibacterial properties, such as the nonantibiotic azithromycin derivative CSY0073 [Balloy et al. 2014].

Statins

Statins have pleiotropic immunomodulatory actions [Greenwood et al. 2006] that may be of value in the treatment of chronic inflammatory diseases [Greenwood et al. 2006; Hothersall et al. 2006; Yeganeh et al. 2014]. In experimental models of allergic asthma [McKay et al. 2004; Zeki et al. 2009] and tobacco-smoke-induced lung inflammation [Lee et al. 2005; Davis et al. 2013] statins reduce inflammatory pathways potentially relevant to the pathogenesis of asthma and smoke-induced airway diseases. Statins might also restore corticosteroid sensitivity in asthma [Samson et al. 2006; Maneechotesuwan et al. 2010]. Taken together, these findings suggest that statin treatment may have anti-inflammatory effects in people with asthma including smokers with asthma. A randomized double-blind parallel group trial undertaken in 71 smokers with mild to moderate asthma associated with noneosinophilic inflammation compared treatment with atorvastatin 40 mg per day with placebo. After 4 weeks of treatment, inhaled beclometasone at 400 μg per day was added to both treatment arms for a further 4 weeks [Braganza et al. 2011]. At 4 weeks, there was an improvement in ACQ and AQLQ scores with atorvastatin, but not in lung function. There was no significant improvement with atorvastatin and inhaled beclometasone compared to inhaled beclometasone alone in clinical outcome measures at 8 weeks. In a follow-up study the effects of atorvastatin alone and in combination with inhaled corticosteroid was investigated on their ability to suppress the concentration of a range of cytokines, chemokines and growth factors in sputum samples collected during the previous clinical trial [Braganza et al. 2011; Thomson et al. 2015]. Sputum mediator concentrations were not reduced by inhaled beclometasone alone. Atorvastatin significantly reduced sputum concentrations of CCL7, IL-12p70, soluble CD40 ligand (sCD40L), fibroblast growth factor (FGF)-2, CCL4, transforming growth factor alpha (TGF-α) and matrix metalloproteinase (MMP)-8 compared with placebo and, when combined with inhaled beclometasone, reduced sputum concentrations of MMP-8, IL-1β, IL-10, MMP-9, sCD40L, FGF-2, IL-7, granulocyte-colony stimulating factor (G-CSF) and CCL7 compared to ICS alone. Improvements in ACQ or AQLQ scores with atorvastatin and inhaled beclometasone were associated with decreases in G-CSF, IL-7, CCL2 and chemokine (C-X-C motif) ligand (CXCL)8. Interestingly, simvastatin suppresses airway IL-17 and upregulated IL-10 in patients with stable chronic obstructive pulmonary disease (COPD) [Maneechotesuwan et al. 2013]. Taken together, these findings suggest that short-term treatment with atorvastatin alone or in combination with inhaled beclometasone reduces several sputum cytokines, chemokines and growth factor concentrations unresponsive to inhaled corticosteroids alone in asthmatic smokers with noneosinophilic inflammation. There is a need for long-term clinical studies examining effect of statins on exacerbations and airway remodelling in chronic noneosinophilic asthma.

Low-dose theophylline

Low dose theophylline has been shown to restore corticosteroid sensitivity in vitro possibly by increasing HDAC-2 activity, which is suppressed in severe asthma and in smokers with asthma and a similar clinical effect might occur in people with severe disease or who are smokers [Barnes, 2009; To et al. 2010]. Theophylline inhibits oxidative stress-dependent PI3K-δ activation and restores corticosteroid sensitivity in peripheral blood mononuclear cells (PBMCs) from patients with COPD [To et al. 2010]. An exploratory clinical trial examined the effects of low dose theophylline added to inhaled beclometasone compared to inhaled beclometasone alone in smokers with asthma associated with noneosinophilic inflammation [Spears et al. 2009a]. The addition of low dose theophylline to inhaled beclometasone, at a dose titrated to provide a ‘subtherapeutic’ concentration, resulted in increased efficacy as measured by lung function and suggested the restoration of corticosteroid sensitivity in those treated with the combination. Clinical trials to date have not investigated the therapeutic effects of adding low-dose theophylline to patients with severe asthma. A fixed combination of ultra-low dose of theophylline with fluticasone proprionate, SKP-2075 (Skyepharma, London, UK), in a dry powder inhaler is under development for the treatment of COPD. This combination would potentially be of benefit in the treatment of severe asthma and smokers with asthma, possibly in those people with noneosinophilic inflammation.

PPARγ agonist

In preclinical studies peroxisome proliferator-activated receptor-γ (PPARγ) agonists exert anti-inflammatory effects potentially relevant to the treatment of inflammatory airway diseases including asthma and COPD [Spears et al. 2006; Belvisi and Hele, 2008; Seidel et al. 2012; Stephen et al. 2013; Bourke et al. 2014; Lakshmi et al. 2014; Lea et al. 2014; Donovan et al. 2015]. For example, PPARγ agonists reduce eosinophilic and neutrophilic lung infiltration in experimental animal models exposed to allergen or tobacco smoke [Bauer et al. 2010; Lea et al. 2014; Zhao et al. 2014; Morissette et al. 2015]. The oral PPARγ agonist rosiglitazone had a modest effect in attenuating the allergen-induced late asthmatic response [Richards et al. 2010]. A further proof-of-concept study reported that rosiglitazone compared with inhaled beclometasone dipropionate resulted in an improvement in lung function and a borderline reduction in sputum IL-8 concentration in smokers with mild to moderate asthma that was associated with noneosinophilic inflammation [Spears et al. 2009b]. The oral PPARγ agonist pioglitazone is not effective in obese asthmatics [Dixon et al. 2015]. Inhaled PPARγ agonist analogues are under development for the treatment of chronic inflammatory airway diseases and potentially might be of benefit in noneosinophilic asthma.

Novel small molecule drugs

Novel small molecule inhibitors have been developed for treating neutrophilic/noneosinophilic asthma including CXCR2 antagonists, 5-lipoxygenase-activating protein (FLAP) inhibitors, PDE₄ inhibitors, dual PDE₃ and PDE₄ inhibitor and various protein kinase inhibitors.

CXCR2 antagonist

CXCR2 receptors are expressed on neutrophils as well as on airway goblet cells, fibroblasts and airway smooth muscle [Chapman et al. 2009]. Ligands for the CXCR2 receptor include the chemokines CXCL8 (IL-8), growth-related protein (Gro)-α, -β, and -γ (CXCL1–3), epithelial-derived neutrophil attractant-78 (ENA-78; CXCL5), granulocyte chemotactic protein-2 (GCP-2; CXCL6) and neutrophil-activating peptide-2 (NAP-2; CXCL7) [Chapman et al. 2009, Campbell et al. 2013]. Activation of CXCR2 receptors result in neutrophil chemotaxis, proteases production, aairway goblet cell hyperplasia, pulmonary blood vessel angiogenesis, collagen deposition and airway smooth muscle contraction and migration [Chapman et al. 2009]. The effects of CXCR2 antagonists have been studied on airway challenges that induce sputum neutrophilia. The CXCR2 antagonist, AZD8309 inhibits lipopolysaccharide (LPS)-induced airway neutrophilic inflammation in healthy volunteers [Leaker et al. 2013] and the CXCR2 antagonist, SB656933 inhibited ex vivo neutrophil activation and ozone-induced airway inflammation in humans [Lazaar et al. 2011]. The CXCR2 antagonist, SCH527123 inhibits ozone-induced neutrophilia in healthy subjects [Holz et al. 2010]. A randomized, placebo-controlled clinical trial of the CXCR2 antagonist SCH527123 administered for 4 weeks to patients with severe asthma and sputum neutrophils > 40% resulted in a reduction of 36.3% in sputum neutrophil percentage, fewer mild exacerbations and a trend towards improvement in ACQ score [Nair et al. 2012]. A clinical trial of the efficacy and safety of a CXCR2 antagonist AZD5069 in severe, uncontrolled persistent asthma reported that the addition of AZD5069 to combination ICS/long acting β₂-agonist (LABA) treatment did not improve clinical outcomes despite a dose-dependent reduction in blood neutrophil counts [O’Byrne et al. 2015]. A lack of improvement in clinical outcome despite a reduction in sputum neutrophil counts was reported with the CXCR2 antagonist AZD5069 in bronchiectasis [De Soyza et al. 2015]. A recent trial of the CXCR2 antagonist Navarixin (SCH527123) in COPD led to significant improvements in FEV₁ and reduction in sputum neutrophil count, particularly in current smokers with COPD [Rennard et al. 2015]. The CXCR2 antagonist AZD8309 administered for 4 weeks to patients with moderate to severe COPD was well tolerated with no increase in the rate of infections [Kirsten et al. 2015]. A small-molecule oral CXCR2 antagonists Danirixin (GSK1325756) is undergoing a clinical trial in patients with COPD at risk of exacerbations [ClinicalTrials.gov identifier: NCT02130193]. Oral CXCR2 antagonists could potentially cause neutropenia, and the therapeutic index of these compounds requires careful assessment.

FLAP inhibitors

Pro-inflammatory cysteinyl leukotrienes (LTs) are synthesized from arachidonic acid in inflammatory cells by 5-lipoxygenase (LO) and 5-lipoxygenase activating protein (FLAP). FLAP inhibitors such as GSK-2190915 [Evans et al. 2008] prevent the formation of LTB₄, which may be of value in the treatment of neutrophilic asthma. GSK2190915 markedly inhibited ex vivo calcium ionophore stimulated blood LTB₄ formation and urinary leukotriene E₄ (LTE₄) formation [Bain et al. 2013]. Pre-treatment with GSK2190915 reduces the early and late phase response to allergen challenge and results in a significant reduction of sputum LTB₄ levels [Kent et al. 2013]. Despite suppressing the target mediator LTB₄, the FLAP inhibitor GSK2190915 has no short-term effect on sputum cell counts or clinical endpoints in smokers and nonsmokers with asthma associated with neutrophilic inflammation (sputum neutrophilia ⩾ 50% for one sample and >45% for the other), suggesting that LTB₄ suppression alone is inadequate in controlling airway neutrophils in asthma [Chaudhuri et al. 2014]. No active clinical trials of 5-lipoxygenase-activating protein (FLAP) inhibitors in asthma are currently registered on ClinicalTrials.gov.

PDE₄ inhibitors and dual PDE₃ and PDE₄ inhibitors

Phosphodiesterase (PDE)₄ inhibitors have immunomodulatory effects on inflammatory cells potentially relevant to the treatment of asthma [Lipworth, 2005; Page and Spina, 2012; Kim et al. 2015b]. In an allergen challenge study the oral PDE₄ inhibitor roflumilast attenuated the rise in sputum eosinophils and neutrophil numbers after the late asthmatic response [Gauvreau et al. 2011]. High doses of PDE₄ inhibitors may be necessary to treat severe asthma, and gastrointestinal side effects limit their use [Lipworth, 2005; Bateman et al. 2006; Bousquet et al. 2006]. The inhaled administration of PDE₄ inhibitors may improve the therapeutic index of PDE₄ inhibitors [Chapman et al. 2010; Singh et al. 2010; Nials et al. 2011; De Savi et al. 2014; Moretto et al. 2015]. Inhaled PDE₄ inhibitors GSK256066 and CHF6001 both inhibit allergen-induced late asthmatic responses [Singh et al. 2010; Dave et al. 2014] and in patients with moderate COPD, GSK256066 inhaled for 4 weeks was well tolerated although there was no inhibitory effect on sputum and blood inflammatory biomarkers [Watz et al. 2013]. The inhaled dual PDE₃ and PDE₄ inhibitor RPL554 (Verona Pharma, London, UK) has bronchodilator effects and is well tolerated in patients with asthma and COPD [Franciosi et al. 2013]. In healthy subjects, inhaled RPL554 attenuates the neutrophilic response to lipopolysaccharide (LPS) challenge [Franciosi et al. 2013]. RPL554 is under development for the treatment of asthma and COPD [ClinicalTrials.gov identifiers: NCT02427165, NCT02542254].

Protein kinase inhibitors

Protein kinases are involved in cellular signalling of pro-inflammatory cytokines in asthma and the inhibition of these kinases may have a role in the treatment of severe asthma associated with noneosinophilic asthma [Bhavsar et al. 2010; Cohen and Fleischmann, 2010; Hammaker and Firestein, 2010; Chung, 2011; Guntur and Reinero, 2012]. Several p38MAPK inhibitors restore corticosteroid sensitivity in PBMCs from patients with severe asthma [Bhavsar et al. 2010; Mercado et al. 2012] and COPD [Khorasani et al. 2015]. Clinical trials of p38MAPK inhibitors oral losmapimod (GW856553) (GlaxoSmithKline, London, UK), and inhaled AZD7624 are registered for the treatment of COPD [ClinicalTrials.gov identifiers: NCT02299375, NCT02238483], although neither are registered for the treatment of asthma. Interestingly, a post-hoc analysis of a 6-month clinical trial of oral losmapimod (GW856553) in COPD reported a reduction in exacerbations in a subgroup of patients with a blood eosinophil count ⩽2% [Marks-Konczalik et al. 2015], which may suggest a preferentially beneficial effect of p38MAPK inhibitors in noneosinophilic inflammation. Imatinib, a specific c-kit tyrosine kinase inhibitor that attenuates airway hyperresponsiveness, inflammation and remodelling in a murine model of asthma [Berlin and Lukacs, 2005; Rhee et al. 2011] is under development for patients with severe refractory asthma [ClinicalTrials.gov identifier: NCT01097694]. A tyrosine kinase inhibitor masitinib targets c-kit and platelet-derived growth factor (PDGF) receptor improved asthma control in patients with severe corticosteroid-dependent asthma [Humbert et al. 2009] and a further clinical trial is underway in patients with severe asthma treated with oral corticosteroids [ClinicalTrials.gov identifier: NCT01449162]. An alternative therapeutic strategy to silencing c-kit with small interference ribonucleic acid (RNA) has been shown to attenuate inflammation in a murine model of allergic asthma [Wu et al. 2012; Wu et al. 2014]. Clinical trials of protein kinase inhibitors have not been studied in patients with sputum inflammatory subtypes such as noneosinophilic asthma.

PI3kinase inhibitors

Low dose theophylline is thought to act, at least in part, through the inhibition of PI3K [Ito et al. 2007; To et al. 2010]. Preclinical studies suggest that PI3K-δ inhibitors could potentially reverse corticosteroid insensitivity by increasing HDAC2 activity [Marwick et al. 2009; Marwick et al. 2010] and by reversing fungal-induced steroid resistant airway inflammation through modulation of endoplasmic reticulum stress [Lee et al. 2016]. Selective PI3K inhibitors are being developed as novel therapies for the treatment of chronic inflammatory airway diseases. An inhaled PI3Kδ inhibitor GSK2269557 is undergoing several clinical trials in asthma and COPD. PI3K δ and γ isoforms are involved in inflammatory cell recruitment and activation and dual PI3Kδ/γ inhibitors, such as TG100-115 and IPI-145 reduces airway inflammation induced by allergen or cigarette smoke in murine models [Doukas et al. 2009; Winkler et al. 2013] and restored corticosteroid sensitivity in the smoke model [Doukas et al. 2009]. RV1729, a PI3Kδ/γ inhibitor has undergone early stage clinical evaluation in asthma and COPD. SH2-containing inositol-50-phosphatase 1 (SHIP1) is an endogenous inhibitor of the PI3K pathway. A SPIP1 activator AQX-1125 reduced the allergen-induced late asthmatic response with a non-significant trend for a reduction in sputum eosinophils and neutrophils [Leaker et al. 2014]. Further development of AQX-1125 is underway for the treatment of COPD [ClinicalTrials.gov identifier: NCT01954628].

Biological agents

Monoclonal antibody blockers of inflammatory cytokines such as IL-17 and TNF-α that activate receptors on the surface of neutrophils have been investigated as treatments for asthma.

IL-17 blockers

In preclinical studies Th17 cells and IL-17 are implicated in causing neutrophilic inflammation and corticosteroid insensitivity [Shen et al. 2011; Newcomb and Peebles, 2013; Chesné et al. 2014]. IL-17 concentrations and expression are increased in BAL, sputum and bronchial biopsy samples from patients with severe asthma that correlate with sputum neutrophils. Monoclonal inhibitors of IL-17 are in clinical development [Miossec and Kolls, 2012]. Brodalumab is a human monoclonal antibody that binds with high affinity to human IL-17RA, blocking the biologic activity of IL-17A, IL-17F, IL-17A/F heterodimer, and IL-25. A randomized clinical trial of brodalumab in adults with inadequately controlled moderate to severe asthma receiving regular inhaled corticosteroids, but not selected for neutrophilic inflammation, reported no improvement in the primary outcome ACQ score or in lung function and symptom-free days [Busse et al. 2013]. A subgroup with high bronchodilator reversibility demonstrated a borderline improvement in ACQ score. A further clinical trial of brodalumab in inadequately controlled asthma patients with high bronchodilator reversibility was recently terminated due to a lack of observed efficacy in a pre-specified interim analysis [ClinicalTrials.gov identifier: NCT01902290]. The results of a preliminary proof of efficacy study of the IL-17A monoclonal antibody blocker secukinumab (AIN457) in patients with uncontrolled asthma was also recently terminated. The investigators report that further investigations would require changes in study design, the use of different endpoints, a different IL-17 antibody or a different patient population [ClinicalTrials.gov identifier: NCT01478360].

TNF-α blockers

Neutralizing TNFα restores corticosteroid sensitivity in a mouse model of neutrophilic airway inflammation [Dejager et al. 2015]. Several small clinical studies in severe asthma of the soluble TNF-α receptor blocker etanercept reported beneficial effects on clinical outcomes [Howarth et al. 2005; Berry et al. 2006], whereas larger studies with etanercept [Holgate et al. 2011] and the TNF-α receptor blocker golimumab [Wenzel et al. 2009] did not confirm a consistent beneficial clinical effect. When combined with concerns over increased risk of severe infections and malignancies with TNF-α receptor blocker treatment [Wenzel et al. 2009] it is unlikely that this target will be developed further for the treatment of asthma.

Other monoclonal antibodies

Monoclonal antibodies that block IL-1β, for example, canakinumab or block the soluble IL-1 receptor, for example, anakinra [Hernandez et al. 2015] might be of benefit in neutrophilic asthma, although no clinical studies are currently registered. An IL-6 monoclonal antibody blocker toclizumab is licensed for the treatment of rheumatoid arthritis. Toclizumab could potentially be of benefit in neutrophilic asthma although no clinical studies are registered in asthma.

Conclusions and future developments

Noneosinophilic airway inflammation is a term used to describe a subtype of asthma associated with normal numbers of sputum eosinophils. Up to 50% of never smokers or exsmoker patients with stable, mild to severe asthma have noneosinophilic inflammation and this inflammatory phenotype is also found in smokers with asthma, and some patients with a high BMI or occupational asthma. The noneosinophilic phenotype is subdivided into neutrophilic inflammation, when neutrophil numbers are raised above a defined cut-off level or paucigranulocytic inflammation, when both eosinophil and neutrophil numbers are normal. The relative proportion of each subtype is uncertain because of variable cut-off points used to define neutrophilia. The most appropriate value that indicates that neutrophils are activated and contributing to the pathogenic processes in asthma is not certain. The severity of current symptoms are in general similar or slightly better in noneosinophilic or neutrophilic subgroups of asthma compared to eosinophilic subgroups. Sputum eosinophilia is a better predictor of future exacerbations and a greater risk factor for more rapid decline in lung function than sputum neutrophilia. Noneosinophilic inflammation is associated with an impaired therapeutic response to inhaled corticosteroids. Neutrophilic inflammation is associated with activation of the innate immune system in asthma and systemic inflammation. Several mechanisms either alone or in combination could explain raised sputum neutrophil counts in asthma including corticosteroids, associated chronic sinopulmonary infection, delayed human neutrophil apoptosis due to epithelial growth factor, impaired macrophage phagocytosis and an altered airway microbiome. Limited information has been published on the immunopathological characteristics of noneosinophilic inflammation compared with other inflammatory airway phenotypes including Th2-low inflammation, Th17-high inflammation or a combination of Th2/Th17 profiles in asthma. Taken together, the finding suggest that noneosinophilic inflammation and Th2-low inflammation in nonsmokers with asthma share some similar immunopathological features including normal eosinophil numbers, submucosal mast cell numbers and subepithelial basement membrane thickness. Due to the lack of effective specific therapies targeting noneosinophilic inflammation, including neutrophilic inflammation, there is currently no definitive evidence for the involvement of these inflammatory phenotypes in chronic asthma. Additional pathways may account for poor asthma control in patients with noneosinophilic asthma including Th1 inflammation, Th17 inflammation, or a combination of Th2 and Th17 inflammation, as well as corticosteroid insensitivity (Figure 2). The role of corticosteroid treatment in causing neutrophilic and Th17 inflammation in severe asthma requires further investigation. Noninflammatory mechanisms may also be important in some individuals including airway hyperreactivity and airway remodelling.

There is an unmet need for novel treatments that will impact favourably on clinical outcomes in patients with noneosinophilic inflammation. Nonpharmacological interventions, ‘off-label’ use of licensed drugs, novel small molecules and biologics agents are being investigated as possible treatments of noneosinophilic inflammation in asthma. Smoking cessation in smokers with asthma and cessation of exposure to occupational agents are associated with a reduction in neutrophilic inflammation. Preliminary data of studies of ‘off-label’ use of licensed drugs suggest that macrolides show efficacy in nonsmokers with noneosinophilic asthma and statins, low-dose theophylline and PPARγ agonists may be beneficial in asthmatic smokers with noneosinophilic inflammation and corticosteroid insensitivity. Further clinical studies are indicated to confirm these findings and to determine the role of these therapies in the management of severe asthma. Novel small molecules targeting neutrophilic inflammation in asthma such as CXCR2 antagonists reduce neutrophil counts, but do not improve clinical outcomes. A FLAP inhibitor did not reduce neutrophils or improve symptoms. Inhaled PDE4 inhibitors and dual PDE3 and PDE4 inhibitors are potential therapies for neutrophilic asthma and a dual PDE3 and PDE4 inhibitors is under development for the treatment of asthma and COPD. Additional small molecule drugs including p38MAPK inhibitors, tyrosine kinase inhibitors and PI3kinase inhibitors are under development for asthma. The development of biological agents to target noneosinophilic inflammation in asthma has been disappointing to date with the termination of clinical programmes of monoclonal antibodies targeting IL-17 and TNF-α. In the future, the selection of patients with severe asthma and evidence of Th17 high inflammation may be more likely to identify a subpopulation that respond to IL-17 blockers. Long-acting bronchodilators or bronchial thermoplasty are possible treatment options for symptomatic patients with paucigranulocytic inflammation in whom there is no evidence of activated inflammatory pathways or corticosteroid insensitivity that could be targeted by specific therapies.

Greater understanding of the mechanisms of noneosinophilic inflammation in asthma should lead to improved therapies. International collaborative programmes of research investigating pathogenic mechanism of severe asthma have focused mainly on type 2 eosinophilic inflammation. The Unbiased Biomarkers for the Prediction of Respiratory Disease Outcome (U-BOPRED) study [Gaga et al. 2015; Shaw et al. 2015] and the UK Refractory Asthma Stratification Programme (RASP-UK) [Heaney et al. 2015 ] are designed to identify new phenotypes/endotypes and treatment targets and will hopefully identify new approaches for the treatment of patients with noneosinophilic asthma.