T-helper type 2-driven inflammation defines major subphenotypes of asthma

Abstract

Rationale: T-helper type 2 (Th2) inflammation, mediated by IL-4, IL-5, and IL-13, is considered the central molecular mechanism underlying asthma, and Th2 cytokines are emerging therapeutic targets. However, clinical studies increasingly suggest that asthma is heterogeneous.

Objectives: To determine whether this clinical heterogeneity reflects heterogeneity in underlying molecular mechanisms related to Th2 inflammation.

Methods: Using microarray and polymerase chain reaction analyses of airway epithelial brushings from 42 patients with mild-to-moderate asthma and 28 healthy control subjects, we classified subjects with asthma based on high or low expression of IL-13-inducible genes. We then validated this classification and investigated its clinical implications through analyses of cytokine expression in bronchial biopsies, markers of inflammation and remodeling, responsiveness to inhaled corticosteroids, and reproducibility on repeat examination.

Measurements and main results: Gene expression analyses identified two evenly sized and distinct subgroups, "Th2-high" and "Th2-low" asthma (the latter indistinguishable from control subjects). These subgroups differed significantly in expression of IL-5 and IL-13 in bronchial biopsies and in airway hyperresponsiveness, serum IgE, blood and airway eosinophilia, subepithelial fibrosis, and airway mucin gene expression (all P < 0.03). The lung function improvements expected with inhaled corticosteroids were restricted to Th2-high asthma, and Th2 markers were reproducible on repeat evaluation.

Conclusions: Asthma can be divided into at least two distinct molecular phenotypes defined by degree of Th2 inflammation. Th2 cytokines are likely to be a relevant therapeutic target in only a subset of patients with asthma. Furthermore, current models do not adequately explain non-Th2-driven asthma, which represents a significant proportion of patients and responds poorly to current therapies.

Figure 1.

Expression levels of three IL-13 induced genes in the airway define two subgroups of patients with asthma. (A) Relative expression levels (normalized fluorescence units) of periostin (POSTN), chloride channel regulator 1 (CLCA1), and serpin peptidase inhibitor, clade B, member 2 (SERPINB2) in healthy control subjects (n = 28) and in patients with (n = 42). (B) Two-way comparisons of expression levels of POSTN, CLCA1, and SERPINB2 in the 42 patients with asthma. Spearman's rank order correlation (ρ) and P values are indicated. (C) Heatmap depicting unsupervised hierarchical clustering (Euclidean complete) of POSTN, CLCA1, and SERPINB2 expression levels in bronchial epithelium across all subjects at baseline. (D) Mean (±SEM) expression levels of IL-4, IL-5, and IL-13 in bronchial biopsy homogenates obtained contemporaneously with bronchial brushings from a subset of subjects depicted in A through C (cluster 1: 18 patients with T-helper type 2 [Th2]-high asthma, red bars; cluster 2: 14 healthy control subjects, gray bars; and 16 patients with Th2-low asthma, blue bars). Two-way correlations across all subjects between IL-4, IL-5, and IL-13 indicated at right (ρ).

Figure 2.

Clinical features of asthma are present in patients with Th2-high and Th2-low asthma. (A) FEV₁, a measure of airway obstruction. (B) Improvement in FEV₁ after four puffs (360 μg) of albuterol (bronchodilator reversibility testing). (C) Provocative concentration of methacholine required to induce a 20% decline in FEV₁ (PC₂₀), a measure of airway hyperresponsiveness.

Figure 3.

Markers of allergy, eosinophilic inflammation, and airway remodeling are increased in Th2-high asthma. (A) Skin prick test (SPT) results using a panel of 12 aeroallergens. (B) Serum IgE. (C) Peripheral blood eosinophil count. (D) Eosinophils as a percentage of total cells in bronchoalveolar lavage (BAL) fluid. (E) Stereologic measurement of reticular basement membrane thickness on bronchial biopsy, a measure of subepithelial fibrosis. (F) Ratio of MUC5AC to MUC5B expression in epithelial brushings as determined by real-time PCR.

Figure 4.

Alveolar macrophage gene expression in Th2-high and Th2-low asthma. Mean (+SEM) expression levels of 15-lipoxygenase (ALOX15) and TNF-α as determined by real-time PCR (*P < 0.03 for subjects with Th2-high asthma vs. healthy control subjects). n = 15 healthy control subjects (gray bars); n = 5 patients with Th2-low asthma (blue bars); and n = 9 patients with Th2-high asthma (red bars).

Figure 5.

Responsiveness of Th2-high asthma to inhaled steroids and reproducibility of phenotypic markers after placebo in a randomized controlled trial. (A) FEV₁ measured at baseline (week 0), after 4 and 8 weeks on daily fluticasone (500 μg BID), and 1 week after the cessation of fluticasone (week 9). *See Table 2 for number of subjects and P values. There was no significant change in FEV₁ in response to placebo at any time point in either group. (B) Heatmap depicting unsupervised hierarchical clustering of POSTN, CLCA1, and SERPINB2 as in Figure 1C in bronchial epithelium of patients with asthma 1 week after the initiation of fluticasone or placebo treatment (n = 19 receiving fluticasone; 13 receiving placebo). Cluster at baseline for individual subjects in Figure 1C and treatment are indicated below the heatmap.