The first two authors contributed equally to this study.
Asia sand dust (ASD) is known to cause various human diseases including respiratory infection. The aim of this study was to examine the effect of ASD on inflammatory response in human middle ear epithelial cells (HMEECs)
Cell viability was assessed using the cell counting kit-8 assay. The mRNA levels of various genes including
We observed dose-dependent decrease in HMEEC viability. ASD exposure significantly increased
Environmental ASD exposure can result in the development of otitis media.
Otitis media (OM) is a multifactorial disease caused by many agents, including viral and bacterial infections, biofilm formation, congenital anomaly, and environmental factors such as smoking, and air pollution [
The invasion of Asian sand dust (ASD) is a seasonal phenomenon in Asia, which arises from the Gobi and Taklimakan desert plains of China. ASD is known to spread over large areas of East Asia every spring, including East China, North and South Korea, and Japan. ASD is composed of PM less than 10 μm in diameter (PM10), as well as several hazardous chemical compounds and microbial agents.
ASD is known to cause various human diseases, including allergies, chronic pulmonary injury, and cardiovascular and respiratory dysfunction [
The major aim of this study was to investigate the potential risk of ASD on the development of OM. To this end, we investigated the cytotoxic effect of ASD and the changes of mucin gene and inflammatory cytokine expression in human middle ear epithelial cells (HMEECs). Additionally, we evaluated the histopathologic changes in the middle ears of rats injected with ASD.
ASD was collected from the atmosphere on March 16, 2009 outside the Gachon University building in Korea, using a high volume air sampler (HV500F; Sibata Scientific Technology, Ltd., Saitama, Japan) at a flow rate of 500 L/min. These ASD samples were prefiltered into filter packs (Prefilter AP, 124 mm; EMD Millipore, Bedford, MA, USA) and sieved through a filter with a 10-μm pore size, after mixing with phosphate buffered saline (PBS) in a 15-mL tube. The filtered ASD particles were sterilized in an autoclave at 121°C for 15 minutes; subsequently, these particles were weighed. Each sample was transferred to a 1.5-mL tube. The filtered PM was stored at –20°C until further use. The particle diameter of the samples was measured (a total of 600 particles) under a scanning electron microscope (JSM-5800; JEOL Ltd., Tokyo, Japan). The mean distribution peak of the ASD particle diameter was observed at 6 μm. Chemical components of the ASD particles were analyzed via X-ray fluorescence spectrometry (PHILIPS pw2404; Philips, Eindhoven, The Netherlands) at the Korea Basic Science Institute. The chemical composition of ASD was determined to be as follows: 78.4% SiO2, 9.35% Al2O3, 2.52% K2O, 2.41% Na2O, 2.06% Fe2O3, and 1.74% CaO. MaO, TiO2, P2O5, and MnO made up less than 0.1% of the total composition.
HMEECs (kindly provided by Dr. David J. Lim, House Ear Institutes, Los Angeles, CA, USA), immortalized with the
Cell viability was measured using the cell counting kit (CCK-8, Dojindo Laboratories, Kumamoto, Japan). HMEECs were seeded in 96-well plates, with each well containing 1×104 cells. The following day, cells were treated with 0, 6.25, 12.5, 25, 50, 100, 200, 300, or 400 μg/mL of ASD. CCK-8 solution was added to each well after 24 hours, and the plates incubated for 150 minutes at 37°C. The contents of the plates were mixed using a shaker (at room temperature for 5 minutes), and the optical density was measured at 450 nm using a microplate reader (Spectra Max plus 384; Molecular devices, Sunnyvale, CA, USA).
Real-time polymerase chain reaction (RT-PCR) was performed using a LightCycler 480 Real-Time PCR System (Hoffmann-LaRoche, Basel, Switzerland). Each reaction mixture contained 10 μL of LightCycler 480SYBR Green I Master (Hoffmann-LaRo che), 1 μL of 4 pmol sense and antisense primers, and 0.4 μL of cDNA, in a final volume of 20 μL. Reaction mixtures were incubated at 95°C for 5 minutes to activate the FastStart Taq DNA Polymerase; this was followed by amplification for 50 cycles (one cycle: 15 seconds at 95°C, 30 seconds at 60°C, and 30 seconds 72°C). The data was analyzed using the LightCycler 480 software 1.5 (Hoffmann-LaRoche). Target mRNA expression was normalized to that of
HMEECs were stimulated with 0, 50, 100, 200, or 400 μg/mL of ASD for 24 hours. After treatment of HMEECs with ASD, the medium was removed and the cells were washed twice in PBS (10 mM, pH 7.4). Lysis buffer (0.2 mL, PRO-PREP; INtRON, Seongnam, Korea) was added, and the cells were incubated for 1 hour at –20°C. The cells were then centrifuged at 13,000 ×g for 10 minutes at 4°C. The supernatant containing the total cell lysate was collected and stored at –80°C. Protein concentration of the lysates was measured using Quick Start Bradford 1 dye reagent (Bio-Rad, Hercules, CA, USA). Equal quantities of protein were analyzed using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein content of the gels was transferred to a polyvinyl difluoride membrane (Hybond P; Amersham Biosciences Corp., GE Healthcare, Buckinghamshire, UK), and the membranes blocked with 25 mM Tris and 120 mM NaCl and 0.05% Tween-20 containing 5% (w/v) skim milk for 1 hour at room temperature. Membranes were probed with antibodies against COX-2 (1:200; Santa Cruz Biotechnology, Dallas, Texas, USA) and β-Actin (1:3,000; Sigma-Aldrich, St. Louis, MO, USA), followed by peroxidase-conjugated antimouse IgG (1:5,000; Vector Laboratories, Burlingame, CA, USA). The membranes were developed using an ECL detection kit (Pierce, Rockford, IL, USA) and the signal captured using an image reader (LAS3000; Fuji Photo Film, Tokyo, Japan).
Fifty pathogen-free, male Sprague Dawley rats weighting 150–200 g (Orient Bio, Seongnam, Korea) were used for this study. The protocols used in this study were approved by the Institutional Animal Care and Use Committee (IACUC); the animals were cared for according to the Guidelines of Animal Experiments of the Korea University Guro Hospital. The rats were assigned randomly to one of 5 groups: 4 groups received a unilateral tympanic membrane ASD injection (on days 1, 3, 5, and 14; each group, n=5) and the control group received a PBS vehicle injection (vehicle control group, n=5). ASD (400 μg/mL) dissolved in PBS was injected into all experimental mice (4 groups) at all time-points using a 27-gauge spinal needle. The rats were injected with ASD and subsequently sacrificed on days 1 (group 1), 3 (group 2), 5 (group 3), or 14 (group 4) postprocedure. Anesthesia was performed with an intraperitoneal injection of ketamine hydrochloride (30 mg/kg) and xylazine hydrochloride (5 mg/kg).
Five rats from each experimental group (groups 1, 2, 3, and 4) and five rats from the control group were sacrificed; the bullae (including the middle and inner ear) were isolated from the ears of all rats. Following dissection, the bullae were fixed overnight in a 4% paraformaldehyde solution, and subsequently decalcified using ethylenediaminetetraacetic acid. The specimens were embedded in paraffin, sectioned into 5-μm slices, and stained with hematoxylin and eosin (Sigma-Aldrich).
All data was expressed as mean±standard deviation. One-way analysis of variance (ANOVA) was used to determine the statistically significant differences between the control and experimental groups at each time or dose point. Scheffe
The effect of ASD on HMEEC viability after exposure to ASD for 24 hours was initially investigated. Microscopic evaluation showed higher cell debris in the ASD-treated groups (100 and 400 μg/mL) than in the control HMEECs; in addition, there was a sharp decrease in the viable cell population in the group treated with 400 μg/mL of ASD (
The CCK-8 cell viability assay demonstrated that ASD caused a dose-dependent decrease in cell viability. Cell viability was considerably decreased in groups exposed to higher concentrations of ASD (>100 μg/mL). Especially, administration of 400 μg/mL of ASD resulted in approximately 60% of viability compared to that of the control group (
The expression of
In addition, we investigated the possible activation of apoptosis and reactive oxygen species (ROS) reactions in HMEECs treated with ASD. Interestingly, the pro-apoptotic gene including
The change in
The morphology of middle ear epithelium was observed at different time points after ASD treatment by light microscopy. Normal middle ear epithelium was thin and free of inflammatory cells as seen in the control groups (
The major air pollutants include sulfur oxides, nitrogen dioxides, ozone, and inhalant PM. Many previous studies have demonstrated that air pollutions cause adverse effects on human health, such as respiratory inflammation, asthma, heart disease, and lung cancer [
ASD has become a serious concern in East Asia over the past decade as it can cause diverse harmful effects on human health; this is particularly of concern as ASD is characterized by long-range transport, and can affect an increasing proportion of the population each year. Several epidemiological studies have shown that the annual ASD event to be responsible for numerous negative effects on human health, including cardiovascular and respiratory problems [
Since the cause of OM is multifactorial and includes environmental factors such as smoking and air pollution [
The inflammatory response in the middle ear significantly contributes to the development and progression of OM. Many studies have implicated tumor necrosis factor-α (TNF-α) and cyclooxygenase 2 (COX-2) as a key inflammatory cytokines in OM [
Mucins, a family of high molecular weight, heavily glycosylated proteins, are produced by mucous glands in the epithelial tissues and perform various functions, from lubrication to cell signaling [
We also explored the effect of ASD on middle ear pathology
In this report, we have demonstrated the effects of ASD on the middle ear using HMEECs (
In conclusion, ASD caused a decrease in cell viability and increase the inflammation, mucin production, apoptosis, and oxidative stress-related genes (mRNA) in HMEECs. Additional studies conducted in animal model clearly demonstrated that ASD exposure caused an inflammatory response in the middle ear of rats. Overall, these findings demonstrated that exposure to ASD can cause damage to the middle ear epithelium, and induce an inflammatory responses. In addition, our data indicates that ASD may be a risk factor for OM, which provides an important link between environmental air pollution and middle ear diseases.
▪ We evaluated the effect of Asia sand dust (ASD) in human middle ear epithelial cells (HMEECs).
▪ ASD exposure decreased the HMEEC viability dose-dependently.
▪ ASD exposure significantly increased inflammation-related genes (
▪ ASD exposure significantly increased mucin-pro ducing genes (
▪ Environmental ASD exposure can result in the development of otitis media.
No potential conflict of interest relevant to this article was reported.
This study was supported by the Korea Ministry of Environment as part of “The Environmental Health Action Program” (2012 001360002).
Microscopic images of human middle ear epithelial cell morphology after exposure to Asia sand dust (ASD) for 24 hours. (A) The control group showed 80% of confluence and unified morphology. In contrast, the ASD-treated groups (B, 100 μg/mL; C, 200 μg/mL; and D, 400 μg/mL) demonstrated ASD particles on the cells and many cells were detached from the culture plate in the ASD groups, compared to what was observed in the control group.
Cytotoxicity testing of human middle ear epithelial cells (HMEECs) exposed to Asia sand dust (ASD). Cells were treated with the indicated concentrations of ASD for 24 hours. Cell viability was calculated as the percentage of the viability of the control. HMEEC viability decreased in a dose dependent manner (0–400 μg/mL) when treated with ASD. Especially, administration of 400 μg/mL of ASD resulted in approximately 60% of viability compared to that of the control group. The results presented in the graph are from three independent experiments; the error bars indicate mean±SD. *
Asia sand dust (ASD) stimulates the expression of the inflammation and mucin production genes in human middle ear epithelial cells (HMEECs). HMEECs treated with ASD for 24 hours were subjected to quantitative real-time polymerase chain reaction analysis. HMEECs displayed an increase in inflammatory response and mucin gene expression upon stimulation with ASD in a dose-dependent manner. (A)
Expression levels of the apoptosis and reactive oxygen species production marker genes were investigated. The treatment of Asia sand dust (ASD) (400 μg/mL) for 24 hours resulted in the elevation of
The levels of COX-2 protein increased when exposed to Asia sand dust (ASD) for 24 hours. Human middle ear epithelial cells were treated with ASD for 24 hours, at various concentrations (0, 50, 100, 200, or 400 μg/mL) and western blot analysis showed an increase of expression of COX-2 proteins in a dose-dependent manner. Results were obtained from three independent experiments. Beta-actin was used as the loading control.
Histological changes in the middle ear are observed resulting from Asia sand dust (ASD) injection. Each panel displays representative images of a middle ear cavity at specifi c time points after ASD treatment. (A) shows the control animals (n=10) that received only phosphate buffered saline (vehicle). (B –E) display images of the middle ears taken at 1 (B), 3 (C), 5 (D), and 14 days (E) after ASD injection (each group, n=10). We observed a considerable thickening of the middle ear epithelium on days 1 and 3, which was gradually reduced by day 5. The thickened middle ear mucosa was normalized by day 14 (H&E, ×100, scale bar 100 μm).
Histopathologic finding of otitis media demonstrated in Asia sand dust (ASD) injected rats. The 5 panels display representative images of the middle ear at a specific time point after ASD exposure: (A) control group, (B) day 1, (C) day 3, (D) day 5, and (E) day 14. Images demonstrate the pathological changes occurring as a result of ASD exposure. In particular, 3 days after ASD injection (C), we observed the appearance of cilia in the epithelium of the middle ear (arrow) and a considerable increase in the mucosal thickness (double-headed arrow) in the middle ear cavity. In addition, the middle ear cavity also displayed infiltration of the inflammatory cells (open arrow) (H&E, ×400, scale bar 100 μm)
Oligonucleotide primer sequences for quantitative reverse transcriptase polymerase chain reaction
Gene | Primer sequence (forward) | Primer sequence (reverse) | Annealing temperature (°C) | Product size (bp) |
---|---|---|---|---|
5-TTGCTGGCAGGGTTGCTGGT-3’ | 5-TTGCTGGCAGGGTTGCTGGT-3’ | 60 | 86 | |
5-GAGGCCAAGCCCTGGWG-3’ | 5-CGGGCCGATTGATCTCAGC-3’ | 60 | 91 | |
5-CAGCACMCCCCTGTTTCAM-3’ | 5’-GCGCACAGAGGATGACAGT-3’ | 60 | 99 | |
5-GCCTACGAGGACTTCAACGT-3’ | 5’-CCTTGATGACCACACGGGTG-3’ | 60 | 79 | |
5’-AGACCTGTGGGAAGCGAAAA-3’ | 5-TCATCCATTGCTTGGGACGG-3’ | 60 | 106 | |
5’-CCAAGGTGCCGGAACTGA-3’ | 5’-CCCGGAGGAAGTCCAATGT-3’ | 60 | 57 | |
5-TCCCTCGCTGCACAAATACTC-3’ | 5’-ACGACCCGATGGCCATAGA-3’ | 60 | 72 | |
5-CTCAGCGGMTCAATCAGCTGTG-3’ | 5-AGGAACACGACAATCAGCCTTA-3’ | 60 | 284 | |
5’-GTAATGGACCAGTGAAGGTGTG-3’ | 5’-CAATTACACCACAAGCCAAACG-3’ | 60 | 375 | |
5-TCGCCCCACTTGATTTTGG-3’ | 5’-GCAAATTCCATGGCACCGT-3’ | 60 | 105 |