Published online Oct 30, 2023.
https://doi.org/10.4168/aair.2024.16.1.22
ST2-Mediated Neutrophilic Airway Inflammation: A Therapeutic Target for Patients With Uncontrolled Asthma
,1,2
Thi Bich Tra Cao,1,2
Jae-Hyuk Jang,1
Yoo Seob Shin,1,2
Youngwoo Choi,1,3
and Hae-Sim Park1,2
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.
INTRODUCTION
Asthma is a chronic airway inflammatory disease with diverse phenotypes, including eosinophilic asthma (EA) and neutrophilic asthma (NA). Among adult asthmatic patients, 5% to 10% showed less responsiveness to conventional therapy and presented the phenotype of uncontrolled asthma (UA), where neutrophil activation profiles in the airways are commonly found and less sensitive to corticosteroid treatment.1 Neutrophils play an important role as protectors in the battle against various invading pathogens.2 However, many triggers, including bacterial endotoxin, air pollution exposure, and work-related factors, induce neutrophilic airway inflammation in asthmatic patients.3 Moreover, the activated status of neutrophils has been highlighted in the pathogenesis of asthma by producing multiple proinflammatory cytokines, reactive oxygen species (ROS), and neutrophil extracellular traps (NETs).4, 5, 6 High levels of myeloperoxidase (MPO) and NETs were found in patients with severe asthma (SA), and most of them were in uncontrolled status and had negative connections with lung function decline parameters.7, 8, 9 The stimulators and regulators of NET induction, however, were not clearly defined.
Macrophages (Mφ) and innate lymphoid cells (ILCs) are well-known as immune cells that collaborate together in the airways to drive the phenotypes of asthma.10 Indeed, Mφ can differentiate into M1Mφ subsets and produce multiple proinflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-6, IL-1β, IL-12, and IL-23) with the aim of recruiting and activating neutrophils.11 These cytokines then also activate ILCs (ILC1 and ILC3, which produce type 1- and type 3-related cytokines, including interferon-gamma [IFN-γ] and IL-17), resulting in prolonged neutrophilia and steroid resistance in patients with SA. Recently, S100 calcium-binding protein A9 (S100A9), a critical component of NETs, was found to have potential effects on Mφ polarization into M1Mφ subsets, implying the involvement of the neutrophils-NETs-Mφ axis in asthma.6 However, precise processes underlying the interactions between ILCs, neutrophils, and Mφ have yet to be clearly determined.
IL-33 belongs to the IL-1 family members, exerting its biological effects in immune cells through binding to receptors composed of suppression of tumorigenicity 2 (ST2) and IL-1 receptor accessory protein (IL-1RAcP).12 Although this cytokine has been shown to be involved in type 2-high immune responses, emerging evidence has revealed that IL-33 is associated with neutrophil activation.13, 14 Eosinophilia as well as neutrophilia in house dust mite-induced chronic airway inflammation was reduced by anti-IL-33 antibody treatment.15 In addition, 2 randomized clinical studies have demonstrated that anti-IL-33 receptor antibody could decrease asthma exacerbations in patients with type 2-low asthma and UA.16, 17 Therefore, IL-33 and its receptors should be considered a target for managing patients with UA accompanied by neutrophilic airway inflammation.
A previous report demonstrated increased CD66+ neutrophils in patients with UA compared to those with controlled asthma (CA), partly-controlled asthma (PA), or healthy controls (HCs).7 Therefore, the present study was conducted to (1) compare the serum levels of IL-33 (as a versatile cytokine) and soluble ST2 (sST2) in adult asthmatics according to asthma control status, (2) investigate the mechanism of IL-33/ST2-mediated neutrophilic airway inflammation ex vivo/in vitro, and (3) evaluate the effects of anti-IL-33/ST2 antibodies on the activation of immune cells (neutrophils, Mφ, and ILC3).
MATERIALS AND METHODS
Materials
Detailed information on the key materials, isolation kits, enzyme-linked immunosorbent assay (ELISA) kits, antibodies, software, and machine used in this study is described in Supplementary Tables S1, 2, 3, 4, 5, 6. The BCA Protein Assay Kit was used to determine protein concentration in this study. The ELISA kits, western blot, immunofluorescence, and flow cytometry antibodies were used according to the manufacturer’s protocols.
Study subjects and clinical parameters
This study was approved by the Institutional Review Board of Ajou University Hospital (AJIRB-GEN-SMP-13-108 and AJIRB-BMR-SUR-15-498), and all the subjects provided written informed consent. In the present study, we enrolled 104 HCs and 105 asthmatic patients to measure the serum levels of sST2 and IL-33. HC subjects had no history of asthmatic symptoms. All asthmatic subjects had maintained anti-asthmatic medications, including medium- to high-dose inhaled corticosteroid plus long-acting β2-agonist with/without leukotriene receptor antagonists, according to their control status. Patients with autoimmune and comorbid diseases affecting asthma outcomes were excluded.
Diagnosis of asthma and evaluation of asthma control status were determined following the Global Initiative for Asthma guideline.18 Patients with UA were considered to have at least 3 of the 4 uncontrolled features, including daytime symptoms, limitations on daily activities, night-time symptoms, and the requirement for reliever/rescue medication, while those with PA were considered to have 1 or 2 of these features. In addition, asthmatic patients in CA were in stable status without suffering from any asthmatic symptoms for the previous 1 month. SA was defined according to the International Guidelines of the European Respiratory Society/American Thoracic Society.19
Clinical parameter collection
The degree of airway obstruction (forced expiratory volume in 1 second [FEV1]% pred. and mid maximal expiratory flow [MMEF]% pred.) was evaluated using spirometry. To collect peripheral bloods from study subjects, ethylenediaminetetraacetic acid-containing tubes were used. Automated hematology analyzers were used to quantify total eosinophil counts (TEC) in blood from a total white blood cell count. Sputum eosinophils/neutrophils were counted as previously described.20 Briefly, trypan blue dye was used to remove dead cells, and then the total number of cells was determined. The Wright-Giemsa stain was used to determine the differential cell counts. The proportion of eosinophils or neutrophils per 100 leukocytes counted was used to calculate the eosinophil/neutrophil counts in sputum samples. Sputum eosinophilia was defined if sputum eosinophil counts were higher than 3%, while sputum neutrophilia was defined if sputum neutrophil counts were higher than 65%. Atopic status was determined by the skin prick test as previously described.20 Serum total immunoglobulin (Ig) E levels were measured using the ImmunoCAP system. Fraction of exhaled nitric oxide levels were obtained by using NIOX. Serum samples were collected into Vacuette tubes (Greiner Bio-One, Monroe, NC, USA) and stored at −70°C for further analysis. The serum levels of IL-33 and sST2 were measured using ELISA.
Peripheral neutrophil stimulation
Immune cells (neutrophils and monocytes) were isolated using isolation kits and purified by using their expressed CD markers as shown in Supplementary Data S1 and Supplementary Fig. S1. After isolation and purification, peripheral neutrophils were suspended in RMPI-1640 medium supplemented with 2% heat-inactivated fetal bovine serum (FBS) and treated with 100 ng/mL of recombinant human IL-33 or thymic stromal lymphopoietin (TSLP) in a time-dependent manner. The doses of IL-33 were based on the basis of preliminary experiment results showing significant effects on NET formation.21 To test the suppression of peripheral neutrophils by therapeutic agents, cells were preincubated with dexamethasone (Dex; 0, 0.1, 1, and 10 µg/mL) or PD98059 (0, 1, 10, and 100 µM) or SB203580 (0, 1, 10, and 100 µM), or anti-ST2 (0, 0.1, 1, and 10 µg/mL) antibody for 1 hour prior to 100 ng/mL of IL-33 treatment for 10 minutes. ROS levels were measured by 2',7' dichlorofluorescein diacetate assay.
Human Mφ activation
Human classic monocytes were suspended in serum-free RMPI-1640 for 2 hours, followed by culture in medium supplemented with 10% heat-inactivated FBS and treated with 20 ng/mL of granulocyte-Mφ colony-stimulating factor for 7 days. Every 2 days, half of granulocyte-Mφ colony-stimulating factor-supplemented new media was replaced.22 Then, cells were treated with 20 ng/mL of IFN-γ and 100 ng/mL of lipopolysaccharide (LPS) or 100 ng/mL of recombinant human IL-33 for 3 days. In some experiments, cells were pretreated with 1 μg/mL of anti-ST2 antibodies for 30 minutes, followed by 100 ng/mL of IL-33 stimulation.23 Cells were harvested for the measurement of inducible nitric oxide synthase (iNOS), arginase 1, and full-length transmembrane form of ST2 (ST2L) mRNA expression and the evaluation of Mφ markers. The supernatant was harvested for IFN-γ, TNF-α, and IL-6 level measurements by using ELISA.
Human ILC activation
In order to evaluate the interaction between Mφ, neutrophils, and ILCs, Mφ were pretreated with or without 10 μg/mL of NETs isolated from the patients with CA, PA, and UA for 48 hours. Then, ILCs were cocultured with activated Mφ in the presence of a transwell with a pore size of 5 µm for 8 hours (migration assay) and in the presence of a transwell with a pore size of 0.4 µm for 48 hours (ELISA) as shown in Supplementary Fig. S2. Regarding migration assay, ILC migration was evaluated by staining with calcein AM as previously demonstrated.6 Briefly, human ILCs (5 × 104 cells) were seeded on the medium and stained with 2 µM of calcein AM for 30 minutes. Cells were then washed 3 times in their medium before being added onto an upper chamber plate with a pore size of 5.0 µm. NETs were induced and isolated from asthmatic patients (Supplementary Data S1). Then, the lower chamber of the transwell plate was incubated for 8 hours at 37°C with a medium containing NET-primed Mφ. Medium-containing stained cells (100 μL) from the lower chamber were transferred a black-well plate and measured the mean fluorescence intensity at 494 and 517 nm for excitation and emission, respectively.
Establishment of asthma mouse models
All the experimental protocols were approved by the Ajou University Institutional Animal Care and Use Committee (IACUC 2021-0007). In this study, 6-week-old female BALB/c and C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, USA) were used under specific pathogen-free conditions.
To induce EA or NA in mice, mouse models were established as previously described with some modifications (Supplementary Fig. S3).24 For the sensitization procedure, mice were intranasally sensitized with 75 µg of ovalbumin (OVA) plus 10 μg of LPS in saline on days 1, 2, 3, and 14 for the NA group or intraperitoneally sensitized with 75 µg of OVA plus Alum on days 1 and 14 for the EA group. From days 21 to 25, the mice from the EA and NA groups were challenged with 3% OVA or saline for 30 minutes using an ultrasonic nebulizer. For the antibody treatment groups, mice were divided randomly into 4 groups as follows: (1) mice were sensitized with OVA and challenged with saline (normal control [NC] group, n = 10); (2) mice sensitized with OVA plus LPS, challenged with OVA, and treated with normal goat IgG isotype control (NA group, n = 10); (3) mice sensitized with OVA plus LPS, challenged with OVA, and treated with mouse anti-IL-33 antibody (NA + anti-IL-33 group, n = 5); and (4) mice sensitized with OVA plus LPS, challenged with OVA, and treated with mouse anti-ST2 antibody (NA + anti-ST2 group, n = 10). Mice were assayed 24 hours after the last challenge. Total and differential cell counts were evaluated in the bronchoalveolar lavage fluid (BALF) and lung tissues.
Confocal microscopic analysis
Human neutrophils were incubated overnight with anti-MPO and -ST2 or -TSLPR antibodies, whereas human Mφ were stained with anti-ST2, -CD68, and -iNOS antibodies. The lung tissues were deparaffinated in xylene and rehydrated in ethanol in a concentration-dependent manner. The slides were blocked with 5% bovine serum albumin in 10% normal donkey serum at room temperature for 1 hour. The sections were incubated overnight with anti-MPO and -ST2 antibodies for the colocalization of NETs in the peripheral lung. The slides were incubated with Alexa fluor 488 donkey anti-rabbit and/or 594 donkey anti-goat antibodies and/or 633 donkey anti-mouse antibodies for 1 hour. Then, 4',6-diamidino-2-phenylindole (DAPI) was used to stain DNA. Fluorescent images were acquired using confocal laser scanning microscopy (LSM710) at the Three-Dimensional Immune System Imaging Core Facility of Ajou University Medical Center.
Quantitative real-time polymerase chain reaction
The human cell pellet was used to extract RNA by using QIAzol® Lysis Reagent. Total RNA was synthesized for cDNA by the SuperScriptTM First-Strand Synthesis System. Gene expression was evaluated with cDNA as a template using the KAPA SYBR® FAST qPCR Master Mix (2x) kit. The expression levels of target genes were quantitated relative to glyceraldehyde-3-phosphate dehydrogenase or actin. The sequences of the primers are displayed in Supplementary Table S7. All primers were purchased from Bioneer Corporation (Daejeon, Korea).
Flow cytometry
Human Mφ markers: cells were stained with human CD11c and CD206, and then stained with intracellular CD68. M1Mφ was defined as CD68+CD206−CD11c+.25 Human ILC3 markers: ILCs were suspended in their media and rested for 2 hours, and then the suspension cells were harvested and stained with human Lineage, CD45, CD127, CD294, and CD117. ILC3s were defined as SSClowLineage−CD45+CD127+CD294−CD117+.15 Mouse Mφ markers: cells were stained with CD45, F4/80, CD11c, and CD206. M1Mφ was defined as CD45+F4/80+CD206−CD11c+, whereas M2Mφ was categorized as CD45+F4/80+CD206+CD11c−.25 Mouse neutrophil markers: cells were stained with CD45, CD11b, Ly6G, Siglec F, and ST2. Neutrophils were categorized as CD45+CD11b+Ly6G+ cells. Eosinophils were categorized as CD45+CD11b+Ly6G−Siglec F+.26 Mouse ILC markers: CD45+ cells were isolated from single cells in the lung tissues using CD45 MicroBeads, mouse. Then, cells were blocked and stained with a hematopoietic lineage antibody cocktail, CD90.2, CD117, and CD278. ILC3 is categorized as SSClowLineage−CD45+CD90.2+CD278−D117+.15
Statistical analysis
The D’Agostino-Pearson omnibus test was used to check for normal distribution. For categorical variables, the Pearson’s χ2 test was used. The Mann-Whitney U test was used to compare data from 2 groups for nonnormally distributed variables. The one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test was used to compare data from several groups for normally distributed variables, whereas rank-based nonparametric Jonckheere-Terpstra test or nonparametric Kruskal Wallis with Dunn’s post hoc test for nonnormally distributed variables. Correlation data are presented as the Spearman’s rank correlation coefficient r (P value). Statistical analyses were performed using SPSS software version 26.0 (IBM Corp., Armonk, NY, USA). Graphs were drawn by using GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). Illustrative images and abstracts were created with Servier Medical Art (https://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).
RESULTS
Comparisons of serum sST2 levels according to the control status of asthma
The demographic data of the study subjects are described in Table 1. The asthmatic patients showed significantly higher age, atopy prevalence, total IgE, and TEC, but significantly lower FEV1% and MMEF% than the HCs (P < 0.010 for all). Moreover, the levels of serum sST2 and IL-33 were significantly higher in the asthmatics than in the HCs (P < 0.01 and P = 0.021, respectively; Fig. 1A and B). When the asthmatics were classified into 3 groups (CA, PA, and UA) according to their control status, the levels of serum sST2 were significantly higher in the UA than in the CA or PA groups (P = 0.021 for CA and P = 0.048 for PA; Fig. 1C), while no differences were noted in serum IL-33 (P > 0.05 for all; Fig. 1D). In addition, the levels of serum sST2 (but not serum IL-33) showed a negative correlation with FEV1% (r = −0.203, P = 0.038; Fig. 1E and F). This study further classified asthmatics into the sST2-high and sST2-low groups (cutoff value: 15.0 ng/mL), as described in Table 2. As a result, the sST2-high group had a significantly higher prevalence of the UA group (P = 0.003) and SA (P = 0.006) as well as higher levels of serum MPO, IL-8, and S100A9 (P = 0.023, P = 0.025, and P = 0.010, respectively), suggesting that ST2 could be associated with asthma severity and neutrophilic airway inflammation in patients with UA.
Table 1
Demographic data of the study subjects
Fig. 1
*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).
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.
Table 2
Comparison of demographic characteristics between the sST2-high and sST2-low groups
Higher expressions of ST2 on the surface of peripheral neutrophils in asthmatics
As IL-33 and TSLP are key cytokines for immune cell activation, this study investigated their receptors on peripheral neutrophils. As a result, IL-33 receptors (ST2 and IL1RAcP), but not TSLP receptors (TSLPR and IL-7Rα), were commonly expressed on the surfaces of the cells (Supplementary Fig. S4). Moreover, the expressions of ST2 receptors on the neutrophils were significantly higher in the UA group than in the CA and PA groups or the HCs (P = 0.026 and P < 0.001, respectively; Fig. 2A and B). Here, we further investigated the effect of IL-33 on neutrophil activation. IL-33 enhanced the expressions of MPO as well as the phosphorylation of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinase (MAPK) in the cells, while TSLP did not (Fig. 2C, Supplementary Fig. S5). Treatment of anti-ST2 antibody, but not Dex, inhibited MPO expression in neutrophils (Fig. 2D and E). Although PD98059 (ERK inhibitor) and SB203580 (p38 MAPK inhibitor) suppressed ERK and p38 MAPK pathways, they could not reduce MPO expression in the cells (Supplementary Fig. S6). The present study further evaluated neutrophil activation according to asthma control status. As a result, the productions of ROS, MPO, and citrullinated histone H3 were significantly increased by IL-33 in 3 study groups (P < 0.05 for all); the UA group showed a greater increase than the CA or PA groups (P = 0.011 for ROS, P = 0.045 for MPO, and P < 0.001 for citrullinated histone H3; Fig. 3A-C). In addition, IL-33 was able to induce the formation of NETs, which was inhibited by anti-ST2 antibody, but not Dex treatment (Fig. 3D).
Fig. 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).
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.
Fig. 3
*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).
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.
LPS plus IFN-γ-mediated expressions of ST2 on the surface of Mφ in asthmatics
When monocytes-derived Mφ were treated with LPS in the presence of IFN-γ, the cells were markedly polarized into M1Mφ (Fig. 4A). Moreover, M1Mφ showed significantly higher expression of iNOS, CD68, and ST2 as evaluated by immunofluorescent assay (Fig. 4B). The levels of ST2L mRNA were significantly increased in M1Mφ (P < 0.001) and had a significant positive correlation with iNOS mRNA expression (r = 0.742, P < 0.001; Fig. 4C and D). As M1Mφ expressed ST2 on their surface, we further investigated the effects of IL-33 on Mφ polarization and activation. As a result, IL-33-induced expression of iNOS mRNA in Mφ was significantly higher in the UA group than in the PA and CA groups or the HCs (P = 0.003 and P < 0.001, respectively; Fig. 4E). Moreover, anti-ST2 antibody treatment significantly reduced IL-33-mediated M1Mφ polarization (Fig. 4F) as well as proinflammatory cytokine production including TNF-α, IL-6, and IFN-γ (P < 0.001 for all; Fig. 4G), but not by Dex treatment (data not shown), indicating that ST2 may be a key cytokine in activating both neutrophils and Mφ in neutrophilic inflammation in the asthmatic airways.
Fig. 4
*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).
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.
NETs-mediated interactions among neutrophils, Mφ, and ILCs
As Mφ activation has been known to be closely associated with increased numbers of ILC1s and ILC3s, this study investigated the effect of NETs on Mφ and ILC activation.10 When the effects of NETs on Mφ were compared among the 3 study groups and HCs, NET-induced changes in Mφ morphology, Mφ polarization, and IL-6 production were significantly greater in the UA group than in the CA and PA groups or the HCs (P < 0.010 for all; Fig. 5A-C). In addition, we found that the proportion of ILC3s was markedly enhanced in the UA group compared to the CA/PA group, although total numbers of ILCs were similar between the 2 groups (P = 0.002 for ILC3 and P = 0.843 for ILCs; Fig. 5D-F). To evaluate the effects of Mφ on ILC activation in the presence of NETs, the transwell system was used, as depicted in Supplementary Fig. S2. As a result, the NETs-activated Mφ significantly enhanced ILC migration (P < 0.001; Fig. 5G). In addition, these activated Mφ could induce IL-17A/IL-22 (but not IL-5 production) from ILCs (P < 0.010 for all; Fig. 5H-J), implying that NETs may be a key player mediating Mφ and ILC3 activation.
Fig. 5
*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).
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.
The effects of anti-ST2 on neutralization in mice with NA
When mice with NA were treated with anti-ST2 or IL-33 antibody, they significantly reduced the numbers of eosinophils and neutrophils (Fig. 6A) as well as M1Mφ in the BALF of the NA group (P < 0.05 for all; Supplementary Fig. S7A and B). In addition, increased levels of multiple mediators, including S100A9, MPO, and IL-17A, in the BALF were significantly decreased by these 2 antibody treatments in the NA group (P < 0.001 for all; Fig. 6B and C). As ILC3s were known to be the counterparts of M1Mφ in the enhancement of neutrophil activation, this study investigated ILC3 subsets in the lung tissues. As a result, the NA group showed higher numbers of CD117+ILC3s compared to the NC group, in which anti-IL-33 or anti-ST2 antibody treatment significantly decreased these numbers of ILC3s (Fig. 6D). Furthermore, a positive correlation between the numbers of ILC3s and M1Mφ was noted (r = 0.843, P < 0.001; Supplementary Fig. S7C). Finally, these antibodies had an effect on inhibiting inflammatory cell infiltration and mucus production in the lungs (Fig. 6E), suggesting ST2 serves as a novel target for managing neutrophilic airway inflammation in NA (Supplementary Data S2, Supplementary Fig. S3, S8, S9, S10, S11).
Fig. 6
*P < 0.050 and **P < 0.001 were obtained by one-way analysis of variance with Bonferroni’s post hoc test for (A-C).
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.
DISCUSSION
This study demonstrated the significance of IL-33 in neutrophil activation via the ST2 pathway. Here, we showed significantly higher levels of sST2 in the serum of patients with UA as well as the expression of ST2 on human peripheral and mouse lung neutrophils. Moreover, IL-33-mediated NET formation could contribute to Mφ and ILC3 activation ex vivo. Anti-ST2 antibody treatment showed a significant effect on the attenuation of neutrophilic airway inflammation in mice, providing new insight into ST2 as a novel target for managing patients who are poorly controlled by conventional anti-inflammatory treatment.
Previously, serum sST2 has been shown to predict asthma exacerbation and to suppress the expression of type 2 cytokines (IL-4, IL-5, and IL-13) from IL-33-treated splenocytes; the sST2-IL-33 complex could contribute to neutrophil activation to produce several mediators including matrix metallopeptidase 9 and C-X-C motif ligand 1/17.4, 27 Moreover, much evidence has shown that IL-33 induces neutrophil migration and activation in multiple diseases such as Candida albicans-related diseases, mast cell-related diseases, and bronchopulmonary dysplasia.28, 29 The present study found that patients with UA had higher levels of serum sST2 with lower MMEF%/FEV1%, even though all the study subjects had maintained inhaled corticosteroids plus long-acting-beta agonists, and that they poorly responded to current anti-asthmatic medications. Moreover, in vitro and ex vivo studies demonstrated that peripheral neutrophils derived from the UA group had higher level of ST2 expression and ROS/MPO/NETs production, which could be suppressed by anti-ST2 antibody (not by Dex treatment), suggesting that IL-33/ST2-mediated airway inflammation is a potential target for overcoming steroid resistance in poorly controlled asthmatics.
In addition to neutrophil activation, numerous studies have demonstrated that IL-33 enhances the releases of M1Mφ-derived proinflammatory cytokines (TNF-α, IL-6, and IL-1β) in the presence or absence of LPS.30, 31 Consistently, the present study demonstrated that in asthmatic patients, IL-33 elevated iNOS mRNA expression and released proinflammatory cytokines (IFN-γ, TNF-α, and IL-6) by binding to its receptors (ST2) on Mφ, which resulted in type 2-low airway inflammation and neutrophil/Mφ activation. Moreover, the expressions of IFN regulatory factor 5 and CD68 (classic Mφ markers) were significantly increased in patients with NA compared to the other phenotypes (mixed granulocytes and EA) and associated with bacteria exposure and low FEV1%.32, 33 In the present study, iNOS and ST2 mRNA expressions were significantly increased in Mφ in response to M1-skewed stimulators (LPS/IFN-γ); ST2 mRNA expression was positively correlated with iNOS mRNA expressions in Mφ. Taken together, our results suggest that ST2 expressed on Mφ contributes to steroid resistance in UA through inducing Mφ polarization and activation.
To date, airway neutrophilia with high NET formation has been shown to induce airway epithelial cell damage, eosinophil activation, and asthma exacerbation,7, 8, 9 although potential biomarkers and therapeutics targeting NET formation remain unclear.34 Nevertheless, the current study found that Mφ was activated and polarized under the stimulation of NETs isolated from patients with UA, which was consistent with previous reports in patients with Behçet’s disease.35 Mφ stimulation is critical because the interaction between Mφ and ILC3s has been highlighted to contribute to airway inflammation and remodeling in NA.11 In addition, we confirmed the increased number of ILC3s in patients with UA; NET-activated Mφ recruited ILCs and activated ILCs to release IL-17A/IL-22. Collectively, IL-33 plays complicated roles in the regulation of Mφ and ILC3 activation via a NET formation, which is enhanced by ST2 on the surface of neutrophils.
Recently, the role of p38 MAPK pathway has been emphasized in the pathogenesis of asthma associated with eosinophilic and neutrophilic inflammatory conditions.36 Particularly, GATA binding protein 3 translocation to the nucleus is regulated by the phosphorylation of p38 MAPK as the main factor for ILC2 activation and T helper 2 cell differentiation in EA.37, 38 In addition, p38 MAPK could directly contribute to eosinophil survival, cytokine secretion, migration, and biological activities.37 The p38 MAPK has been shown to be closely associated with NA, because it enhances the expression of intercellular adhesion molecule-1 on pulmonary microvascular endothelial cells.39, 40 Moreover, the induction of TNF-α released from natural killer cells was mediated by the phosphorylated p38 MAPK pathway under the stimulation of IL-33 and IL-12.41 The current study suggests that IL-33-mediated phosphorylation of ERK and p38 MAPK may play an important role in neutrophilic inflammation of NA, although ERK and p38 MAPK pathways were not directly linked to expression of neutrophil granule proteins.
Steroids and biologics targeting T2 inflamamation, including anti-IgE and anti-IL-5/IL-5R antibodies, are not suitable for the treatment of NA; further pharmacological and biological interventions are required.42 These patients can be partly alleviated by avoidance of smoking or environmental/ occupational factors.42 There have been numerous studies on potential therapeutics for NA, including (1) chemokine receptor inhibitors (C-X-C chemokine receptor 2 antagonists),43, 44 (2) inhibitors for activated neutrophil-related proinflammatory cytokines (TNF-α, IL-6, IL-17, and IL-1β blockers),45, 46, 47 and (3) inflammatory signaling pathway inhibitors (phosphodiesterase 4 and 5-lipoxygenase-activating protein inhibitors);48 however, these results were inconsistent.49 More recently, astegolimab (anti-ST2 antibody) reduced asthma exacerbation rates in patients with inadequately CA and those with eosinophil-low type.16 Therefore, our results support that anti-ST2 antibody may have potential benefits for managing patients with UA by inhibiting neutrophilic airway inflammation.
Although the present study suggested that IL-33 and ST2 had a crosslinking with UA, some limitations remain unsolved. First, this is an observational single-center study; further replication studies with larger sample sizes are needed to confirm our results. In addition, examination of the outcomes of serum sST2 changes along with inflammatory cell profiles in patients with UA or NA would be valuable in interventional or subsequent studies of anti-ST2 antibody treatment. Secondly, the concentrations of sST2 and IL-33 in the nasal or sputum of asthmatic patients need to be measured, which may provide direct evidence to explain IL-33/ST2 roles in the lungs. Thirdly, the number of asthmatics, particularly those with UA, was not sufficient to categorize into various endotypes (EA, NA, mixed granulocytic asthma, and pauci granulocytic asthma) based on sputum inflammatory cell profiles, which may be associated with different clinical outcomes. Despite these, we provided new insights on IL-33/ST2-mediated neutrophilic airway inflammation in UA as summarized in Fig. 7.
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.
In conclusion, the results of this study suggest that IL-33 could play a pivotal role in neutrophil activation to produce NETs via ST2, leading to M1Mφ and ILC3 stimulation. Therefore, ST2 blockage can be a possible therapeutic option for managing patients with UA accompanied by steroid resistance.
SUPPLEMENTARY MATERIALS
Materials and methodsSupplementary Data S1
ResultsSupplementary Data S2
Key materialsSupplementary Table S1
Isolation kits and recombinant proteinsSupplementary Table S2
Enzyme-linked immunosorbent assay kitsSupplementary Table S3
Antibodies for western blot and immunofluorescenceSupplementary Table S4
Antibodies for flow cytometrySupplementary Table S5
The used machines and software in this studySupplementary Table S6
The sequences of the primersSupplementary Table S7
Purification of human neutrophils and classical monocytes from asthmatic patients. (A) Gating strategy to identify neutrophils (CD45+CD16+CD66b+) before and after magnetic isolation. (B) Gating strategy to identify classical monocytes (CD45+CD14+CD16-) before and after magnetic isolation.Supplementary Fig. S1
Schematic diagram of the coculture and treatment procedure for ILCs.Supplementary Fig. S2
The in vivo treatment protocol in mouse models.Supplementary Fig. S3
Comparison of receptor expression between IL-33 and TSLP in peripheral neutrophils of asthmatics and HCs. (A) The expressions of IL-33 receptors and TSLP receptors on neutrophils evaluated by western blot. (B) The expressions of ST2 and TSLPR in peripheral neutrophils of asthmatics and HCs observed by confocal microscopy.Supplementary Fig. S4
The effects of TSLP on the expression of MPO and the phosphorylation of ERK and p38 MAPK in peripheral neutrophils of asthmatics with extending incubation time.Supplementary Fig. S5
The effects of PD980549 (EKR inhibitor) and SB203580 (p38 inhibitor) on MPO expression and ERK/p38 signaling pathways in peripheral neutrophils of asthmatics.Supplementary Fig. S6
The effects of anti-IL-33 and anti-ST2 antibodies on the activations of macrophages (Mφ). (A) Detection of M1Mφ in the BALF. (B) The numbers of M1Mφ in the lung tissues evaluated by flow cytometry (n = 5 for each group). (C) Correlations between M1Mφ numbers and ILC3 numbers in the lung tissue. Values are expressed as median with interquartile range for (B).Supplementary Fig. S7
Comparisons of IL-33, sST2, and S100A9 levels in the 2 mouse models of asthma (NA vs. EA). (A) Total cell counts, (B) neutrophils, and (C) eosinophils (n = 10 for each group). (D-F) concentrations of sST2, S100A9, and IL-33 in the BALF (n = 10 for each group). (G) Expression of ST2 and MPO in the lung tissues. (H) Confocal microscopic images of the lung tissues stained with DAPI (blue), ST2 (red), and MPO (green). Scale bar, 200 μm. Values are expressed as means ± SD for (A) & (D), and median with interquartile range for (B-C) & (E-F).Supplementary Fig. S8
Comparisons of macrophage (Mφ) subsets between the 2 mouse asthma models (EA and NA). (A) Flow cytometric plots of CD45+F4/80+CD206-CD11c+ M1Mφ and CD45+F4/80+CD206+CD11c- M2Mφ. (B, C) Comparisons of the numbers of M1 and M2 subsets between the EA and NA groups (n = 5 for each group). (D) Comparisons of TNF-α in the BALF between the EA and NA groups (n = 10 for each group). Values are expressed as means ± SD for (B-C) and median with interquartile range for (D).Supplementary Fig. S9
Flow cytometric plots of ST2 expression on CD45+CD11b+Ly6G-Siglec F+ eosinophils and CD45+CD11b+Ly6G+ neutrophils in the 2 mouse models of asthma (NA vs. EA). (A) Comparison of the number of eosinophils and neutrophils between the EA and NA mouse models. (B) Comparisons of ST2 expression on eosinophils and neutrophils between the EA and NA mouse models (n = 5 for each group). Values are expressed as median with interquartile range for (B).Supplementary Fig. S10
Correlations between the levels of sST2 and the numbers of various inflammatory cells. Correlations between sST2 levels in the BALF and (A) ST2+ Eosinophils or (B) ST2+ Neutrophils in the lung tissues. Data are presented as the Pearson correlation coefficient r (P value) for (A, B).Supplementary Fig. S11
Disclosure:There are no financial or other issues that might lead to conflict of interest.
ACKNOWLEDGMENTS
This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A1A01073900) and a grant from the Korean Health Technology R & D Project, Ministry of Health & Welfare, Republic of Korea (HR16C0001).
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Funding Information
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National Research Foundation of Korea
2020R1I1A1A01073900
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Ministry of Health and Welfare
HR16C0001
