ST2-Mediated Neutrophilic Airway Inflammation: A Therapeutic Target for Patients With Uncontrolled Asthma

Abstract

Purpose: Suppression of tumorigenicity 2 (ST2) has been proposed as the receptor contributing to neutrophilic inflammation in patients with type 2-low asthma. However, the exact role of ST2 in neutrophil activation remains poorly understood.

Methods: A total of 105 asthmatic patients (classified into 3 groups according to control status: the controlled asthma [CA], partly-controlled asthma [PA], and uncontrolled asthma [UA] groups), and 104 healthy controls were enrolled to compare serum levels of soluble ST2 (sST2) and interleukin (IL)-33. Moreover, the functions of ST2 in neutrophils and macrophages (Mϕ) were evaluated ex vivo and in vivo.

Results: Serum sST2 levels were significantly higher in the UA group than in the CA or PA groups (P < 0.05 for all) with a negative correlation between serum sST2 and forced expiratory volume in 1 second % (r = -0.203, P = 0.038). Significantly higher expression of ST2 receptors on peripheral neutrophils was noted in the UA group than in the PA or CA groups. IL-33 exerted its effects on the production of reactive oxygen species, the formation of extracellular traps from neutrophils, and Mϕ polarization/activation. In neutrophilic asthmatic mice, treatment with anti-ST2 antibody significantly suppressed proinflammatory cytokines (tumor necrosis factor-alpha and IL-17A) as well as the numbers of immune cells (neutrophils, Mϕ, and group 3 innate lymphoid cells) in the lungs.

Conclusions: These results suggest that IL-33 induces the activation of neutrophils and Mϕ via ST2 receptors, leading to neutrophilic airway inflammation and poor control status of asthma. ST2 could be a therapeutic target for neutrophilic airway inflammation in patients with UA.

Keywords: Asthma; IL-33; IL1RL1 protein; cytokines; inflammation; macrophages; neutrophils; therapeutics.

Fig. 1. Increased serum sST2 levels in patients with UA. Comparisons of serum (A) sST2 or (B) IL-33 levels between the HCs and the asthmatic patients. Comparisons of serum (C) sST2 or (D) IL-33 levels according to control status, patients with CA, those with PA, and those with UA. Correlations between serum (E) sST2/(F) IL-33 level and FEV1% value. Correlation data are presented as the Spearman correlation coefficient r (P value) for (E, F). Values are expressed as median with interquartile range for (A-D).

UA, uncontrolled asthma; sST2, soluble suppression of tumorigenesis 2; IL, interleukin; HC, healthy control; PA, partly-controlled asthma; CA, controlled asthma; FEV1, forced expiratory volume in 1 second. ^*P < 0.050 and ^**P < 0.001 were obtained by the Mann-Whitney U test for (A, B) and the nonparametric Jonckheere-Terpstra test for (C, D).

Fig. 2. Expressions of IL-33 receptor (ST2) in peripheral neutrophils of the asthmatics compared to the HCs. (A) Confocal microscopic images of MPO and ST2. Scale bar, 50 µm. (B) The expressions of ST2L mRNA. (C) The effect of IL-33 on the expression of MPO and phosphorylation of ERK/p38 MAPK in neutrophils. The effects of (D) anti-ST2 antibody and (E) Dex on MPO expression and ERK/p38 signal pathways in a time-dependent manner. Values are expressed as means ± standard deviation for (B).

IL, interleukin; HC, healthy control; MPO, myeloperoxidase; ST2, suppression of tumorigenicity 2; ERK, extracellular-signal-regulated kinase; p38 MAPK, p38 mitogen-activated protein kinase; Dex, dexamethasone; CA, controlled asthma; PA, partly-controlled asthma; UA, uncontrolled asthma; DAPI, 4′,6-diamidino-2-phenylindole; GADPH, glyceraldehyde 3-phosphate dehydrogenase; ST2L, full-length transmembrane form or suppression of tumorigenicity 2. ^*P < 0.050, ^**P < 0.010, and ^***P < 0.001 by one-way analysis of variance with Bonferroni’s post hoc test for (B).

Fig. 3. The effects of IL-33 on the activation of peripheral neutrophils in asthmatics. Comparisons of (A) ROS, (B) MPO, and (C) citrullinated histone H3 production among patients with UA, CA/PA, and HCs. (D) The effects of IL-33 and anti-ST2 antibody on neutrophil activation as evaluated by confocal microscopy with DAPI (blue), MPO (green), and NE (yellow) staining. Scale bar, 50 µm. Values are expressed as means ± standard deviation for (A, B), and median with interquartile range for (C).

IL, interleukin; ROS, reactive oxygen species; MPO, myeloperoxidase; UA, uncontrolled asthma; CA, controlled asthma; PA, partly-controlled asthma; HC, healthy control; ST2, suppression of tumorigenicity 2; DAPI, 4',6-diamidino-2-phenylindole; NE, neutrophil elastase; Dex, dexamethasone; PMA, phorbol 12-myristate 13-acetate; PBS, fetal bovine serum. ^*P < 0.050, ^**P < 0.010, and ^***P < 0.001 were obtained by the 2-way analysis of variance with Bonferroni’s post hoc test for (A, B), and Mann-Whitney U test and nonparametric Jonckheere-Terpstra test for (C).

Fig. 4. The effects of IL-33 on the activation and polarization of Mφ in asthmatics. (A) The effects of LPS plus IFN-γ on Mφ polarization as evaluated by flow cytometry. (B) The effects of LPS plus IFN-γ on the expressions of CD68, iNOS, and ST2 in asthmatic Mφ using confocal assay. Scale bar, 50 µm. (C) The effects of LPS plus IFN-γ on the expressions of Arginase 1 or ST2L or iNOS mRNA in Mφ. (D) Correlations between the expressions of ST2L and iNOS mRNA in Mφ. The data are presented as the Spearman correlation coefficient r (P value) for (D). (E) The effects of IL-33 on the expression of iNOS mRNA in Mφ. (F) The effects of IL-33 on the polarization of M (derived from patients with UA) as evaluated by flow cytometry. (G) The effects of IL-33 on proinflammatory cytokine releases (IFN-γ, TNF-α, and IL-6) in asthmatic Mφ. Values are expressed as median with interquartile range for (C, G), and means ± standard deviation for (E).

IL, interleukin; Mφ, macrophages; LPS, lipopolysaccharide; IFN-γ, interferon-gamma; iNOS, inducible nitric oxide synthase; ST2, suppression of tumorigenesis 2; ST2L, full-length transmembrane form of suppression of tumorigenicity 2; TNF-α, tumor necrosis factor alpha; UA, uncontrolled asthma; CA, controlled asthma; PA, partly-controlled asthma; HC, healthy control; PBS, fetal bovine serum. ^*P < 0.050, ^**P < 0.010, and ^***P < 0.001 were obtained by Mann-Whitney U test for (C), 2-way analysis of variance with Bonferroni’s post hoc test for (E), and Kruskal-Wallis test and Dunn’s post hoc test for (G).

Fig. 5. The effects of NETs on the activation and polarization of Mφ. Mφ was stimulated with NETs for 48 hours. (A) Morphological changes in Mφ in response to 10 µg/mL of NETs (upper panel). The expressions of CD11c in human M1Mφ (lower panel). (B) The number of M1Mφ and (C) the levels of IL-6 production from NETs-stimulated or untreated Mφ (n = 5–6 for each group). (D-F) The number of total ILCs and ILC3 in human peripheral blood mononuclear cells was evaluated by flow cytometry. (G) The percentage of migrated ILCs toward NETs-primed Mφ was evaluated by transwell migration assay. The assays were performed in duplicate in the 3 independent experiments (n = 6 for each group). The concentrations of (H) IL-5, (I) IL-17A, and (J) IL-22 released from ILCs in the presence of transwell. The assays were performed in duplicate in 3 independent experiments (n = 6 for each group). Values are expressed as means ± standard deviation for (B-H), and median with interquartile range for (I, J).

NET, neutrophil extracellular trap; Mφ, macrophages; IL, interleukin; ILC, innate lymphoid cell; HC, healthy control; CA, controlled asthma; PA, partly-controlled asthma; UA, uncontrolled asthma. ^*P < 0.010 and ^**P < 0.001 were obtained by the one-way analysis of variance with Bonferroni’s post hoc test for (B-H), and Kruskal-Wallis test and Dunn’s post hoc test for (I, J).

Fig. 6. The effects of anti-IL-33 and anti-ST2 antibody treatment in the mouse model of NA. (A) Changes in the counts of eosinophils and neutrophils in BALF (n = 5–10 for each group). (B, C) Changes in S100A9, MPO, and IL-17A in the BALF (n = 5–10 for each group). (D) Multicolor flow cytometry analysis of innate lymphoid cell populations in the lung tissues. (E) The lung tissues were stained with H&E (left panel) or PAS (right panel). Scale bar, 200 µm. Values are expressed as means ± standard deviation for (A-C).

NA, neutrophilic asthma; BALF, bronchoalveolar lavage fluid; S100A9, S100 calcium-binding protein A9; MPO, myeloperoxidase; IL, interleukin; H&E, hematoxylin and eosin stain; NC, normal control; PAS, periodic acid–Schiff; ST2, suppression of tumorigenesis 2; ILC, innate lymphoid cell. ^*P < 0.050 and ^**P < 0.001 were obtained by one-way analysis of variance with Bonferroni’s post hoc test for (A-C).

Fig. 7. Summary of key findings of IL-33/ST2 in neutrophilic inflammation of uncontrolled asthma. Uncontrolled asthma was characterized by the activation of neutrophils, where serum levels of soluble suppression of tumorigenicity 2 may be potential biomarkers of neutrophilic inflammation. In vitro and ex vivo experiments demonstrated that IL-33 induced neutrophil and Mφ activation as the cells highly expressed IL-33 receptors. These activated cells induced the migration and activation of group 3 ILCs amplifying type 2-low inflammation in uncontrolled asthma. Therefore, blockage of ST2 showed a potential benefit for suppressing neutrophilic inflammation in the airways.

IL, interleukin; Ab, antibody; AEC, airway epithelial cell; IFN-γ, interferon-gamma; ILC, innate lymphoid cell; MPO, myeloperoxidase; Mφ, macrophages; NET, neutrophil extracellular trap; ROS, reactive oxygen species; S100A9, S100 calcium-binding protein A9; ST2, suppression of tumorigenesis 2; TNF-α, tumor necrosis factor alpha; HC, healthy control; CA, controlled asthma; PA, partly-controlled asthma; UA, uncontrolled asthma; sST2, soluble suppression of tumorigenesis 2.