Plasma proteins elevated in severe asthma despite oral steroid use and unrelated to Type-2 inflammation

Abstract

Rationale: Asthma phenotyping requires novel biomarker discovery.

Objectives: To identify plasma biomarkers associated with asthma phenotypes by application of a new proteomic panel to samples from two well-characterised cohorts of severe (SA) and mild-to-moderate (MMA) asthmatics, COPD subjects and healthy controls (HCs).

Methods: An antibody-based array targeting 177 proteins predominantly involved in pathways relevant to inflammation, lipid metabolism, signal transduction and extracellular matrix was applied to plasma from 525 asthmatics and HCs in the U-BIOPRED cohort, and 142 subjects with asthma and COPD from the validation cohort BIOAIR. Effects of oral corticosteroids (OCS) were determined by a 2-week, placebo-controlled OCS trial in BIOAIR, and confirmed by relation to objective OCS measures in U-BIOPRED.

Results: In U-BIOPRED, 110 proteins were significantly different, mostly elevated, in SA compared to MMA and HCs. 10 proteins were elevated in SA versus MMA in both U-BIOPRED and BIOAIR (alpha-1-antichymotrypsin, apolipoprotein-E, complement component 9, complement factor I, macrophage inflammatory protein-3, interleukin-6, sphingomyelin phosphodiesterase 3, TNF receptor superfamily member 11a, transforming growth factor-β and glutathione S-transferase). OCS treatment decreased most proteins, yet differences between SA and MMA remained following correction for OCS use. Consensus clustering of U-BIOPRED protein data yielded six clusters associated with asthma control, quality of life, blood neutrophils, high-sensitivity C-reactive protein and body mass index, but not Type-2 inflammatory biomarkers. The mast cell specific enzyme carboxypeptidase A3 was one major contributor to cluster differentiation.

Conclusions: The plasma proteomic panel revealed previously unexplored yet potentially useful Type-2-independent biomarkers and validated several proteins with established involvement in the pathophysiology of SA.

FIGURE 1

Study overview. Two independent cohorts, U-BIOPRED and BIOAIR, were investigated in this study. In a first screening, the U-BIOPRED cohort including 525 baseline plasma samples from 525 subjects was profiled using antibody bead arrays detecting 177 proteins with 377 antibodies. In the validation stage, the same array was used to profile the BIOAIR cohort comprising 351 plasma samples from 142 subjects. The BIOAIR cohort included a double-blind placebo-controlled oral corticosteroid intervention trial where the placebo group received additional open steroid treatment. These samples were used to study the influence of steroids on plasma protein levels. Asthmatic subjects from the U-BIOPRED cohort were used to identify potential phenotypes using consensus clustering of protein profiles. COPD: chronic obstructive pulmonary disease; HC: healthy non-smoking controls; MMA: non-smokers with mild-to-moderate asthma; SAn: non-smokers with severe asthma; SAs/ex: smokers or ex-smokers with severe asthma.

FIGURE 2

a) Volcano plots of pairwise group comparisons in U-BIOPRED highlight multiple proteins elevated in subjects with severe asthma, but not in mild-to-moderate. Each dot represents a protein measured by the antibody array. Dashed lines represent adjusted p-values <0.05. Highlighted in red are proteins significantly different in the respective pairwise group comparison as well as in the multiple group comparison (i.e. any of the 110 proteins). If multiple antibodies for the same target protein were significant, they all needed to show the same sign of log2 fold change. b) Group comparisons in U-BIOPRED when limited to subjects where oral corticosteroid (OCS) use was not reported and confirmed negative by urinary analysis (non-smokers with severe asthma (SAn) n=103, smokers or ex-smokers with severe asthma (SAs/ex) n=42, non-smokers with mild-to-moderate asthma (MMA) n=63, healthy control (HC) n=90). Highlighted in red are the proteins found to be significantly different in the respective pairwise group comparison based on all subjects (i.e. proteins that were significantly different in the respective comparisons in figure 2a). The majority of the proteins were still significantly different, seen by enrichment of red circles above the p-value threshold. (c) Analysis of steroid effect in U-BIOPRED. SAn and SAs/ex stratified by reported OCS use and OCS metabolite detection. OCS use defined as prescribed and confirmed positive in urine (SAn n=51, SAs/ex n=21) and no OCS defined as not prescribed and confirmed negative in urine (SAn n=103, SAs/ex n=42).

FIGURE 3

Proteins validated in two independent cohorts. Boxplots of the 10 proteins showing significantly different plasma levels between non-smokers with severe asthma (SAn) and non-smokers with mild-to-moderate asthma (MMA) in both studied cohorts (adjusted p<0.05). The screening cohort U-BIOPRED and the validation cohort BIOAIR are shown side by side for each protein. COPD: chronic obstructive pulmonary disease; HC: healthy non-smoking control; MMA: non-smokers with mild-to-moderate asthma; SAn: non-smokers with severe asthma; SERPINA3: alpha-1-antichymotrypsin; APOE: apolipoprotein E; C9: complement component 9; CCL23: macrophage inflammatory protein 3; CFI: complement factor I; IL6: interleukin-6, SMPD3: sphingomyelin phosphodiesterase 3; TNFRSF11A: receptor activator of nuclear factor kappa B (RANK); TGF-β1: transforming growth factor beta-1; GSTP1: glutathione S-transferase P.

FIGURE 4

Influence of oral corticosteroids. a) Volcano plots of comparisons in BIOAIR show that multiple proteins were affected by the steroid treatment, with the majority being decreased. Each dot represents a protein measured by the antibody array, with significantly changing proteins highlighted in red (above the dashed lines representing adjusted p-values <0.05). The two most significant proteins on the decreasing and increasing side are labelled. Fold change calculated as log2 of the median of individual ratios (post/pre). b) List of proteins where the signal of at least one of the multiple antibodies targeting that protein in the array was affected by oral corticosteroid (OCS). Decreasing levels indicated by no mark and increasing levels marked with (+). Mixed directions provided by multiple antibodies are marked with (*) and mixed directions of effect in different subject groups are marked with (−,+). c) The plasma levels of apolipoprotein E (APOE) and alpha 1-antichymotrypsin (SERPINA3) increased after OCS in BIOAIR subject groups. In U-BIOPRED, APOE and SERPINA3 were associated with the severity of asthma as well as with the use of OCS among the severe asthmatics. Adjusted Wilcoxon signed-rank (BIOAIR) and rank-sum test (U-BIOPRED) p-values: *: p<0.05, **: p<0.01, ***: p<0.001. COPD: chronic obstructive pulmonary disease; HC: healthy non-smoking control; MMA: non-smokers with mild-to-moderate asthma; SAn: non-smokers with severe asthma. Full definitions of each protein name are listed in table S2.

FIGURE 5

Phenotypes of asthma. a) Six clusters were identified in a consensus clustering of asthmatic subjects (n=434, mild-to-moderate or severe asthma) from the U-BIOPRED cohort. The clustering was driven by the profiles of 110 proteins (139 antibodies). b) The three proteins CPA3 (carboxypeptidase A3), TRIM33 (tripartite motif containing protein 33) and TRAF3IP2 (TRAF3 interacting protein 2) with the greatest importance for cluster classification (for ranked importance of all proteins, see figure S3) and a selection of variables with a significant association with clusters. Boxes show the first quartile, median and third quartile. p-values are from the Kruskal–Wallis test. ACQ-7: Asthma Control Questionnaire-average of seven; AQLQ: Asthma Quality of Life Questionnaire–average; BMI: body mass index; FEV₁: forced expiratory volume in 1 s; HC: non-smoking healthy controls; hsCRP: high-sensitivity C-reactive protein; tetranor-PGDM; 9α-hydroxy-11,15-dioxo-13,14-dihydro-2,3,4,5-tetranor-prostan-1,20-dioic acid.