1. Introduction
Extracellular traps (ETs) serve as a key component of the innate immune response against external pathogens. They consist of dense DNA, histones, granule proteins, and several inflammatory mediators [
1]. Not only neutrophils but also other cells, including eosinophils and mast cells, produce ETs [
2,
3]. Neutrophils and eosinophils are both granulocytes and are known to act as effector cells in the immune response against infections and pathological conditions. Neutrophil ETs (NETs) and eosinophil ETs (EETs) are beneficial for host defense owing to their ability to eliminate pathogens, while the released nuclear-derived DNA and granular enzymes can have cytotoxic effects on airway epithelial cells. This can increase secretion viscoelasticity and promote local inflammation and tissue damage [
4]. The characteristic features of type 2 chronic rhinosinusitis (CRS) include the presence of tissue eosinophilia, neutrophilia, and increased NETs and EETs [
5]. EETs in CRS are closely related to disease severity and CRS prognosis. NETs are found in the subepithelial layer of sinonasal mucosa and are associated with refractoriness in non-eosinophilic CRS (NECRS) [
5,
6]. NETs and EETs play a crucial role in the development of CRS or CRS with nasal polyps (CRSwNP) and can be targeted for therapeutic interventions.
Neutrophils and eosinophils act as an effector cell in the removal of fungal pathogens. Neutrophils eliminate fungi through degranulation, phagocytosis, reactive oxygen species (ROS) generation, cytokine production, and NET formation [
7,
8]. Fungi induce NET formation through nicotinamide adenine dinucleotide phosphate (NADPH)–oxidase and the ROS-dependent response or through NADPH–oxidase-independent opsonization by human serum for non-phagocytic effector mechanisms. NETs demonstrate fungicidal properties against many pathogenic fungi as well as the fungistatic effect against
Aspergillus, thereby preventing their further spread [
9,
10,
11]. Fungi induce eosinophil degranulation through interaction with β-glucan and β2-integrin or fungal protease with protease-activated receptors [
12,
13]. EETs can develop in both ROS-dependent and ROS-independent manner. The response promotes epithelial cell-derived cytokine production, such as thymic stromal lymphopoietin (TSLP), which induces the release of EETs in a NADPH–oxidase-dependent manner [
14].
Aspergillus induces the release of EETs in an NADPH–oxidase-independent manner [
15].
Airborne fungi are ubiquitous and prominently associated with airway diseases. Among various pathogenic fungi,
Aspergillus and
Alternaria are frequently isolated from airway secretions, and they interact with airway mucosa and inflammatory cells [
16]. Patients with CRS tend to have a higher fungal burden and exhibit abnormal or inappropriate immune responses to fungi [
17]. Clearance of inhaled fungal conidia in the airway mucosa by neutrophils or eosinophils is a crucial innate defense mechanism. Neutrophils and eosinophils play a crucial role in antifungal immunity and immunomodulatory function in fungal-associated inflammatory diseases, and they release ETs in response to fungal pathogens [
11]. This study aims to investigate the effects of
Aspergillus or
Alternaria on neutrophils and eosinophils migration as well as the release of ETs by these cells in response to fungi.
3. Discussion
Eosinophils and neutrophils have a similar biological activity, acting as effector cells against fungal pathogens and modulating adaptive immune responses through extracellular trap formation [
11].
Alternaria and
Aspergillus are commonly found in the nasal secretion of both healthy volunteers and patients with CRS [
20]; thus, this study elucidated the role of these airborne fungi in EET and NET formation in CRS. Sinonasal tissue from patients with ECRS exhibited a higher number of NETs and EETs compared to NECRS.
Aspergillus, but not
Alternaria, induced EET and NET formation, which were more pronounced in patients with ECRS compared to those with NECRS and to healthy volunteers.
Airborne fungi enter the airways through inhalation, and most of them are removed by the innate mucosal defense system, including mucociliary clearance, fungicidal enzymes, and innate immune cells. Airway epithelial cells induce the production of chemical mediators and facilitate pathogen access to the target tissue or the migration of inflammatory cells into the airway lumen in pathological conditions with increased fungal contact [
19,
21]. Fungal cell wall components, especially protease, can induce epithelial barrier dysfunction with ROS overproduction [
18]. The protease activity of 10
6 conidia of
Alternaria and
Aspergillus was similar to that of 2.0 μg/mL of trypsin. Epithelial TER decreased after 4 h of fungal conidia treatment. TER experienced a more substantial decrease in ECRS patients compared to the other two groups (30–35%, 17–25%, and 15–20% in the ECRS, NECRS, and control groups, respectively), although failing to attain statistical significance.
Alternaria and
Aspergillus enhanced both eosinophil and neutrophil migration in the ECRS and control groups. However, only the neutrophil migration increased as a result of fungi in the NECRS group. These discrepancies may be associated with the strength of molecular interactions within the apical junctional complex or with the difference in the effect of fungi on eosinophil-associated chemokine production from nasal epithelial cells. Furthermore, eosinophil migration was more strongly induced by fungi in the ECRS group. We cannot explain the reasons for these differences, but the pronounced eosinophil migration induced by fungi is thought to result from barrier dysfunction, which may involve a molecular or structural change of tight or apical epithelial cell junctions due to fungal protease or other components, as well as the abundant production of chemical mediators from epithelial cells compared to other conditions.
Aspergillus strongly induced neutrophils migration compared to
Alternaria. This phenomenon may be attributable to the strong induction of neutrophilic innate immune responses by
Aspergillus with respect to the identification and elimination of fungal organisms, especially in normal nasal epithelial cells. Increased intracellular ROS is generally associated with nasal epithelial barrier dysfunction [
18]. ROS production was inhibited by treatment with the ROS scavenger glutathione, indicating that intracellular ROS is a contributing factor to epithelial barrier dysfunction and inflammatory cell migration in the nasal mucosa.
Eosinophils modulate the immune response in various infectious diseases and act as effector cells to protect the host against non-phagocytizable pathogens, such as parasites and fungi [
22]. Eosinophils can remove fungi through both physical contact with β-glucan components of fungal cell walls or without physical contact through pattern-recognition receptors (PRRs) associated with eosinophil activation [
12,
23]. PRRs, particularly the β2-integrin Mac-1, respond to fungi and mediate fungal-induced EET release [
15].
Alternaria can induce activation and release of toxic granule protein from eosinophils through physical interactions between CD11b and the β-glucan of fungi [
12]. However, this study revealed that
Alternaria could not influence EET development, while
Aspergillus strongly induced EETs in vitro. Fungi exhibit remarkable diversity in terms of structure and components, and they interact with the host and immune cells in various ways. While
Alternaria can activate eosinophils through various way, it lacks the ability to actively participate in the development of ETs as observed in
Aspergillus.
Aspergillus-induced EET formation was much more pronounced in ECRS. This indicates that, in ECRS, eosinophils may be more prone to respond strongly and quickly to
Aspergillus. In this study, approximately half of ECRS patients had asthma. Although, we did not directly compare the impact of asthma comorbidity on EET formation, the biological properties of eosinophils in asthmatic patients may predispose to the development of EET. The ROS inhibitor DPI treatment, but not the mitochondrial ROS inhibitor MitoTempo, strongly inhibited
Aspergillus-induced EET formation. This result differs from the previous study which indicated that
A. fumigatus induces the release of EETs in an NADPH–oxidase- and mitochondrial-ROS-independent manner [
15]. This discrepancy may be due to the use of eosinophils isolated from patients with ECRS, whose biological condition may be different from those of healthy volunteers.
Neutrophils are essential for the clearing of fungi through the phagocytosis of conidia, degranulation, and oxidative burst [
8,
10]. NET formation is involved in fungal killing and entrapment [
11]. The intensity of
Aspergillus-fumigatus-induced NET formation was highest in ECRS, but
A. fumigatus also induced NET formation in patients with NECRS and in heathy controls.
Alternaria only induced NET formation in patients with NECRS. NETs were found to be abundant in sinonasal tissue with fewer EETs in NECRS, and they may play a critical role as part of an innate immune response against airborne fungi, such as killing or inhibiting fungal growth and germination. NADPH–oxidase activation plays a crucial role in NET formation and in the effector response to external pathogens [
24]. This study revealed that both NADPH–oxidase and mitochondrial ROS inhibitors significantly inhibited
Aspergillus-fumigatus-induced NET formation.
EET and NET accompany rapid cell death characterized by cell membrane rupture and the release of intracellular granule proteins and chemical mediators. We measured IL-8 and ECP for eosinophils and elastase for neutrophils. However, due to the low sensitivity of commercially available ELISA kits for ECP and elastase in the ng/mL range, measuring and comparing them statistically was challenging.
Aspergillus strongly induced IL-8 production compared to
Alternaria both in EETs and NETs. Because
Aspergillus strongly enhance the development of EET and NET compared to
Alternaria,
Aspergillus strongly induced the release of a larger amount of intracellular IL-8 from eosinophils and neutrophils. IL-8 levels were significantly higher in eosinophils or neutrophils from patients with CRS than in those from healthy volunteers, and IL-8 levels within CRS were much higher in ECRS patients than in NECRS patients. The IL-6 and tumor necrosis factor (TNF)-α levels in EETs were also higher in ECRS patients than in NECRS patients (
Figure S2). Eosinophils and neutrophils that migrated to the fungi may have been in a preactivated state or prone to producing larger amounts of intracellular contents in patients with CRS. Eosinophils and neutrophils might be more strongly activated by fungi in ECRS patients than in NECRS patients. ET formation by eosinophils and neutrophils is known to contribute to pathogenic fungi removal and may influence fungal metabolic activity. The IL-8 study revealed that
Aspergillus only induced a small amount of IL-8 production from eosinophils or neutrophils derived from healthy volunteers compared to patients with CRS. This indicates that
Aspergillus could not strongly induce the release of granule protein and chemical mediators compared to patients with CRS, which have fungicidal or fungistatic properties.
4. Materials and Methods
4.1. EET and NET Detection in CRSwNP
This study collected sinonasal polyp tissues from 30 patients with CRSwNP during endoscopic sinus surgery. We excluded patients who were younger than 18 years of age, had active inflammation, and had used antibiotics, antihistamines, or other medications for at least 4 weeks prior to the operation. The Institutional Review Board of Daegu Catholic Medical Center approved this study, and all the patients signed a consent form that outlined the study objectives. We classified ECRS as eosinophilic when more than an average of 70 eosinophils were found in the three densest areas on high-power field (×400) images.
Table 1 shows the clinical and demographic characteristics of patients with CRSwNP. The average counts of EETs and NETs were analyzed in three representative fields characterized by the highest degree of cell infiltration throughout the sinus mucosa.
Immunofluorescence staining was used to detect EETs and NETs in CRSwNP. The deparaffinized tissue slides were treated with 0.3% Triton-X100 for 15 min and blocked with 5% bovine serum albumin (Merck, Darmstadt, Germany). The slides were incubated with histone H3 (citrulline R17) antibody (Abcam, Cambridge, UK) and neutrophil elastase (Santacruz, Dallas, TX, USA) to identify NET. The slides were incubated with histone H3 antibody and galactin-10 (Santacruz) for EET. After being washed with phosphate buffer saline (PBS), the section slides were incubated with Alexa Fluor® 488-conjugated IgG (Invitrogen, Carlsbad, CA, USA) and cy3-conjugated IgE (Invitrogen). The cell nucleus was stained with Hoechst 33342 (Invitrogen). The Zeiss LSM 900 confocal microscope (Zeiss, Oberkochen, Germany) was used to determine image capture and analyses.
4.2. Isolation of Fungal Conidia
Aspergillus fumigatus (ATCC 46645) and Alternaria alternata (KCTC 26781) conidia were inoculated on potato dextrose/corn meal agar plates with cycloheximide for 5–7 days at 25 °C. Conidia were collected by scrapping the plate with sterile PBS that contained 0.05% tween-20 (Bio-Rad, Hercules, CA, USA). Eluates were centrifuged at 1000 rpm for 10 min, and pellet suspensions were filtered through a 40 μm cell strainer. Conidia suspensions at 2 × 107/mL were left to dry at 45 °C, after which they were stored at −80 °C until required.
4.3. Isolation of Eosinophil and Neutrophil
Cells were isolated from heparinized venous blood from patients with ECRS, NECRS, and also from normal healthy volunteers. The Institutional Review Board of Daegu Catholic Medical Center approved this study, and all subjects signed a consent form outlining the study objectives. Neutrophils were isolated using the density gradient centrifugation method with 1.077 g/mL of percoll (Sigma-Aldrich, St. Louis, MO, USA). After collecting granulocyte and red blood cells (RBCs), the RBCs were removed using hypotonic water lysis. The purity and viability of neutrophils determined using trypan blue staining was >95%.
Negative immunomagnetic bead selection was used to isolate eosinophils. Heparinized blood was layered over 1.085 g/mL of percoll (Sigma-Aldrich). After RBC lysis, the granulocytes were mixed with anti-CD16 antibody conjugated with magnetic particles (Miltenyl Biotechnology, Sunnyvale, CA, USA) and passed through a magnetic field (Variomax, Miltenyl Biotechnology). The purity of the eosinophils determined using Randolph staining was >95%.
4.4. Primary Nasal Epithelial Cell Inverted Air-Liquid Interface (ALI) Culture
Primary nasal epithelial cells were isolated from the NP of subjects with ECRS (six men and two women, aged 51.3 ± 16.2 years), NECRS (five men and two women, aged 48.3 ± 13.5 years), and inferior turbinates of septal deviation (five men and three women, aged 47.6 ± 12.7 years). The Institutional Review Board of Daegu Catholic Medical Center approved this study, and all subjects signed a consent form that outlined the study objectives.
The specimens were placed in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with antibiotic–antimycotic (100×). Nasal mucosa was rinsed with RPMI 1640 supplemented with antibiotics and incubated with 0.1% protease (Sigma-Aldrich) for 16 h at 4 °C. The epithelial cells were isolated by gentle agitation, and cell suspensions were filtered through a No. 60 mesh cell dissociation sieve. The cells were suspended in defined keratinocyte serum-free medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with antibiotics. Cell suspensions (106 cells/mL) were plated in six-well culture plates and placed in a 5% CO2 humidified incubator at 37 °C. The culture medium was changed after 24 h and every 2 days thereafter. The cells were seeded on 3.0 μm 12 mm transwell inserts (Corning Costar, Cambridge, MA, USA) when the epithelial cells cultures reached 80–90% confluence to make ALI culture model at a density of 1 × 105 cells/well mixed with the 0.5% GrowDex® (UPM Biochemicals, Helsinki, Finland) in a medium. The medium was retrieved to adapt the inverted ALI culture in the PneumaCult ALI basal medium (Stemcell, Vancouver, BC, Canada) once the epithelial cells grew to complete confluence. The transwell plates were positioned upside down to place the epithelial cells at the apical side. After the cell was seeded, it was incubated overnight in a medium, and the transwells were flipped back to the normal position in 24-well plates. PneumaCult-ALI Maintenance medium was added to an apical camber and PneumaCult Ex Basal Medium to the basal chamber of the wells when cell cultures reached 80% confluence. The medium was changed every second day after 14 days in ALI culture.
4.5. Transepithelial Migration of Eosinophil and Neutrophil
ALI-cultured apical nasal epithelial cells were treated with 2 × 106 conidia/cm2 for 2 h and then excess conidia were washed out to determine the effect of A. fumigatus on cell migration. ALI-cultured cells were positioned upside down to place the apical nasal epithelial cells on the underside, and one hundred microliters of the eosinophils or neutrophils (5 × 106/mL) were added at the basolateral side to enable the migration. After 4 h, apically migrated cells were counted by using light microscopy. To investigate the effect of eosinophil and neutrophil migration in the epithelial cells of the ECRS, NECRS, and normal control groups, cells isolated from the peripheral blood of each ECRS, NECRS, and normal control group were utilized.
The capacity of the fungi to disrupt epithelial barrier function was determined by measuring transepithelial resistance (TER) using EVOM-II (World Precision Instruments, New Haven, CT, USA). The values for cell-covered filters were calculated in standard units of ohms × square centimeter (Ω × cm2) after subtracting the resistance of blank filters, and TER was expressed as a percentage compared with the non-stimulated condition at time zero. The effect of ROS on the migration was determined by epithelial pretreatment with 5 mmol/L of glutathione.
4.6. Confocal Microscopic Evaluation of EETs and NETs
Purified eosinophils or neutrophils were cultured with A. fumigatus or A. alternata at a cell-to-fungi conidia ratio of 1:10 for 6 h at 37 °C. ETs were detected using Sytox Green (Life Technologies, Gaithersburg, MD, USA) staining in a FLUOstar Optima (BMG Labtech, Ortenberg, Germany) with a wavelength combination of excitation at 485 nm and emission at 538 nm. Cells, as a positive control, were treated with 50 nM of phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) for 2 h. Cells were pretreated with 30 μM of diphenyleneiodonium (DPI, Sigma-Aldrich) or 500 μM of MitoTempo (Invitrogen, Carlsbad, CA, USA) as a ROS inhibitor to determine the role of ROS in ET development.
4.7. Quantification of ECP, Neutrophil Elastase, and IL-8
Cell-free supernatants were collected after a 6 h incubation with eosinophils or neutrophils and A. fumigatus or A. alternata or 2 h treatment with PMA at a cell-to-fungi conidia ratio of 1:10. ECP (MyBioSource, San Diego, CA, USA) and IL-8 (Invitrogen) were measured for EETs using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. Neutrophil elastase (Invitrogen) and IL-8 for NETs were measured following the manufacturer’s directions. The detection limits were 0.39 ng/mL for ECP, 0.16 ng/mL for neutrophil elastase, and 2 pg/mL for IL-8.
4.8. Quantification of ROS in Eosinophils and Neutrophils
This study collected sinonasal polyp tissues from 30 patients with CRSwNP during endoscopic sinus surgery. We excluded patients who were younger than 18 years of age, had active inflammation, and had used antibiotics, antihistamines, or other medications for at least 4 weeks prior to surgery. The Institutional Review Board of Daegu Catholic Medical Center approved this study, and all the patients signed a consent form that outlined the study objectives. We classified ECRS as eosinophilic when more than an average of 70 eosinophils were found in the three densest areas on high-power field (×400) images.
Table 1 shows the clinical and demographic characteristics of patients with CRSwNP.
4.9. Determination of Fungal Protease
The protease activity of A. fumigatus and A. alternata was determined using a protease activity assay kit (Cayman, Ann Arbor, MI, USA). Fungal conidia were placed in 96-well black plates with a protease substrate for 20 min at RT. A total of 2.0 μg/mL trypsin was used as a standard sample. The value of protease activity was determined with an excitation wavelength of 485 nm and an emission wavelength of 520 nm using FLUOstar Optima.
4.10. Statistical Analysis
The Statistical Package for the Social Sciences version 25.0 (IBM Corp., Armonk, NY, USA) was used to analyze data obtained in the experiments, with data presented as the mean ± standard deviation. The clinical profiles of patients with CRS were compared using the Mann–Whitney U test and the two-sample t-test. Cell migration, EET and NET formation, IL-8, and fungal metabolic activity were analyzed using a one-way analysis of variance, followed by a Dunnett’s test. Differences with a p-value of ≤0.05 were considered statistically significant.