Cluster analysis of sputum cytokine-high profiles reveals diversity in T(h)2-high asthma patients

Abstract

Background: Asthma is characterized by a heterogeneous inflammatory profile and can be subdivided into T(h)2-high and T(h)2-low airway inflammation. Profiling of a broader panel of airway cytokines in large unselected patient cohorts is lacking.

Methods: Patients (n = 205) were defined as being "cytokine-low/high" if sputum mRNA expression of a particular cytokine was outside the respective 10th/90th percentile range of the control group (n = 80). Unsupervised hierarchical clustering was used to determine clusters based on sputum cytokine profiles.

Results: Half of patients (n = 108; 52.6%) had a classical T(h)2-high ("IL-4-, IL-5- and/or IL-13-high") sputum cytokine profile. Unsupervised cluster analysis revealed 5 clusters. Patients with an "IL-4- and/or IL-13-high" pattern surprisingly did not cluster but were equally distributed among the 5 clusters. Patients with an "IL-5-, IL-17A-/F- and IL-25- high" profile were restricted to cluster 1 (n = 24) with increased sputum eosinophil as well as neutrophil counts and poor lung function parameters at baseline and 2 years later. Four other clusters were identified: "IL-5-high or IL-10-high" (n = 16), "IL-6-high" (n = 8), "IL-22-high" (n = 25). Cluster 5 (n = 132) consists of patients without "cytokine-high" pattern or patients with only high IL-4 and/or IL-13.

Conclusion: We identified 5 unique asthma molecular phenotypes by biological clustering. Type 2 cytokines cluster with non-type 2 cytokines in 4 out of 5 clusters. Unsupervised analysis thus not supports a priori type 2 versus non-type 2 molecular phenotypes. www.clinicaltrials.gov NCT01224938. Registered 18 October 2010.

Keywords: Airway inflammation; Endotype; Phenotype; Precision medicine; Type 2 inflammation.

Figures

Fig. 1

Absolute sputum cytokine levels among different clusters of asthmatics. Patients were clustered based on their sputum cytokine-high or cytokine-low profile. Asthmatics are divided into 5 clusters: cluster 1: n = 24, IL-5-high and IL-17 F-high; cluster 2: n = 15, IL-5-high and IL-17 F-low; cluster 3: n = 8, IL-6-high; cluster 25: n = 15, IL-22-high; cluster 5: n = 132. Absolute sputum cytokine levels were shown as 10-90th percentile box and whiskers plots. The dotted line represents the 10th or 90th percentile value of control individuals

Fig. 2

Lung function and airway inflammatory parameters. Asthmatics are divided into 5 clusters: cluster 1: n = 24, IL-5-high and IL-17 F-high; cluster 2: n = 15, IL-5-high and IL-17 F-low; cluster 3: n = 8, IL-6-high; cluster 25: n = 15, IL-22-high; cluster 5: n = 132. Data are represented as mean ± standard deviation (a-c) or median ± interquartile range (d-f). Data are compared between the 5 clusters by Kruskal-Wallis and Dunn’s Multiple comparison test (∞: p < 0.05). Data of each cluster was compared to the mean (a-c) or median (d-f) of the total group (*: p < 0.05, **:p < 0.01; ***:p < 0.001)

Fig. 3

Decision tree with patient clusters of all asthmatics. Patients with an “IL-17 F-high and IL5-high” profile, irrespective of expression of other cytokines, were labelled as cluster 1 (n = 24). Patients with an “IL-5-high or IL-10-high” but not an “IL-17 F-high” profile, were identified as cluster 2 (n = 16). In the next step, patients who had normal levels of the previous cytokines but were “IL-22-high”, were assigned to cluster 3 (n = 25). Patients with an “IL-6-high profile” were labelled as cluster 4 (n = 8). All other patients were grouped in cluster 5 (n = 132). *: This group consists of patients with normal levels of the previous cytokines with or without an “IL-1β- or TNF-low” profile (n = 49 and n = 83, respectively)

Fig. 4

Absolute sputum cytokine levels among different clusters of steroid-naive asthmatics. Patients were clustered based on their sputum cytokine-high or cytokine-low profile. Absolute sputum cytokine levels were shown as 10–90th percentile box and whiskers plots. The dotted line represents the 10th or 90th percentile value of control individuals

Fig. 5

Lung function and airway inflammatory parameters of steroid-naive asthmatics. Steroid-naive asthmatics are divided into 6 clusters: cluster I: n = 2, IL-5-high and IL-17 F-high; cluster II: n = 2, IL-5-high or IL-10-high and IL-17 F-low; cluster III: n = 5, IL-4-high; cluster IV: n = 10, IL-4-high and IL-13-high; cluster V: n = 3, IL-22-high; cluster VI: n = 20. Data are represented as mean ± standard deviation (a-b) or median ± interquartile range (c-e). FEV1% predicted levels of each cluster were compared to the mean of the total group (*: p < 0.05)