A common IL-4 receptor variant promotes asthma severity via a Treg cell GRB2-IL-6-Notch4 circuit

Abstract

Background: The mechanisms by which genetic and environmental factors interact to promote asthma remain unclear. Both the IL-4 receptor alpha chain R576 (IL-4RαR576) variant and Notch4 license asthmatic lung inflammation by allergens and ambient pollutant particles by subverting lung regulatory T (T_reg ) cells in an IL-6-dependent manner.

Objective: We examined the interaction between IL-4RαR576 and Notch4 in promoting asthmatic inflammation.

Methods: Peripheral blood mononuclear cells (PBMCs) of asthmatics were analyzed for T helper type 2 cytokine production and Notch4 expression on T_reg cells as a function of IL4R^R576 allele. The capacity of IL-4RαR576 to upregulate Notch4 expression on T_reg cells to promote severe allergic airway inflammation was further analyzed in genetic mouse models.

Results: Asthmatics carrying the IL4R^R576 allele had increased Notch4 expression on their circulating T_reg cells as a function of disease severity and serum IL-6. Mice harboring the Il4ra^R576 allele exhibited increased Notch4-dependent allergic airway inflammation that was inhibited upon T_reg cell-specific Notch4 deletion or treatment with an anti-Notch4 antibody. Signaling via IL-4RαR576 upregulated the expression in lung T_reg cells of Notch4 and its downstream mediators Yap1 and beta-catenin, leading to exacerbated lung inflammation. This upregulation was dependent on growth factor receptor-bound protein 2 (GRB2) and IL-6 receptor.

Conclusion: These results identify an IL-4RαR576-regulated GRB2-IL-6-Notch4 circuit that promotes asthma severity by subverting lung T_reg cell function.

Keywords: GRB2; Notch4; asthma; interleukin 4 receptor; regulatory T cells.

Fig.1,. Notch4, Chemokine and cytokine profiles of circulating CD4⁺ T cells in asthmatics segregated by the IL4R genotype.

A-B, flow cytometric analysis (A) and cell frequencies (B) of IL-13⁺ and CRTH2 expression on circulating T_reg or T_eff cells (A, B) in healthy controls and subjects with moderate and severe asthma homozygous for the dominant IL4R^Q576 allele or heterozygous or homozygous for the IL4R^R576 allele. C-D, flow cytometric analysis (C) and cell frequencies (D) of IL17⁺ and CCR6⁺ expression on circulating T_reg or T_eff cells (C, D) in healthy controls and subjects with moderate and severe asthma homozygous for the dominant IL4R^Q576R allele or heterozygous or homozygous for the IL4R^R576 allele. (E) Notch4 expression on T_reg cells of healthy controls and mild persistent, moderate persistent and severe persistent asthmatics segregated by IL4R genotype (QQ versus QR and RR). (F) Pearson correlation analysis of Notch4 expression of T_reg cells of asthmatics with IL4R alleles. (G) IL-6 concentrations in the serum of control and asthmatic subjects. (H) Pearson correlation between Notch4 expression of T_reg cells of asthmatic subject with serum IL-6 concentrations. Each symbol represents one patient. Numbers in flow plots indicate percentages. Error bars indicate SEM. Statistical tests: One-way ANOVA with Dunnett’s post hoc analysis *P<0.05, ***P<0.001, ****P<0.0001. simple linear regression analysis (F,H); ***P<0.001, ****P<0.0001.

Fig. 2.. Notch4 expression on lung T_reg cells licenses allergic airway inflammation in Il4Rα^R576 mice.

(A, B and C) AHR in the respective mouse groups in response to methacholine. (D) serum OVA-specific IgE concentrations. (E and F) absolute numbers of lung eosinophils and CD4⁺ T cells. (G) IL-13 and IL-17 expression in lung T_eff cells (H and I) Notch4 expression in T_reg cells of the respective mouse groups. Each symbol represents an independent sample. Numbers in flow plots indicate percentages. Error bars indicate SEM. Statistical tests: two-way ANOVA with Sidak’s post hoc analysis (A-I). *P<0.05, ***P<0.001, ****P<0.0001. Data representative of two or three independent experiments.

Fig. 3.. Anti-Notch4 neutralizing mAb inhibit allergic airway inflammation in Il4Rα^R576 mice.

(A) scheme of antibody therapy (10μg) and airway sensitization and challenge. (B) airway hyperresponsivness in the respective mouse groups in response to methacholine. (C) serum OVA-specific IgE concentrations. (D - F) absolute numbers of lung CD4⁺ T cells, eosinophils and neutrophils. (G) IL-13 and IL-17 expression in lung T_reg cells. (H) IL-13 and IL-17 expression in lung T_eff cells. Each symbol represents an independent sample. Numbers in flow plots indicate percentages. Error bars indicate SEM. Statistical tests: two-way ANOVA with Sidak’s post hoc analysis (B-G). **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 4.. GRB2 drives the super induction of allergic airway inflammation in Il4Ra^R576 mice.

(A and B) airway hyperresponsivness in the respective mouse groups in response to methacholine. (C) peak airway resistance in the respective groups of panels A and B. (D) serum OVA-specific IgE concentrations. (E-F) absolute numbers of lung neutrophils and eosinophils. (G) IL-4 and IL-17 expression in lung T_reg cells (H) IL-4 and IL-17 expression in lung T_eff cells. Each symbol represents an independent sample. Error bars indicate SEM. Statistical tests: two-way ANOVA with Sidak’s post hoc analysis (A-H). **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 5.. GRB2 drives the super induction of Notch4 and downstream molecules expression in Il4Ra^R576 mice.

(A) Notch4 expression in Treg cells in the respective mouse groups in Sham and HDM sensitized and challenged mice. (B) IL-6 expression in Treg cells in the respective mouse groups in Sham and HDM sensitized and challenged mice. (C) Yap1 expression in Treg cells in the respective mouse groups in Sham and HDM sensitized and challenged mice. (D) β-Catenin expression in Treg cells in the respective mouse groups in Sham and HDM sensitized and challenged mice. Each symbol represents an independent sample. Numbers in flow plots indicate percentages. Error bars indicate SEM. Statistical tests: two-way ANOVA with Sidak’s post hoc analysis (A-D). ***P<0.001, ****P<0.0001.