Improvement of glucocorticoid sensitivity and attenuation of pulmonary allergic reactions by exogenous supplementation with betaine in HDM and LPS-induced allergic mouse model

Abstract

Background: Childhood asthma is a heterogeneous disease that exhibits different characteristics and varying severity; however, the metabolite alterations underlying the difference in asthma severity, especially in severe asthma, are not well understood. The aim of this study was to identify the plasma metabolic profile of children with different asthma severity and explore the potential intervention targets in severe asthma and glucocorticoid resistance.

Methods: Untargeted liquid chromatography mass spectrometry was utilized to analyze plasma metabolites in 54 children with mild-to-moderate asthma, 50 children with severe asthma and 39 healthy controls. Multivariate statistical analyses were used to explore plasma metabolic alterations that were strongly associated with asthma severity. Meanwhile, the severe allergic airway inflammation mice with glucocorticoid resistance were constructed to validate the potential therapeutic capacity of metabolites.

Results: The plasma metabolic profiles of children with mild to moderate asthma and severe asthma exhibited significant alterations. The distinct plasma metabolite shifts were accompanied by functional alterations in lipid metabolism, particularly choline metabolism, glycerophospholipids and sphingolipid metabolism. 11-cis-retinol, LysoPC (20:4 [8Z,11Z,14Z,17Z]), and glycerophosphatidylcholine were associated with exacerbated airway inflammation and lung function. Furthermore, 2-Hydroxyestradiol, LysoPC (18:3 [6Z,9Z,12Z]), zeaxanthin, and betaine were shifted exclusively in the severe asthma group and may serve as potential biomarkers. Subsequent in vivo studies demonstrated that betaine supplementation partially improved glucocorticoid resistance.

Conclusions: Overall, children with different asthma severity displayed distinct plasma metabolic patterns. These may contribute to the difference in response to glucocorticoids in childhood asthma and could be potential targets and interventions.

Keywords: betaine; choline metabolism; glucocorticoid resistance; severe asthma; therapeutic targets.

FIGURE 1

Plasma metabolomic features in children with different asthma severity. (A) PLS‐DA score plot of children with different asthma severity and healthy controls. (B) PLS‐DA score plot between children with severe asthma and mild‐to‐moderate asthma. (C–E) Representative heatmap of significantly altered metabolites in children with different asthma severity and healthy controls. VIP > 1 and p‐value < 0.05. PLS‐DA, partial least‐squares‐discriminant analysis.

FIGURE 2

Analysis of metabolic pathways in children with different asthma severity. (A) Bubble map of differential metabolite enrichment pathways between mild‐to‐moderate asthma and healthy controls. (B) Bubble map of differential metabolite enrichment pathways between severe asthma and healthy controls. (C) Bubble map of differential metabolite enrichment pathways between mild‐to‐moderate asthma and severe asthma. The enrichment factor is the ratio of metabolites with significant differences in the pathway to the total number of metabolites. A larger enrichment factor indicates a higher enrichment. The color gradient from green to red represents a sequential decrease in p‐value.

FIGURE 3

Identification of plasma metabolites associated with asthma severity in children. (A) Venn diagram displays the number of differentially expressed metabolites between the three groups. (B) Hierarchical clustering analysis of the metabolites shared by the three groups. (C) Box plot of the relative abundance of metabolites shared by the three groups. (D) Heat map of the correlation intensity between trends in metabolites with trends in clinical indicators in children with different asthma severity. (E–H) Violin plots of the relative expression of four differential metabolites that were specific in severe asthma. (I) Heat map of the correlation intensity between the four plasma metabolites and clinical indicators in severe asthma children. (J) ROC curves analysis of the four metabolites for discriminating severe asthma patients from mild‐to‐moderate asthma children and healthy children. ROC, receiver operating characteristic.

FIGURE 4

Exacerbated airway inflammation and steroid resistance in HDM and LPS induced mice. (A) Schematic diagram of the experimental design. (B) Total and differential (C) BALF cell numbers in mice with different treatments detected by flow cytometry. (D) Representative images of HE‐stained slides (Scale bar = 400 μm), and PAS‐stained slides for mucus (purple stain) in the airway (Scale bar = 100 μm). Immunohistochemistry for Ly6G to identify neutrophils in lung tissue (Scale bar = 400 μm). Inflammation score and PAS score. Data are shown as means ± SEM (n = 4‐6 mice/group) from 2 to 3 independent experiments. BALF, bronchoalveolar lavage fluid; HDM, house dust mite; HE, hematoxylin and eosin; LPS, lipopolysaccharide; PAS, periodic acid‐Schiff; ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 5

Exogenous betaine supplementation enhances glucocorticoid sensitivity in severe asthma. (A) Schematic diagram of the experimental design of exogenous betaine supplementation for the treatment of mice with severe asthma. (B) Total BALF cell numbers and differential (C) BALF cell percent or numbers in mice with different treatments detected by flow cytometry. (D) Representative images of HE‐stained slides (Scale bar = 400 μm), and PAS‐stained slides for mucus (purple stain) in the airway (Scale bar = 100 μm). Immunohistochemistry for Ly6G to identify neutrophils in lung tissue (Scale bar = 400 μm). Inflammation and PAS scores were quantified. (E) Detection of serum Ig E expression by ELISA in mice. (F) Assessment of methacholine‐induced airway hyperresponsiveness (AHR) in mice. Central airway resistance (Newtonian resistance, Rn) values are shown. Data are shown as means ± SEM (n = 4–6 mice/group) from 2 to 3 independent experiments. BALF, bronchoalveolar lavage fluid; PAS, periodic acid‐Schiff; ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.