Contribution of monocyte and macrophage extracellular traps to neutrophilic airway inflammation in severe asthma

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Original Article

Contribution of monocyte and macrophage extracellular traps to neutrophilic airway in ﬂ ammation in severe asthma

Quang Luu Quoc

^a^,^b

, Thi Bich Tra Cao

^a^,^b

, Ji-Young Moon

^a

, Jae-Hyuk Jang

^a

, Yoo Seob Shin

^a

, Youngwoo Choi

^a

, Min Sook Ryu

^a

, Hae-Sim Park

^a^,^b^,^*

aDepartment of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, South Korea

bDepartment of Biomedical Sciences, Ajou University School of Medicine, Suwon, South Korea

a r t i c l e i n f o

Article history:

Received 17 November 2022 Received in revised form 29 April 2023

Accepted 16 May 2023 Available online 24 June 2023

Keywords:

Asthma Macrophages Monocytes Neutrophils Severe asthma

Abbreviations:

AECs, Airway epithelial cells;

BALF, Bronchoalveolar lavageﬂuid;

BM, Bone marrow; CMs, Classical monocytes; DAPI, 4⁰,6-diamidino-2- phenylindole; EA, Eosinophilic asthma;

ETs, Extracellular traps; FBS, Fetal bovine serum; FEV1, Forced expiratory volume in theﬁrst second; HCs, Healthy controls;

IFN-g, Interferon-gamma;

IgE, Immunoglobulin E; ILCs, Innate lymphoid cells; M4, Macrophage;

MoETs, Monocyte extracellular traps;

M1ETs, M1 macrophage extracellular traps;

MCP-1, Monocyte chemoattractant protein- 1; MMEF, Mid maximal expiratoryﬂow;

MMP-9, Matrix metallopeptidase-9;

MPO, Myeloperoxidase; NE, Neutrophil elastase; NEA, Noneosinophilic asthma;

NETs, Neutrophil extracellular traps;

NSA, Nonsevere asthma; PAD, Peptidyl arginine deiminase; PBNs, Peripheral blood neutrophils; ROS, Reactive oxygen species;

SA, Severe asthma; sST2, Soluble suppression of tumorigenicity 2; TEC, Total eosinophil counts; TNF-a, Tumor necrosis factor-a; ZO-1, Zonula occludens-1

a b s t r a c t

Background: Increased blood/sputum neutrophil counts are related to poor clinical outcomes of severe asthma (SA), where we hypothesized that classical monocytes (CMs)/CM-derived macrophages (M4) are involved. We aimed to elucidate the mechanisms of how CMs/M4induce the activation of neutrophils/

innate lymphoid cells (ILCs) in SA.

Methods: Serum levels of monocyte chemoattractant protein-1 (MCP-1) and soluble suppression of tumorigenicity 2 (sST2) were measured from 39 patients with SA and 98 those with nonsevere asthma (NSA). CMs/M4were isolated from patients with SA (n¼19) and those with NSA (n¼18) and treated with LPS/interferon-gamma. Monocyte/M1M4extracellular traps (MoETs/M1ETs) were evaluated by western blotting, immunoﬂuorescence, and PicoGreen assay. The effects of MoETs/M1ETs on neutrophils, airway epithelial cells (AECs), ILC1, and ILC3 were assessedin vitroandin vivo.

Results: The SA group had significantly higher CM counts with increased migration as well as higher levels of serum MCP-1/sST2 than the NSA group. Moreover, the SA group had significantly greater production of MoETs/M1ETs (from CMs/M1M4) than the NSA group. The levels of MoETs/M1ETs were positively correlated with blood neutrophils and serum levels of MCP-1/sST2, but negatively correlated with FEV1%.In vitro/in vivostudies demonstrated that MoETs/M1ETs could activate AECs, neutrophils, ILC1, and ILC3 by increased migration as well as proinflammatory cytokine production.

Conclusions: CM/M4-derived MoETs/M1ETs could contribute to asthma severity by enhancing neutrophilic airway inﬂammation in SA, where modulating CMs/M4may be a potential therapeutic option.

*Corresponding author. Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Ajou University Medical Center, Suwon, South Korea.

Worldcup-ro 164, Yeoungtong-gu, Suwon-si, South Korea.

E-mail address:[email protected](H.-S. Park).

Peer review under responsibility of Japanese Society of Allergology.

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Introduction

Severe asthma (SA) is characterized by persistent airway inflammation (mainly eosinophilic asthma [EA]), followed by frequent asthma exacerbation and lung function decline even on maintenance medications including the medium-to-high doses of inhaled corticosteroids.^1,2Although its prevalence is estimated at 5%e10% in adult asthma cohorts, the cost of anti-asthmatic medications has required more than three times compared to nonsevere asthma (NSA).³ According to inflammatory phenotypes, SA has traditionally been classified into two types: type 2-high/EA and type 2-low/neutrophilic asthma (NA).⁴Current biomarkers (blood total eosinophil counts [TEC] and sputum eosinophil counts) are used to define type 2-high asthma; however, type 2-low asthma is less well characterized, and their biomarkers and biologics are lacking.⁴

Key functions of monocytes and macrophages (M4) include antigen recognition, presentation, and removal of invading allergens as well as immune homeostasis, linking between innate and adaptive immune responses.⁵ If these modulating functions are impaired, they may contribute to airway inflammation and remodeling in patients with SA.⁶The accumulation of monocytes was noted in the lower airway mucosa and alveoli of the patients with fatal asthma.⁶Monocytes are classified into the subsets of classical monocytes (CMs, CD14^þþCD16^-), intermediate monocytes (CD14^þCD16^þ), and nonclassical monocytes (CD14^þCD16^þþ) according to their surface CD markers.⁷CMs could be co-recruited with neutrophils in the airway mucosa of patients with NA.⁸In addition, monocytes are differentiated into M4, which are divided into the M1M4 and M2M4 groups according to environmental stimulations.⁵ In particular, M1M4 is closely linked to neutrophilic inflammation in asthma, which is mediated by proinflammatory cytokines (tumor necrosis factor-a[TNF-a], IL-6, and IL-1b) and the production of reactive oxygen species (ROS).^5,9 Recently, increased TNF receptor levels were found in the sputa of patients with NA, which were positively correlated with monocyte/neutrophil counts.¹⁰ The increased level of soluble suppression of tumorigenicity 2 (sST2) was noted in SA, contributing to type 2-low/neutrophilic airway inflammation.^11,12 In addition, CMs and M4have recently been proven to be able to release extracellular traps (ETs), as noted in neutrophils and eosinophils in SA^13,14; however, their underlying pathophysiological mechanisms in asthma are still unclear. In the present study, we hypothesized that monocyte ETs (MoETs) and M1M4ETs (M1ETs) released from CMs/M4contribute to the severity of neutrophilic inflammation in SA, and investigated their inflammatory pathways and therapeutic interventions.

Methods Materials

Detailed information on the key materials, isolation kits, ELISA kits, and antibodies used in this study is described in Supplementary Tables 1AeG. The BCA Protein Assay Kit was used to determine the protein concentration in this study. The ELISA kits, Western blot, immunoﬂuorescence, andﬂow cytometry antibodies were used according to the manufacturer's protocols.

Study subjects

Twenty-two healthy controls (HCs) and 137 asthmatic patients were recruited from the Department of Allergy and Clinical

Immunology at Ajou University Hospital in Suwon, South Korea to measure the serum levels of monocyte chemoattractant protein-1 (MCP-1), sST2, and neutrophil-associated cytokines. Our asthmatic patients had maintained anti-asthmatic medications including the medium-to-higher doses of inhaled corticosteroids plus long- acting beta 2 agonists (with no systemic steroid exposure at least 4 weeks prior to enrollment) for at least 2 years because they presented moderate-to-SA. Among them, 18 patients with NSA and 19 with SA were recruited in order to compare the levels of MoETs and M1ETs released from CMs/M1M4. The effects of MoETs/M1ETs on peripheral blood neutrophils (PBNs), airway epithelial cells (AECs), and innate lymphoid cells (ILCs) were evaluatedin vitro and/or ex vivo. This study was approved by the Institutional Review Board of Ajou University Hospital (AJIRB-GEN-SMP-13-108; AJIRB-BMR- SUR-15-498), and all the subjects provided written informed con- sent. This study was conducted in accordance with the Declaration of Helsinki.

According to the Global Initiative for Asthma guideline, asthma was diagnosed by recurrent episodes of wheezing, dyspnea, cough, and sputum production; airway hyperresponsiveness to methacholine; and reversible airway obstruction improved by a short-actingb2-agonist.¹⁵Among patients with asthma, SA was defined according to the International European Respiratory So- ciety/American Thoracic Society Guidelines.² Atopy status was defined as at least 1 positive result in skin prick tests using commonly inhaled allergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae, cat, dog, cockroach, tree pollen mixture, grass pollen mixture, mugwort, ragweed, andAspergillus, and Alternariaspp). The ImmunoCAP system (ThermoFisher Sci- entific, Waltham, CA, USA) was used to assess serum total IgE.

Spirometry was performed to evaluate the degree of airway obstruction (FEV1% and MMEF% predicted values).¹⁶ TEC and spontaneous sputum eosinophils values were measured as pre- viously described.¹⁷EA was deﬁned as TEC (>300 cells/mL) and/or sputum eosinophil counts (>3%).

The treatment protocols of human CMs and m4ex vivo

Immune cells were isolated using isolation kits and puriﬁed by using their expressed CD markers as shown in theSupplementary Methods and Results, and Supplementary Figure 1. Following isolation, CMs derived from both the SA and NSA groups were treated with 100 ng/mL of LPS plus 20 ng/mL of interferon-gamma (IFN-g) for 2 h to evaluate TNF-arelease. CMs and CM-derived M4 (M0M4, M1M4, and M2M4) were treated with 100 ng/mL of TNF-a or 100 ng/mL of LPS plus 20 ng/mL of IFN-gfor 6 h. To assess the effects of peptidyl arginine deiminase (PAD) inhibitor (YW3-56) or anti-IL-33/ST2 antibodies on ET production, CMs and M1M4were pretreated with 2mM of YW3-56 or 10mg/mL of anti-IL-33/ST2 antibodies for 30 min, followed by treating with LPS/IFN-gfor 6 h.

We selected the doses of YW3-56 and antibodies based on the pilot study with variable doses (data not shown). In some experiments, CMs were pretreated with 20 ng/mL of IL-4 and 20 ng/mL of IL-13 for 6 h. To remove the remaining LPS/IFN-g and ET-dissociated molecules, each well was washed three-times with serum-free no- phenol-red RPMI. The ﬁnal wash supernatants (media) were collected and utilized as controls for treating target cells and mice.

No-phenol-red RPMI containing 1 U/mL of micrococcal nuclease (MNase) was added to each well to digest ETs at 37C in an incu- bator for 20 min, followed by centrifugation to remove cell or cell debris.¹⁸

To determine the concentration of dsDNA using Picogreen assay in the supernatant, the inhibition step of MNase effects was

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processed by adding ethylene glycol bis (2 amino ethyl ether) NeNeN⁰-N⁰tetraacetic acid. To measure the release of myeloperoxidase (MPO)-DNA complexes from activated CMs and M1M4, we used an ELISA assay developed from the literature and expressed the data as the OD values at the wavelength of 450 nm.¹⁹ For confocal analysis, isolated human CMs were seeded on a 24-well plate (coated with poly-^L-Lysine solution), and stained with MPO and neutrophil elastase (NE). In contrast, human M1M4 were seeded on a 24-well plate without poly-L-Lysine solution, and stained with matrix metallopeptidase-9 (MMP-9) and ST2 to detect M1ETs. For migration assay, CMs were seeded on the upper chamber with or without 1mg/mL of MoETs/M1ETs added to the lower chamber. They were incubated for 6 h.

The treatment protocols of human PBNs and AECs

PBNs collected from asthmatics were cultured in media containing RPMI-1640 supplemented with 2% fetal bovine serum (FBS), 100 U/mL of penicillin G sodium, and 100mg/mL of streptomycin sulfate. For migration assay, PBNs isolated from patients with NSA and those with SA were seeded on the upper chamber with or without MoETs/M1ETs in the lower chamber and were incubated for 2 h. PBNs were pretreated with 20 ng/mL of IL-4 and 20 ng/mL of IL-13 for 1 h to evaluate the effects of IL-4 and IL-13 on MoET- induced neutrophil activation. For ELISA measurement, PBNs from asthmatics were treated with MoETs/M1ETs for 2 h.

A549 cells (human AECs) were cultured at 37C with 5% CO2in humidiﬁed air in complete media RPMI-1640 supplemented with 10% FBS, 100 U/mL of penicillin G sodium, and 100 mg/mL of streptomycin sulfate. A549 (210⁵) cells were treated with 1mg/

mL of ETs for 24 h (for analyzing shape changes, ELISA, and tight junction-related protein). The alterations in tight junction-related proteins zonula occludens-1 (ZO-1) and occludin were assessed by western blotting analysis.

The treatment protocols of human and mouse ILCs

Human: human ILCs were cultured in their media, including 1%

heat-inactivated human AB serum, 10 U/mL of human IL-2, and 50 ng/mL of human IL-7 in X-Vivo™ 15 medium.²⁰ Regarding migration, ILC1/3 were seeded on the upper chamber, whereas, MoETs/M1ETs were added on the lower chamber. The plate was incubated for 24 h. To test the effects of IL-4 and IL-13, ILCs were preincubated with 20 ng/mL of IL-4 and 20 ng/mL of IL-13 for 1 h.

ETs were used to stimulate ILC1 and ILC3 for 24 h for ELISA. In some tests, ILC1 and ILC3 were activated by 50 ng/mL of IL-12 and 50 ng/

mL of IL-23 for 4 days, and were subsequently cocultured with PBNs for 24 h in their media and in the absence of IL-12/IL-23. MPO expression in the form of neutrophil ETs (NETs) was observed by immunoﬂuorescence. Supernatants were collected for ELISA.

Mouse: mouse ILC media contains serum-free StemSpan SFEM II, 10 U/mL of mouse IL-2, and 50 ng/mL of mouse IL-7.²¹ILC1 was cultured with or without 50 ng/mL of IL-12, while ILC3 was cultured with or without 50 ng/mL of IL-23 after being isolated from different mouse groups. For the other days, cells were gently pipetted. Supernatants were collected after 4 days for ELISA

Immunoﬂuorescence assay

Isolated human CMs, mouse bone marrow (BM)-derived monocytes, and human PBNs were seeded on a 24-well plate

coated with the poly-L-Lysine solution, whereas human/mouse M4 and A549 were seeded on a 24-well plate without the poly-L-Lysine solution.

After incubation of the appropriate primary antibody, cells were stained for 1 h with Alexa Fluor 488 donkey anti-rabbit, and/or Fluor 594 donkey anti-mouse, and/or Fluor 633 donkey anti-goat antibodies. Then, the slides were coated with 4⁰,6-diamidino-2- phenylindole (DAPI, 1:1.000) for 5 min. Fluorescent images were acquired using confocal laser scanning microscopy at the Three- Dimensional Immune System Imaging Core Facility (LSM710, Cal Zeiss Microscopy GmbH, Jena, Germany) of Ajou University.

Migration, ROS, and apoptosis assays

Human ILCs were seeded on ILC media, whereas isolated CMs and PBNs were seeded on RPMI and stained with 2mM of calcein AM for 30 min.²²The cells were then washed three times in their medium before being added to an upper chamber plate with a pore size of 3.0mm for neutrophils, and a pore size of 5.0mm for CMs and ILCs. The lower chamber of Transwell plate was incubated for 2 h for PBNs, 6 h for CMs, and 24 h for ILCs at 37C with stimulators (MoETs and M1ETs)-containing media.

For ROS, the positive fluorescent signal of 2⁰7⁰ dichloro- fluorescein diacetate (H2DCFDA) was used to determine ROS concentrations. Afluorescence microplate reader (Synergy HT; BioTek Instrument, Winooski, VT, USA) was used to measure the mean fluorescence intensity at 480 and 520 nm for excitation and emis- sion, respectively. The FITC Annexin V Apoptosis Detection Kit I was used to assess cell apoptosis using BD FACS Canto II (BD Biosciences, Moutain View, CA, USA) according to the manufacturer's instructions.

Mouse experiments

Six-week-old female BALB/c and C57BL/6 J mice (Jackson Lab- oratory, Bar Harbor, ME, USA) were raised in pathogen-free conditions. All experimental protocols were approved by Ajou University's Institutional Animal Care and Use Committee (IACUC 2021e0007). In order to evaluate the effects of MoETs/M1ETs, C57BL/6 J mice were administered MoETs/M1ETs at doses of 25mg/

kg and 50mg/kg through the intranasal route for 6 consecutive days.

Body weight was checked every day. After 24 h of the last treatment, mice were sacriﬁced. The bronchoalveolar lavageﬂuid (BALF) was collected using a cannula and washed with PBS plus 1% bovine serum albumin before centrifugation to obtain the supernatant for ELISA and cells for evaluating total and differential cell counts. The lung tissues were trimmed and digested in 10 mL of RPMI containing 1.5 mL of DNase I and 1 mL of collagenase/hyaluronidase per 5 pairs of lungs.²¹Single cells were stained with appropriate antibodies for ILC and alveolar M4sorting.

Statistical analysis

The D'Agostino-Pearson omnibus test was used to check for normal distribution. For nonnormally distributed variables for the comparisons between two variables, the ManneWhitney U test was used, and for categorical variables, the Pearson's chi-squared test was used. The one-way ANOVA with Bonferroni'spost hoctest was used to compare data from several groups for normally distributed variables, whereas, rank-based nonparametric Jonck- heereeTerpstra test or nonparametric Kruskal Wallis with Dunn's

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post hoc test for nonnormally distributed variables. Spearman's rank correlation coefﬁcient was used to calculate correlations between continuous clinical variables. SPSS software version 22.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. The graphs were created using the GraphPad Prism 6.0 software (GraphPad Inc., San Diego, CA, USA). Flow cytometry data were analyzed by using FlowJo software (Tree Star Inc., Ashland, Oregon, USA). Illustrative images were drawn by using pictures from 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

Higher serum MCP-1 levels in patients with SA

The clinical characteristics of the study subjects enrolled to compare serum MCP-1/sST2 levels are summarized in Supplementary Tables 2 and 3 The SA group had significantly poorer FEV₁% and MMEF % values (P ¼ 0.002 for FEV₁% and P¼0.001 for MMEF%), whereas had higher TEC than the NSA group (P¼0.004). Sputum eosinophils exhibited a higher tendency in severe asthmatics than in nonsevere ones, although the difference was not significant (P¼0.291). The recruitment of monocytes from the BM or other monocyte-producing organs to target tissues is mainly regulated by MCP-1.²³ê²⁵The serum sST2 levels are an in- direct marker of ST2 expressions on neutrophils.^11,12Therefore, we evaluated the levels of serum MCP-1/sST2 and their associations with clinical parameters of adult asthmatics. Significantly higher levels of serum MCP-1/sST2 were noted in the SA group than in the NSA group, as well as in asthmatics than in HCs (P<0.05 for all).

Severe asthmatic patients were further classified into the EA and noneosinophilic asthma (NEA) groups based on TEC (>300 cells/mL) and/or sputum eosinophil counts (>3%), no significant differences in serum MCP-1 and sST2 levels (P¼0.848 for MCP-1 andP¼0.054 for sST2) were observed between two groups (data not shown). In addition, when the SA or NSA groups were classified according to the cutoff value of serum MCP-1 at 18.5 pg/mL (71.8% sensitivity and 60.2% specificity; data not shown), the MCP-1-high group had significantly higher serum MPO/sST2/MMP-9 levels (P ¼ 0.034, P¼0.020, andP¼0.014, respectively) within the SA group, but not within the NSA group, indicating that increased MCP-1 levels are associated with increased neutrophil activation markers in the SA group.

In a sub-cohort of adult asthmatics, the SA group showed signiﬁcantly higher CM numbers than the NSA group (P¼0.002;

Fig. 1A). When CMs were treated with LPS plus IFN-g, greater TNF-a release was noted in the SA group than in the NSA group (P<0.001;

Fig. 1B). Moreover, monocyte-derived M4 and its transformed forms (M0, M1, and M2) were included, as CMs are known as supplementary source cells for alveolar M1M4.²⁶Released dsDNA and proteins were used to evaluate ET formation from CMs, M0M4, M1M4, and M2M4. It was found that CMs and M1M4 (but not M0M4 or M2M4) could form ETs called MoETs and M1ETs (Supplementary Fig. 2AeC). When potential biomarkers for predicting the presence of MoETs/M1ETs were evaluated, signiﬁcantly higher levels of serum MCP-1 and sST2 were noted in the SA group than in the NSA group (P<0.05 for all;Fig. 1C and E). In addition, positive correlations were noted between the levels of MoETs/

M1ETs and serum MCP-1/sST2 in asthmatics (Fig. 1D and F), suggesting that increased levels of serum MCP-1/sST2 may be potential markers for MoET/M1ET-mediated airway inﬂammation in SA.

Comparisons of MoET/M1ET formation between the SA and NSA groups

MPO and NE were used to detect MoETs; MMP-9 and ST2 were used to detect M1ETs.¹⁴For components of ETs released from CMs and M1M4, IL-8, MPO, and MCP-1 were the substantial components of MoETs, while IL-8, chitinase-3-like protein, and MMP-9 were major ones of M1ETs (Supplementary Fig. 3A and B).

Furthermore, ST2 was more significantly expressed in M1ETs in response to LPS/IFN-g (Supplementary Fig. 2D). Significantly greater levels of ETs and dsDNA were found in the CMs/M1M4of severe asthmatics than in those of nonsevere asthmatics (at baseline, and after treatment of LPS/IFN-gor TNF-a), which were observed by confocal microscopy (Fig. 2A) and PicoGreen assay (P< 0.05;Fig. 2B and C). The increased levels of MoETs/M1ETs were negatively correlated with FEV1% (r¼- 0.685,P<0.001;r¼- 0.762,P<0.001; respectively;Fig. 2D and E). Furthermore, MoETs and M1ETs, as evaluated by migration assay, recruited CMs in severe asthmatics than in nonsevere asthmatics (P<0.001 for all, Fig. 2F), suggesting that MoETs/M1ETs contribute to chronic neutrophilic inflammation in SA via autocrine functions on CMs and inflammatory cytokines.

Signaling pathways of CM/M1M4extracellular trap (MoETs/M1ETs) releases

Recent studies have demonstrated that anti-ST2 antibody could improve asthma control in patients with type 2-low asthma.²⁷; PAD promotes histone citrullination, chromatin projectiles, and neutrophil extracellular trap formation.²⁸In this study, the severe asthma group showed a greater expression of ST2 on LPS/IFN-g- treated CMs and M1M4than the NSA group (one patient in each group) (Supplementary Fig. 4A and B), and released more MoETs/

M1ETs, which were signiﬁcantly suppressed by both anti-IL-33 and anti-ST2 antibodies (Supplementary Fig. 4C and D). Furthermore, YW3-56 treatment reduced the levels of MoETs/M1ETs as well as the MPO-DNA complex released from activated CMs/M1M4, indicating that the releases of MoETs/M1ETs may be controlled by the PAD pathway (Supplementary Fig. 5AeD). Taken together, these results suggest that anti-IL-33/ST2 antibody as well as PAD inhibitor may have the potential therapeutics in asthmatic patients with MoETs/M1ETs-mediated inﬂammation.

Moreover, the effects of type 2-high-related cytokines (IL-4 and IL-13) on the formation of MoETs were also investigated in this study. As a result, MoETs released from LPS/IFN-gor TNF-a-treated CMs, as well as their effects on CM/PBN migration, were suppressed by IL-4 and IL-13 treatment (Supplementary Fig. 6AeC).

The effects of MoETs/M1ETs on neutrophil activation in SA

Neutrophils are known as key inﬂammatory cells to induce steroid insensitivity in SA.²⁹; therefore, the present study evaluated the effect of MoETs/M1ETs on neutrophil migration and activation in SA. Signiﬁcantly positive correlations between MoETs/M1ETs and blood neutrophil counts were noted (r¼0.607,P<0.001 for MoETs andr¼0.602, P<0.001 for M1ETs; Fig. 3A and D). The migrated rates of PBNs towards MoETs/M1ETs were greater in the SA group than in the NSA group (P<0.05 for all;Fig. 3B and E). After migration, the direct effects of MoETs/M1ETs on the development of NETs were evaluated in a time-dependent manner (Supplementary Fig. 7AeC). The peak points for ROS production and dsDNA release (after subtraction of dsDNA levels of MoETs/

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Fig. 1.Classical monocyte activation in severe asthma (SA).A,Comparison of classical monocytes between patients with SA, and those with nonsevere asthma (NSA).B,The levels of TNF-areleased from classical monocytes in patients with SA and those with NSA.C,Comparison of serum MCP-1 levels among the study groups.D,Correlation between LPS/IFN-g- induced MoETs/M1ETs and the levels of serum MCP-1 in asthmatics.E,Comparison of serum sST2 levels among the study groups.F,Correlation between LPS/IFN-g-induced MoETs/

M1ETs and serum levels of sST2 in asthmatics. Boxes denote the median and 25th to 75th percentiles with whiskers extending to the farthest points within 1.5 times the box height.

#P<0.050,^##P<0.010, and^###P<0.001 between patients with SA and those with NSA.***P<0.001 between PBS and treated groups.Pvalue was obtained using the ManneWhitneyUtest for A, C, and E, and using the JonckheereeTerpstra test for B. Correlation data are represented as Spearman correlation coefﬁcientr(Pvalue) for D and F. IFN- g, interferon-gamma; MCP-1, monocyte chemoattractant protein-1; MoETs, monocyte extracellular traps; M1ETs, M1 macrophage extracellular traps; sST2, soluble suppression of tumorigenicity 2; TNF-a, tumor necrosis factor-a.

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M1ETs at the same time) were noted in MoET/M1ET-treated groups (Supplementary Fig. 7B and C), indicating that MoETs/M1ETs could contribute to chronic neutrophilic inﬂammation via activating neutrophils and NET formation.

PBNs incubated with MoETs/M1ETs released signiﬁcantly higher MPO levels than those incubated with media controls (P<0.001 for all; Fig. 3C and F). Moreover, apoptosis percentages of PBNs increased in the MoET- and M1ET-stimulated groups at the time point of 12 h in comparison to those at the time point of 0 h (Supplementary Fig. 7D), which suggested that MoETs/M1ETs could induce NET formation, enhancing neutrophil activation, similar to various stimuli (pathogens, antibodies, immune complexes, cytokines, and phorbol 12-myristate 13-acetate)-induced NETs.³⁰

The effects of MoETs/M1ETs on AECs in vitro

AECs are well known as the first-line cells in response to invading pathogens and release a multitude of cytokines, driving various asthma phenotypes.³¹ The present study evaluated the capacity of MoET/M1ET-derived activated CMs/M4to induce type 2-low cytokine production from AECs. When A549 cells, an AEC cell line, were treated with MoETs/M1ETs for 24 h, pro-inflammatory cytokines, including MCP-1, TNF-a, IL-6, IL-8, and MMP-9, were more significantly released as shown inFigure 4A and B. In addition, the morphology of A549 cells was changed; the expressions of ZO-1 and occludin were decreased by Western blot analysis (Fig. 4CeE), indicating that MoETs/M1ETs lead to neutrophilic Fig. 2.Comparison of MoETs/M1ETs between patients with severe asthma (SA) and those with nonsevere asthma (NSA).A,The formation of MoETs/M1ETs as observed by using confocal microscopy in asthmatics. Scale bar, 100mm.BeC,The levels of MoETs/M1ETs in patients with SA and NSA using Picogreen assay.D-E,Negative correlations between FEV1% and the levels of LPS/IFN-g-induced MoETs/M1ETs.F,Classical monocyte migration toward MoETs, M1ETs, and IL-33. Boxes denote the median and 25th to 75th percentiles with whiskers extending to the farthest points within 1.5 times the box height.^#P<0.050,^##P<0.010, and^###P<0.001 between patients with SA and those with NSA.**P<0.010 and

***P<0.001 between PBS/media and treated groups.Pvalue was obtained using the JonckheereeTerpstra test for B, C, and F. Correlation data are represented as Spearman correlation coefﬁcientr(Pvalue) for D and E. DAPI, 4⁰,6-diamidino-2-phenylindole; FEV1, forced expiratory volume in 1s; IFN-g, interferon-gamma; M4, macrophage; MoETs, monocyte extracellular traps; M1ETs, M1 macrophage extracellular traps; MPO, myeloperoxidase; MMP-9, matrix metallopeptidase-9; NE, neutrophil elastase; ST2, suppression of tumorigenicity 2; TNF-a, tumor necrosis factor-a.

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inﬂammatory responses by affecting airway epithelium to release neutrophil activation-related cytokines and by causing tight junction disruption.

The effects of MoETs/M1ETs on human ILC activation

Considering that the numbers of ILC1 and ILC3 are limited in peripheral blood of adult asthmatics, we evaluated the effects of MoETs/M1ETs on the combination of both ILC1 and ILC3ex vivo (Fig. 5A). After treatment with MoETs/M1ETs for 24 h, migrated numbers of ILCs were increased (P<0.010;Fig. 5B), and signiﬁ- cantly increased levels of IFN-g, IL-17 A, and IL-22 were noted compared to those without MoETs/M1ETs (P<0.010;Fig. 5CeE).

ILC3 (releasing IL-17 A or IL-22) was shown to be closely associated with neutrophil activation in asthma.²⁹Therefore, the present study evaluated the interactions between activated ILC3 and neutrophil activation. In addition, M4released IL-23 in response to pathogen-associated recognization molecules,³² ILC1/ILC3 were

primed with IL-12 (ILC1 activator) and IL-23 (ILC3 activator) as well as IL-2 and IL-7 (ILC survival stimulants) and cocultured with SA- derived PBNs. As a result, PBNs released signiﬁcantly higher levels of MPO and MCP-1 when they were cocultured with activated ILC1/

ILC3 (P<0.001 for all;Supplementary Fig. 8A and B). Furthermore, the formation of NETs, as evaluated by the expressions of DAPI and MPO using confocal analysis, was increased (Fig. 5F), suggesting that MoETs/M1ETs induce ILC1/ILC3 activations, affecting NETs- mediated airway inﬂammation, and activating neutrophils and M4. The effects of MoETs/M1ETs in vivo

To validate the effects of MoETs/M1ETs on airway inﬂammation in vivo, mice were intranasally administrated with MoETs and M1ETs for 6 days (Fig. 6A). MoETs- or M1ETs-treated mice showed a reduction in body weight (P<0.001 for all;Supplementary Fig. 9A and B), while increased total and neutrophil cell counts were noted in the BALF in a dose-dependent manner compared to media- Fig. 3.The effects of MoETs/M1ETs on neutrophil activation in severe asthma (SA).A,Correlation between the concentrations of LPS/IFN-g-induced MoETs and blood neutrophil counts.B,Comparison of neutrophil migration towards MoETs between patients with SA and those with nonsevere asthma (NSA).C,The levels of MPO released from neutrophils of asthmatic patients in response to MoETs (n¼14 for each group).D,Correlation between the concentrations of LPS/IFN-g-induced M1ETs and blood neutrophil counts.E,Com- parison of neutrophil migration towards M1ETs between patients with SA and those with NSA.F,The levels of MPO released from neutrophils of asthmatic patients in response to M1ETs (n¼14 for each group). Boxes denote the median and 25th to 75th percentiles with whiskers extending to the farthest points within 1.5 times the box height.^#P<0.050 and

###P<0.001 between SA and NSA.***P<0.001 between media and treated groups.Pvalue was obtained using the JonckheereeTerpstra test for B and E, and using the ManneWhitneyUtest for C and F. Correlation data are represented as Spearman correlation coefﬁcientr(Pvalue) for A and B. MoETs, monocyte extracellular traps; M1ETs, M1 macrophage extracellular traps; MPO, myeloperoxidase.

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treated mice (P<0.001 for all;Fig. 6B and C). Moreover, signiﬁ- cantly higher MPO, TNF-a, MCP-1, and MMP-9 levels were noted in the BALF after treatment with MoETs/M1ETs compared to media- treated mice (P < 0.050 for all; Fig. 6D and Supplementary Fig. 9CeE). Hematoxylin-eosin staining demonstrated increased numbers of inﬂammatory cells in BALF and lung tissues (Supplementary Fig. 10), suggesting the contribution of MoETs/

M1ETs to neutrophilic inﬁltration in asthmatic lung tissuesin vivo.

To demonstrate the effect of MoETs/M1ETs on ILC1/ILC3 activation, the subsets of ILCs (ILC1 or ILC3) were sorted from the lung tissues of media- and MoET/M1ET-treated mice (Fig. 6E). Increased numbers of total and subsets of ILCs (ILC1 and ILC3) were observed in the lung tissues after MoET/M1ET treatment (Fig. 6F). Moreover, in activated conditions by IL-12/IL-23, signiﬁcantly higher levels of IFN-gand IL-17 A were noted in both ILC1 and ILC3 isolated from MoET/M1ET-treated mice compared to those from PBS-treated Fig. 4.The effects of MoETs/M1ETs on airway epithelial cells. A549 cells were treated with or without MoETs/M1ETs derived from severe asthmatic patients.A-B,Analysis of cytokine proﬁles in MoETs/M1ETs as well as those released from A549 cells in response to MoETs/M1ETs. Data were obtained from 6 dependent experiments and are presented as mean values.C,Changes in the morphology of A549 cells after incubation with MoETs/M1ETs.D,Changes in tight junction-related proteins in A549 cells after incubation with MoETs/M1ETs.E,Representative data of ZO-1 and occludin expression was investigated by Western blot analysis (n¼4 for each group). Boxes denote the median and 25th to 75th percentiles with whiskers extending to the farthest points within 1.5 times the box height.***P<0.001 was obtained using the one-way ANOVA with Bonferroni'spost hoctest for E. MoETs, monocyte extracellular traps; M1ETs, M1 macrophage extracellular traps; MCP-1, monocyte chemoattractant protein-1; MMP-9, matrix metallopeptidase-9; TNF-a, tumor necrosis factor-a; ZO-1, zonula occludens-1.

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Fig. 5.The effects of MoETs/M1ETs on the activation of human ILC1 and ILC3. ILC1/3 cells were isolated from asthmatic patients and treated with or without MoETs/M1ETs derived from severe asthmatic patients.A,Schematic for ILC1/ILC3 treatment.B,The migrations of ILCs toward MoETs/M1ETs (n¼6 for each group). The levels ofC,IFN-g,D,IL-17 A, andE, IL-22 were released from ILCs after treatment with MoETs/M1ETs (n¼6 for each group).F,Neutrophils from asthmatic patients were stained with MPO and DAPI. The levels of NETs were evaluated by using confocal microscopy after coculture with activated ILC1 and ILC3. Scale bar, 20mm. Boxes denote the median and 25th to 75th percentiles with whiskers extending to the farthest points within 1.5 times the box height.**P<0.010 and***P<0.001 were obtained using the JonckheereeTerpstra test for B, and using the ManneWhitney Utest for C-E. DAPI, 4⁰,6-diamidino-2-phenylindole; ILC, innate lymphoid cell; IFN-g, interferon-gamma; MoETs, monocyte extracellular traps; M1ETs, M1 macrophage extracellular traps; MPO, myeloperoxidase; Neu, neutrophils.

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Fig. 6.The effects of MoETs/M1ETs on the activation of neutrophils and ILC1 and ILC3in vivo.A,Schematic for MoET/M1ET treatment.B,Total cell counts in the BALF.C,Neutrophil counts in the BALF.D,The levels of MPO in the BALF (n¼5 for each group).E,Schematic diagram of ILC1 and ILC3 isolation.F,The increased numbers of total ILCs, ILC1, and ILC3 (%) in the lung tissues were evaluated byﬂow cytometry.G-H,The levels of IFN-greleased from isolated ILC1, and IL-17 A were released from isolated ILC3 (n¼5 for each group).

#P<0.050,^##P<0.010, and^###P<0.001 between PBS and IL-12/IL-23-treated groups.*P<0.050,**P<0.010, and***P<0.001 between media and MoET/M1ET-treated groups.P value was obtained using one-way ANOVA with Bonferroni'spost hoctest for B and C, using the JonckheereeTerpstra test for D, and using the Kruskal Wallis with Dunn'spost hoc test for G and H. BALF, bronchoalveolar lavageﬂuid; i. n, intranasal; ILC, innate lymphoid cell; IFN-g, interferon-gamma; MoETs, monocyte extracellular traps; M1ETs, M1 macrophage extracellular traps; MPO, myeloperoxidase.

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mice (P<0.050 for all;Fig. 6G and H), indicating ILC1/ILC3 activation in neutrophilic inﬂammation.

Discussion

To the best of our knowledge, this is theﬁrst study to demonstrate the contribution of CMs and M1M4(through the release of ETs) to neutrophilic airway inﬂammation in SA. LPS/IFN-gand TNF- a could stimulate the production of MoETs/M1ETs from CMs/

M1M4, which were greater in patients with SA than in those with NSA. Moreover, in vitro and ex vivo studies demonstrated that MoETs/M1ETs could enhance neutrophilic airway inﬂammation in SA by directly recruiting and activating neutrophils, and by indirectly activating them through the activation of AECs, ILC1, and ILC3, which was also validatedin vivostudy. Anti-IL-33/ST2 antibody treatment had protective effects by inhibiting the formation of MoETs/M1ETs. Taken together, MoETs/M1ETs (derived from CMs/

M1M4) could contribute to the severity of airway inﬂammation by regulating neutrophil functions in SA.

Monocytes are essential for the maintenance of allergic inflammatory reactions.³³ Mice (with depleted monocytes) have considerably lower eosinophil recruitment and activation in response to the allergen challenge.²⁶Recent studies have demonstrated that CM dysfunction in sputum contributes to the asthma severity and phenotype of NA.⁸The genetic polymorphism ofMCP- 1-2518 A>Ghas been linked to asthma severity.³⁴In addition, the IL-33/ST2 signaling pathway plays an important role in the recruitment of monocytes as well as the polarization and activation of M4.^35,36Treatment with clodronate liposomes (monocyte inhibitor) could suppress the production of IL-33 and recruitment of inflammatory cells in BALF as well as airway hyperreactivity in the mouse model of house dust mite-induced asthma.³⁷The present study demonstrated significantly higher serum MCP-1 levels as well as sST2 levels in severe asthmatics than in nonsevere asthmatics, both of which showed a positive correlation with MoETs/

M1ETs levels, suggesting that serum MCP-1/sST2 levels may be potential biomarkers for predicting MoETs/M1ETs formation in SA.

Moreover, asthmatics with higher MCP-1 levels had higher MMP-9/

MPO as well as sST2 levels, indicating that monocytes are more markedly activated in SA than in NSA, which are associated with neutrophilic inﬂammation and IL-33/ST2 signaling pathways. The ex vivo study demonstrated that severe asthmatics had higher blood monocytes (total and classic phenotypes) and MoET/M1ET levels, mediated by ST2-expressed CMs/M1M4, which was significantly suppressed by anti-IL-33/ST2 antibody. ST2 was found as a major component of M1ETs, and BM-derived M4 isolated from ST2^/mice less produced cytokines in response to LPS than those from wild-type mice,³⁸suggesting that the IL-33/ST2 pathway plays a major role in the recruitment and activation of CMs/M4in SA.

Taken together, activated CMs/M4could contribute to activating neutrophils in the airway inﬂammation of SA by releasing MoETs/

M1ETs, which are mediated by the IL-33/ST2 signaling pathway.

Anti-IL-33/ST2 antibody may be a potential therapeutic target for CM/M4-mediated neutrophilic airway inﬂammation in SA.

Patients with NA less respond to steroids and duplimab,³⁹which may be explained by the different regulatory roles of IL-4 and IL-13 in EA or NA. IL-4/IL-13 could enhance eosinophil migration and activation, and inhibit neutrophils to release NETs.⁴⁰ When the present study evaluated the effects of IL-4 and IL-13 on MoETs and M1ETs, M1M4produced significantly more ETs upon stimulation with LPS and IFN-gthan M2M4, the latter of which were differentiated using M-CSF, IL-4, and IL-13. IL-4/IL-13-conditioned CMs could prevent MoET formation in response to LPS/IFN-gor TNF-a. Furthermore, IL-4/IL-13-treated PBNs and CMs migrated less toward MoETs. Thesefindings imply that these cytokines (IL-4/IL-13) play a major role in the regulation of CMs and M1M4in severe NA, and their antibodies may have limitations to control CMs/M4- induced airway inflammation.

The mechanism of airway neutrophilia in asthma is associated with the activation of ILC1/ILC3 and proinﬂammatory cytokine

Fig. 7.Underlying mechanisms of MoETs/M1ETs in severe asthma. Severe asthmatics had increased monocyte activities with the greater release of MoETs/M1ETs compared to nonsevere asthmatics.In vitro/in vivostudies demonstrate that MoETs/M1ETs could directly activate neutrophils, and indirectly activate them via ILC1/ILC3 and AECs. IFN-g, interferon-gamma; ILC, innate lymphoid cell; MoETs, monocyte extracellular traps; M1ETs, M1 macrophage extracellular traps; NETs, neutrophil extracellular traps.

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production.^41,42 NA is associated with steroid insensitivity, contributing to the progression of SA.^29,43However, the pathways of ILC activation have not yet been completely clarified in SA. The increased number of ILC1/ILC3 is strongly correlated with M1M4 activation.⁴¹Patients with NA had more activated M4in sputa as evaluated by transcriptome analysis and more increased phagocy- tosis/efferocytosis factors than those with nonneutrophilic asthma.⁴⁴Previous studies have revealed that activated M4could produce IL-12/IL-23 in response to inflammatory environments, activating ILC/ILC3, while ILC1/ILC3 could release IFN-gand IL-17 A, inducing migration and activation of neutrophils in NA.^29,45ê⁴⁷The present study demonstrated a negative correlation between MoETs/M1ETs and FEV1%, and positive correlations between MoETs/M1ETs and blood neutrophil counts as well as activated ILCs (ILC1/ILC3) after MoET/M1ET treatment, leading to neutrophilic airway inflammation through the production of IFN-g, IL-17 A, and IL-22 in SA. In addition, the levels of NETs measured by dsDNA and confocal assay were higher in severe asthmatics than in nonsevere asthmatics.¹⁸NETs could induce epithelial dysfunction as well as neutrophil activation.^18,31,48The present study demonstrated that MoETs/M1ETs (especially from severe asthmatics) affected the disruption of tight junctional proteins in AECs (ZO-1 and occludin).

CMs/M4 in severe asthmatics released more TNF-athan in nonsevere asthmatics with MoET/M1ET formation. TNF-a is a key epithelial molecule to drive neutrophilic airway inﬂammation along with activated monocytes in NA.¹⁰Taken together, increased CMs/M4enhance MoETs/M1ETs and TNF-arelease, activate neutrophils (directly and indirectly via activated ILCs), and dampen epithelial integrity, leading to airway neutrophilia and steroid resistance in SA (the M4-AECs-neutrophil axis).

Moreover, theseﬁndings were validatedin vivoby using two kinds of asthma mouse models (EA and NA) in the present study.

Naïve BM-derived monocytes/M4 released MoETs/M1ETs in response to LPS/IFN-gand TNF-a(Supplementary Fig. 11). Airway neutrophilia was closely linked to M1M4 by releasing M1ETs, while airway eosinophilia was related to M2M4.^8,49 Our NA mouse model showed signiﬁcantly higher levels of M4 (especially M1M4), dsDNA (representing M1ETs derived from alveolar M4), MPO, MCP-1, MMP-9, and TNF-ain BALF compared to EA models (Supplementary Fig. 12). MoET/M1ET-treated mice showed higher numbers of neutrophils, MPO, MCP-1, MMP-9, and TNF-a levels in BALF as well as total ILCs, and ILC1/ILC3 subsets in the lung tissues than in control mice, indicating that activated M4could induce neutrophilic airway inﬂammation in NA via activating ILCs as well as MoET/M1ET formation, contributing to steroid insensitivity in SA.

This study has some limitations. First, the function of activated CMs/M4 was compared between the SA and NSA groups in the adult asthmatic cohort, which needs to be confirmed in larger cohorts. Secondly, we could compare MoET/M1ET-induced airway inflammation between the SA and NSA groups, which could not be confirmed in sputum samples. However, the number and per- centage of alveolar M4 are inconsistent according to the sample collection methods and demographic factors.^41,50,51 Thirdly, it is unclear whether the characteristics of alveolar M4may be similar to CM-derived M4 in the aspect of ET formation. Finally, the function of MoET/M1ET-derived sST2 in aspects of forming stimu- latory complexes with IL-33 should be further investigated in NA.¹¹ Despite these limitations, we suggest two pathogenic pathways of CMs/M4-induced neutrophilic inflammation (the axis of CMs/

M1M4-MoETs/M1ETs-AECs-neutrophils and CMs/M1M4-MoETs/

M1ETs-ILC1/ILC3-neutrophils) as shown inFigure 7.

In conclusion, theseﬁndings suggest the role of activated CMs/

M4 in persistent neutrophilic airway inﬂammation and steroid

insensitivity in SA by releasing MoETs/M1ETs, where anti-IL-33/ST2 antibodies may be a potential therapeutic target.

Acknowledgements

This study was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health&Welfare, Republic of Korea (HR16C0001). The confocal laser scanning microscope (LSM710) used in this study was supported by a Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education. (grant no. 2019R1A6C 1010003).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.alit.2023.06.004.

Conﬂict of interest

The authors have no conﬂict of interest to declare.

Authors’contributions

QLQ and TBTC conceived the original idea, carried out the experiments, and wrote the manuscript. YSS and YC supported preparing the protocols and running in vivostudies. JHJ collected and analyzed clinical parameters. JYM measured the levels of serum cytokines using ELISA. MSR supportedin vitroexperiments. HSP supervised the project.

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