Oxidative stress enhances the expression of IL-33 in human airway epithelial cells
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Interleukin-33 (IL-33) is a cytokine belonging to the IL-1 family, and its possible involvement in the pathophysiology of COPD and viral-induced exacerbations has been demonstrated. IL-33 has been shown to be increased in the airway epithelial cells from COPD patients, but the regulating mechanism of IL-33 expression in airway epithelial cells remains largely unknown. In the current study, we examined whether oxidative stress, which participates in the pathogenesis of COPD, affects the expression of IL-33 in airway epithelial cells and also evaluated the effect during viral infection.
The involvement of oxidative stress in the expression of IL-33, and its signal pathway was examined after stimulation with hydrogen peroxide (H2O2), with or without stimulation by polyinosinic-polycytidylic acid [poly (I:C)], a synthetic analogue of dsRNA that mimics viral infection, or rhinovirus infection in NCI-H292 cells and primary human bronchial epithelial cells (HBECs). In addition, the effect of antioxidant, N-acetylcysteine (NAC) in the expression of IL-33 was compared between HBECs from healthy subjects and those from COPD patients.
Treatment with H2O2 significantly potentiated IL-33 expression in NCI-H292 cells, and the potentiation was reversed by NAC treatment. Mitogen-activated protein kinase (MAPK) inhibitors, but not nuclear factor-kappa B inhibitors, also significantly decreased the H2O2-potentiated IL-33 expression. In addition, H2O2 significantly potentiated the poly (I:C)- or rhinovirus-stimulated IL-33 expression. In HBECs from healthy subjects, H2O2-potentiated IL-33 expression and its reversal by NAC was also confirmed. Under the condition without H2O2-stimulation, treatment with NAC significantly decreased the expression of IL-33 in HBECs from COPD patients, but not in those from healthy subjects.
These results demonstrate that oxidative stress involves in the expression of IL-33 in airway epithelial cells via MAPK signal pathway and it augments IL-33 expression during viral infection. This mechanism may participate in the regulation of IL-33 expression in airway epithelial cells in COPD and the viral-induced exacerbations. Modulation of this pathway could become a therapeutic target for viral-induced exacerbations of COPD.
KeywordsCOPD Exacerbation IL-33 Oxidative stress Viral infection
A disintegrin and metalloproteinase 17
Analysis of variance
Chronic obstructive pulmonary disease
CXC motif chemokine ligand 8
Diffusing capacity of the lung for carbon monoxide
Dual oxidase 1
Epidermal growth factor receptor
Extracellular signal-regulated kinase
Fetal bovine serum
Forced expiratory volume in one second
Forced vital capacity
The global initiative for chronic obstructive lung disease
Human bronchial epithelial cells
HANK’s balanced salt solution
c-jun N-terminal kinase
Mitogen-activated protein kinase
Multiplicity of infection
Nicotinamide adenine dinucleotide phosphate
Nuclear factor-kappa B
Polymerase chain reaction
- poly (I:C)
Reactive oxygen species
Type 14 rhinovirus
Sodium dodecyl sulfate- polyacrylamide gels
Standard error of the mean
T helper 2 cell
Tumor necrosis factor alpha
Chronic obstructive pulmonary disease (COPD) was ranked sixth as a cause of death in 1990, but it is currently the third leading cause of death in the world . Exacerbations of COPD triggered by respiratory infections or environmental pollutants cause an acute deterioration in airway inflammation and lung function, and also lead to an increase in mortality and healthcare costs [2, 3, 4, 5]. The prevention of exacerbations remains a key therapeutic goal, but current treatments have only modest benefits and considerable adverse effects. Therefore, since new treatments are urgently needed, a better understanding of the mechanisms of COPD exacerbations is needed.
Interleukin-33 (IL-33) is a cytokine belonging to the IL-1 family, which includes IL-1α, IL-1β and IL-18. It is mainly a nuclear protein in several types of cells including endothelial cells, epithelial cells and fibroblasts [6, 7]. Once released into the extracellular compartment in response to tissue damage or inflammation, extracellular IL-33 has been shown to act as a potent inducer of inflammation [6, 7]. Especially, IL-33 promotes T helper 2 cell (Th2) immunity, which leads to allergic pathological changes via ST2 receptor, a member of the IL-1/Toll-like receptor family . IL-33 has been extensively characterized as functionally important in Th2-associated inflammatory diseases including asthma, atopic dermatitis and helminth infections . Although higher expression of IL-33 has been reported in patients with asthma , it has also been demonstrated in bronchial epithelial cells from those with COPD, especially in a severe stage of COPD [10, 11]. In a COPD mouse model and in vitro airway epithelial cells, increases of IL-33 have been also shown, especially during viral infection or after cigarette smoke exposure . Despite the increasing evidence suggesting the contribution of IL-33 to the pathophysiology of COPD and its exacerbations , the regulating mechanism of the expression of IL-33 in airway epithelial cells remains largely unknown.
Reactive oxygen species (ROS), including hydrogen peroxide (H2O2) derived from cigarette smoke or released by inflammatory cells, are reported to contribute to the pathogenesis of COPD and its exacerbations [13, 14]. In fact, oxidative stress markers such as H2O2 are elevated in the airways of COPD patients [15, 16, 17, 18], and higher amounts of H2O2 are detected in the airways during the exacerbations than in those in a stable condition [19, 20]. Pretreatment with H2O2 has been reported to augment the production of pro-inflammatory cytokines and chemokines in airway epithelial cells and inflammatory cells [21, 22]. Our previous report has also demonstrated that H2O2 augments viral-derived double-stranded RNA (dsRNA)-stimulated CXC motif chemokine ligand (CXCL) 8 release in human airway epithelial cells via the activation of Toll-like receptor 3 (TLR3) signal pathway . Concerning IL-33, an antioxidant, N-acetylcysteine (NAC) or glutathione (GSH), has been reported to inhibit the alternaria extract-augmented IL-33 release in an allergic mouse model, which suggests the possible involvement of oxidative stress in the regulation of IL-33 production . However, it has not been determined whether oxidative stress affects the expression of IL-33 and which signal pathways are participated in the regulating mechanisms in airway epithelial cells.
The present study, therefore, was designed to determine, using H2O2 and an experimental viral infection model, the following: (1) whether oxidative stress potentiates IL-33 expression in human airway epithelial cells and which signal pathways participate in the regulating mechanisms; (2) whether oxidative stress augments IL-33 expression in the dsRNA-treated or viral infected cells; and (3) whether antioxidant treatment decreases the expression of IL-33 in airway epithelial cells from COPD patients.
Poly (I:C) (polyinosinic-polycytidylic acid, sodium salt, double-stranded), SB203580, SP600125, U0126, SC-514 and BAY 11-7085 were purchased from Calbiochem (La Jolla, CA). H2O2, NAC and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) were from Sigma-Aldrich (St. Louis, MO).
Characteristics of healthy subjects and patients with COPD
68.0 ± 3.4
68.0 ± 3.8
45.6 ± 8.5**
86.8 ± 3.3
62.7 ± 3.0***
FEV1% of predicted (%)
108 ± 6.7
75.5 ± 6.7**
114 ± 4.2
85.2 ± 3.7***
Preparation of epithelial cells
NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line (ATCC, Manassas, VA) and primary human bronchial epithelial cells (HBECs), acquired from lobes resected from patients at surgery, the details of which are shown in Table 1, were cultured as previously described . Cells were grown to 80% confluence in culture plates and incubated in fetal bovine serum (FBS)- or growth factor-free medium for 24 h before treatment.
To investigate the effect of H2O2 on the expression of IL-33, the cells were harvested at 4 h, unless indicated otherwise, after treatment with H2O2 and stored at − 80 °C until the measurement. To examine the effect of H2O2 on the poly (I:C) or rhinovirus infection-induced IL-33 expression, H2O2 was added to the media 30 min before the treatment with poly (I:C) or rhinovirus . To evaluate the effects of inhibitors, various concentrations of NAC, p38-MAPK inhibitor (SB203580), JNK inhibitor (SP600125), ERK inhibitor (U0126), IKK-2 inhibitor (SC-514) or IκBα inhibitor (BAY11-7085) were added to the media 1 h before H2O2 treatment [27, 28]. In the control group, the cells were treated with medium in the absence of H2O2, poly (I:C), or inhibitors.
A stock solution of type 14 rhinovirus (RV14) [1.0 × 107 tissue culture infectious dose (TCID50)/ ml] was obtained from a patient with a common cold and the rate of RV14 release was quantified in the same manner as previously described in methods . NCI-H292 cells in culture plates were infected with RV14 at a multiplicity of infection (MOI) of 1 for 90 min in RPMI-1640 medium at 33 °C before the virus was removed and replaced with RPMI-1640 medium [26, 30, 31]. NCI-H292 cells were treated with H2O2 or vehicle 30 min prior to RV14 infection. Cells were infected with RV14 for 90 min and the virus was removed and replaced with medium containing 200 μM H2O2 or vehicle. In some experiments, cells were pretreated with NAC 1 h prior to infection. After the cells were incubated for 1 to 48 h at 33 °C, the whole cell extractions were harvested and stored at − 80 °C until required.
Cell viability assay
Cell viability in NCI-H292 cells were evaluated by use of the MTT assay and in HBECs were evaluated by the lactate dehydrogenase (LDH) activity using Cytotoxicity Detection KitPLUS (Sigma-Aldrich) as previously described . Cell viability was calculated as the percentage of viable cells among vehicle-treated cells.
Detection of mitogen-activated protein kinase (MAPK) phosphorylation by immunoblotting
Cells were seeded in 6-well plates at a density of 1 × 105/ml. At 80% confluence, and maintained in FBS-free medium for 24 h before stimulation. To evaluate the inhibitory effect of NAC on the expression of phospho-p38 (p-p38), c-jun N-terminal kinase (p-JNK) and Extracellular Signal-regulated Kinase 1/2 (p-ERK1/2), the cells were treated with NAC 60 min prior to H2O2 stimulation. After 15 min H2O2 stimulation, cells were washed with ice-cold HANK’s balanced salt solution (HBSS) and homogenized in cell lysis buffer (0.05% TritonX, 35 mM Tris-HCl, pH 7.4, 0.4 mM EGTA, 10 mM MgCl2, 1 μM phenylmethylsulfonyl fluoride, 100 μg/ml aprotinin and 1 μg/ml leupeptin) at 4 °C. Samples were solubilized in sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) sample buffer. Equal amounts of protein were loaded and the separated by electrophoresis on 12.5% SDS-PAGE. After electrophoresis, the separated proteins were blotted on a PolyVinylidene DiFluoride (PVDF) membrane (Bio-Rad Laboratories, Herculer, CA). Rabbit monoclonal anti-phospho-p38 MAPK antibody, rabbit monoclonal anti-p38 MAPK antibody, rabbit monoclonal anti-phospho-JNK antibody, rabbit monoclonal anti-JNK antibody, rabbit polyclonal anti-phospho-ERK1/2 antibody, rabbit polyclonal anti-ERK1/2 antibody (1:1000 dilution, all from Cell Signaling Technology, Danvers, MA) and mouse monoclonal anti–β-actin antibody (1:10000 dilution; Sigma) were used to detect the target proteins. Appropriate peroxidase-conjugated secondary antibodies were used. Binding antibodies were detected using ECL-prime (Amersham Biosciences, Buckinghamshire, UK) and visualized with a chemiluminescence imaging system (LAS-4000 mini; Fujifilm, Tokyo, Japan). Each band intensity was quantified by densitometry (Quantity One software, Bio-Rad).
Detection of IL-33 by nuclear extraction and immunoblotting
Cells were treated with 200 μM H2O2 for 0 to 12 h. After washing with HBSS, cells were homogenized in cell lysis buffer to obtain the nuclear fraction using Nuclear Extraction Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions. The following separation, blotting, detection and visualization procedures were performed in the same manner as for MAPK phosphorylation immunoblotting. Target proteins were detected with mouse monoclonal anti-IL-33 antibody (1:1000 dilution, Enzo Life Sciences, Exeter, UK) or mouse monoclonal anti-lamin A/C antibody (1:400 dilution; Santa Cruz Biotechnology, Santa Cruz, CA).
Real-time polymerase chain reaction (PCR)
Total RNA was isolated from the cells to prepare cDNA using High capacity of RNA to cDNA kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. cDNA was amplified with specific primers to IL-33 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the 7500 Real-Time PCR System (Applied Biosystems). Primers used to amplify cDNA were from TaqMan Gene Expression Assays (Applied Biosystems): IL-33 (catalogue number Hs00369211_m1), GAPDH (Hs99999905_ml). Relative quantification of different transcripts was determined with the comparative threshold cycle (CT) method using GAPDH as the endogenous control. Results were calculated as fold change over control.
Data are expressed as the mean ± SEM. GraphPad Prism (GraphPad Software Inc., SanDiego, CA) was used to perform statistical tests. Experiments with multiple comparisons were evaluated using one way analysis of variance (ANOVA) by Bonferroni’s test to adjust for multiple comparisons. Wilcoxon matched-pairs signed rank test or Mann-Whitney U test was used for single comparison. Significance values were defined as p < 0.05.
Effect of H2O2 on the expression of IL-33 in NCI-H292 cells
Involvement of MAPK (p38, JNK, ERK1/2) and NF-κB signaling pathways in H2O2-potentiated IL-33 expression
Effect of H2O2 on a synthetic viral dsRNA analogue, poly (I:C)- or viral-potentiated IL-33 expression
Involvement of oxidative stress in the expression of IL-33 in HBECs from COPD patients
The effects of the treatments on cell viability were assessed with the MTT assay in NCI-H292 cells and with the LDH assay in HBECs. Cell viability in the NCI-H292 cells was more than 77.2% after the treatment with H2O2, excluding the concentration at 500 μM, poly (I:C), rhinovirus infection and the inhibitors. In the HBECs, cell viability was more than 79.8% after the treatment with H2O2 and NAC.
In the present study, we showed that exogenous H2O2 treatment potentiates the expression of IL-33 in NCI-H292 cells and primary HBECs and the potentiation is reversed by treatment with an antioxidant, NAC. In addition, we demonstrated that NAC treatment decreased the expression of IL-33 in HBECs from COPD patients, but not in those from healthy subjects. These results suggest that oxidative stress is involved in the expression of IL-33 in airway epithelial cells. Previously, IL-33 has been reported to be upregulated in human airway epithelial cells by cigarette smoke exposure [11, 37]. As cigarette smoke contains ROS including H2O2 [38, 39], cigarette smoke-potentiated IL-33 expression is consistent with the current findings. In the present study, we also demonstrated that oxidative stress potentiates the amount of IL-33 protein in the nucleus in airway epithelial cells. In a previous study, Uchida M, et al. demonstrated that an antioxidant, NAC or GSH, inhibits the alternaria extract-augmented IL-33 release in an allergic mice model, and they also demonstrated that NAC or GSH prevents extracellular secretion of IL-33 from IL-33-overexpressed human bronchial epithelial cells exposed to alternaria extract . These reports support our current findings that the expression of IL-33 is regulated by oxidative stress.
H2O2 has been reported to activate not only the MAPK signal pathway, but also the NF-κB signal pathway in various cells including airway epithelial cells [23, 28, 34]. In the present study, we clarified the involvement of MAPK signal pathway in the mechanism of H2O2-augmented IL-33 expression in airway epithelial cells by showing the inhibitory effect of p38 MAPK, JNK or ERK1/2 inhibitors. In addition, we demonstrated that these inhibitors also decreased the expression of IL-33 in HBECs from COPD patients, which suggest that MAPK signaling is involved in the regulation of IL-33 expression in airway epithelial cells in COPD. Using murine alveolar epithelial cells or macrophage cells, the involvement of the MAPK signal pathway has been demonstrated in the IL-6 group of cytokines, oncostatin M- or respiratory syncytial virus infection-augmented IL-33 expression and production [40, 41]. These reports support our current results. On the other hand, in our present study, pretreatment with NF-κB inhibitors (an IKK-2 inhibitor, SC-514, or an IκBα inhibitor, BAY 11-7085) did not inhibit the H2O2-augmented IL-33 expression, suggesting that the NF-κB signal pathway is not involved in the mechanism of H2O2-augmented IL-33 expression in airway epithelial cells. In murine mast cells, IκB-α inhibitor (BAY11-7085) did not also inhibit immunoglobulin E (IgE) activation-induced IL-33 expression , which is consistent with our results. However, in human corneal epithelial cells, cardiac cells or murine bone marrow-derived dendritic cells, IκB-α inhibitor (BAY11-7082) and NF-κB inhibitor (quinazoline or dimethylfumarate) was reported to block TLR ligands-, tumor necrosis factor alpha (TNF-α) or ragweed pollen-potentiated IL-33 expression and production [43, 44, 45]. These reports are inconsistent with our present results. This discrepancy might result from differences in the stimulating substance, in the cell types used or in the experimental conditions including the inhibitor used.
In the present study, we showed that combination treatment of H2O2 and a synthetic viral dsRNA analogue and a TLR3 ligand, poly (I:C) or rhinovirus-infection significantly increased the expression of IL-33 compared with each treatment alone. Although the molecular mechanisms by which H2O2 potentiates poly (I:C)- or rhinovirus-induced IL-33 expression remain uncertain, previous reports have proposed possible mechanisms. TLR3 stimulation or rhinovirus infection has been demonstrated to increase the expression of IL-33 in human airway and corneal epithelial cells, and smooth muscle cells [43, 46, 47]. In addition, the MAPK signal pathway has been reported to be activated by TLR3 stimulation or rhinovirus infection in human airway epithelial cells [28, 48, 49]. In our previous studies, we demonstrated that oxidative stress enhances the expression of TLR3, and that cigarette smoke potentiates TLR3-ERK signal pathway in human airway epithelial cells [23, 27]. These results suggest that the MAPK signal pathway is involved in the H2O2-potentiated IL-33 expression induced by poly (I:C) or rhinovirus infection. Recently, Hristova M, et al. have reported that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase homolog, dual oxidase 1 (DUOX1)-derived H2O2, is involved in the alternaria-induced IL-33 release in airway epithelial cells. Activated DUOX1-induced H2O2 has been shown to stimulate A disintegrin and metalloproteinase 17 (ADAM17), which is responsible for the shedding of EGFR pro-ligand and the activation, and leads to the activation of the EGFR-ERK signal pathway . These reports may suggest that oxidative stress augments the rhinovirus-stimulated IL-33 expression via TLR3-EGFR signal pathway. Further studies are necessary to prove this hypothesis.
In the comparison of the expression of IL-33 in the disease status of COPD, there was no increase in the expression of IL-33 in HBECs from COPD patients compared to HBECs from healthy subjects, which is inconsistent with a result previously reported that higher expression of IL-33 appeared in the airway epithelial cells from patients with COPD . However, this higher expression of IL-33 is shown in severe COPD in GOLD stage IV. Another group has also reported that an increase of the IL-33 protein level in the whole lung appears only from severe COPD in GOLD stage III / IV . In our current study, all of the patients evaluated were mild COPD in GOLD stage I-II, which might explain the discrepancy. In the current study, we also showed that NAC treatment decreased the expression of IL-33 in HBECs from COPD patients, but not in those from healthy subjects, suggesting that oxidative stress is involved in the expression of IL-33 in airway epithelial cells in patients with COPD. We have previously reported an increased level of oxidative stress in HBECs of COPD patients compared to those in healthy subjects  and, therefore, it could be thought that NAC treatment decreased the increased level of oxidative stress in the HBECs of COPD patients, which affects the expression of IL-33. However, there remains the discrepancy that an increased level of oxidative stress is suggested in the HBECs from COPD patients and NAC treatment decreased the IL-33 expression from the basal levels only in COPD patients, but there is no increase in the expression of IL-33 in the HBECs from COPD patients compared to healthy subjects. Several negative regulations of IL-33 expression have been demonstrated, such as by lipopolysaccharide treatment via TLR signaling in NCI-H292 cells, or by one of short noncoding RNAs, microRNA-487b in human monocytes [52, 53]. These unknown negative regulatory mechanisms might affect the expression of IL-33 in the airway epithelial cells of COPD patients. Further studies are needed to clarify this point.
As a limitation of the present study concerning HBECs, there is a possibility that sex differences and smoking status might affect the results because HBECs were derived from 5 female healthy subjects and 1 female COPD patient. In addition, all of the healthy subjects were non-smokers, whereas all of the COPD patients were ex-smokers. Also, in the experiments in HBECs from COPD patients and healthy subjects, the evaluation of IL-33 expression was performed by measuring the levels of mRNA expression and was not confirmed by examining the protein expression.
We demonstrated that oxidative stress is involved in the expression of IL-33 in airway epithelial cells via the MAPK signal pathway and that it augments IL-33 expression during viral infection. This mechanism may participate in the regulation of IL-33 expression in airway epithelial cells in COPD and in viral-induced exacerbations. Modulation of this pathway could be a therapeutic target for viral-induced exacerbations of COPD.
We thank Mr. Brent Bell for reading this manuscript.
This work was supported by Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number 16 K09569), Research Grant 2015 from Terumo Foundation for Life Sciences and Arts, and Research Grant 2016 from Novartis Pharma K.K.
Availability of data and materials
Source data and material will be made available upon reasonable request.
HA, AK, YS, SY and MY analyzed the data and HA and AK drafted the manuscript. HA, AK, HS and MI contributed to the conception and design of the original study and contributed substantially to the manuscript. YS, TN and MN assisted with the recruitment of patients and obtaining their informed consent. MY, TI, NF and YO assisted with technical advice and the interpretation of results. All authors approved the final version for publication.
Ethics approval and consent to participate
All experiments in the study were approved by ethics committee of Tohoku University Graduate School of Medicine and written informed consent was obtained from all participating patients.
Consent for publication
The authors declare that they have no competing of interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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