Basic research has established IL-13 and related Th2 cytokines as central regulators of allergic inflammation and many of the pathophysiologic changes associated with asthma (1
). Our work challenges these current concepts of asthma pathogenesis by showing that a gene signature for Th2-driven inflammation in airway epithelial cells is prominent in only half of patients with asthma; non–Th2-driven mechanisms therefore operate in the remaining half. This finding leads us to propose that asthma can be divided broadly into Th2-high and Th2-low molecular phenotypes. We validate this classification scheme through confirmatory analyses of gene expression in another tissue compartment (bronchial biopsies); through analysis of reproducibility on repeat examination; and through comprehensive characterization of the distinct clinical, inflammatory, pathological, and treatment-related characteristics of these two molecular phenotypes of asthma. These findings are timely in that they provide a mechanistic framework for the emerging clinical observation that asthma is a complex and heterogeneous disease (26
Molecular phenotyping of asthma based on Th2 inflammation has important therapeutic implications. First, airway obstruction improves with inhaled steroids in the Th2-high sub-group, whereas it does not in the Th2-low subgroup. These findings identify subjects with Th2-low asthma as a subgroup with a clinical need that is poorly met by current therapies. Second, blockade of IL-13 and related Th2 cytokines is under active clinical development as a therapeutic strategy in asthma (5
). Our data suggest that clinical response to these therapies may be limited to the specific subgroup of patients with Th2-high asthma. It has been noted that patient-specific treatments for asthma depend on the development of discriminatory markers of distinct asthma subtypes (27
), and the markers of Th2-high asthma identified here should have direct application in patient selection for clinical trials. Whether patient heterogeneity has influenced recently completed clinical trials of IL-13 blockade (5
) is uncertain, but these data provide a basis for investigating that possibility.
Our data suggest that Th2-driven inflammation is the molecular mechanism underlying the cellular phenotype of asthma known as “eosinophilic asthma” (28
) because of the airway eosinophilia that we demonstrate in Th2-high asthma and because eosinophilic asthma and Th2-high asthma are characterized by subepithelial fibrosis (8
), ALOX15 production by alveolar macrophages (24
), and lung function responses to inhaled corticosteroids (30
). In addition to these recognized features of eosinophilic asthma, we have identified further clinical features of Th2-high asthma, including increased mucin stores, which occur because of increases in MUC2 and MUC5AC and despite a marked decrease in MUC5B expression. We found increased expression of TNF-α in macrophages in Th2-high asthma. TNF-α is not considered a Th2 cytokine, but it has been previously associated with severe asthma (32
). We predict that all of these features will also be found in eosinophilic asthma because it is likely that IL-5 is a major contributor to the airway and systemic eosinophilia in Th2-high asthma, and we found that IL-5 expression is significantly co-regulated with IL-13 expression and is increased in Th2-high asthma (see
). IL-5 is a major stimulus of eosinophil differentiation, recruitment, activation, and survival (33
). Two recent clinical trials of mepolizumab in patients with relatively severe asthma highlight the importance of IL-5 and eosinophilia in asthma exacerbations (34
). Despite reducing the frequency of asthma exacerbations, these studies did not show a reduction in airway hyperresponsiveness, suggesting that IL-5 blockade may leave residual tissue inflammation, that other Th2 cytokines (e.g., IL-13) drive airway hyperresponsiveness, or that airway hyperresponsiveness is an epiphenomenon. Other studies indicate that IL-13 can strongly induce the expression of eosinophil chemoattractants such as CCL11, CCL24, and CCL26 in the airway (36
) and thus represents an IL-5–independent pathway that promotes eosinophil infiltration, activation, and survival in the airways. Residual IL-13 activity may therefore explain the incomplete tissue depletion of eosinophils observed in previous clinical trials of IL-5 blockade in asthma (37
Molecular phenotyping of asthma has some advantages over phenotyping by sputum cell counts or measures of airway pathology. First, underlying molecular mechanisms likely drive the inflammatory and pathological processes and thus represent proximal targets for directed therapy. Second, phenotyping by gene expression identifies the genes that may contribute to this complex disease based on DNA sequence variation and will help reduce phenotypic heterogeneity in genetic studies. Third, these gene expression array data facilitate the identification of secreted proteins that mark the phenotype and may be developed into diagnostic tests.
One strength of our approach to molecular phenotyping is the use of multiple sample types to provide a broad view of gene expression in the airway. Our preparations of epithelial cells and alveolar macrophages are valuable for gene expression profiling because they represent relatively homogeneous populations of cells. On the other hand, bronchial biopsies are valuable because the mix of cell types they contain (including inflammatory cells) permits direct measurement of Th1 and Th2 cytokines. However, because of the cell type heterogeneity inherent to bronchial biopsies, the cellular source of these cytokines remains uncertain.
Finally, these data reveal that a significant percentage of patients with asthma have a Th2-low phenotype that manifests clinical features of asthma, airway obstruction, airway hyperresponsiveness, and bronchodilator reversibility despite a paucity of Th2-driven inflammation. The causes of Th2-low asthma remain obscure, but possibilities include neutrophilic inflammation, IL-17–driven inflammation, intrinsic defects in barrier function and chronic subclinical infection by viruses, and atypical intracellular bacteria. Whatever the mechanism, our data suggest that it does not relate to uncontrolled allergic inflammation but rather to alternative mechanisms that are not steroid responsive. Future work should be directed at identifying noninvasive biomarkers that correlate with these molecular phenotypes in the airway and at developing a deeper understanding of the pathophysiologic mechanisms underlying asthma that is not driven by Th2 inflammation.
There are limitations inherent to our study design. First, our subjects were not treated with inhaled or oral steroids for at least 4 weeks before enrollment, and the use of leukotriene antagonists was excluded. Although these steps facilitated identification of this Th2 signature in the absence of the confounding effects of antiinflammatory medications, these inclusion/exclusion criteria limit the generalizability of the study to patients with more severe asthma. Whether these markers can be used to detect Th2-driven inflammation in patients who are poorly controlled despite corticosteroid treatment remains to be determined. Second, repeat bronchoscopy was performed 1 week after initiation of treatment, which was continued for a total of 8 weeks. Thus, the timing of the second bronchoscopy may underestimate the extent to which inhaled steroids influence inflammation and our molecular signature. Third, we excluded healthy control subjects who had a history of allergic rhinitis. Thus, it is possible that our Th2 signature may also be detectable in the upper or lower airway of atopic nonasthmatic subjects. Finally, our study design does not definitively rule out the possibility that the Th2-low phenotype in part represents patients with well-controlled asthma. However, the majority of our subjects had not been using inhaled steroids for a long period before enrollment (i.e., we did not stop the use of inhaled steroids in recruitment of our subjects but rather sought patients who were not using inhaled steroids chronically of their own accord), minimizing the effect of prior steroid therapy in our analyses. Furthermore, patients with Th2-high and Th2-low asthma did not significantly differ with regard to airway obstruction or reversibility at baseline in this study, which lends further credence to the notion that the severity of their disease was comparable and not due to differences in asthma control.