International Journal of Pediatric Otorhinolaryngology 121 (2019) 109–113

Contents lists available at ScienceDirect

International Journal of Pediatric Otorhinolaryngology

journal homepage: www.elsevier.com/locate/ijporl

Expression of endoplasmic reticulum stress-related mRNA in otitis media T

with effusion
Dae Woong Kanga, Sung Hwa Donga, Sang Hoon Kima, Young Il Kimb, Dong Choon Parkc,
Seung Geun Yeoa,b,d,∗
a
Department of Otorhinolaryngology-Head and Neck Surgery, Kyung Hee University School of Medicine, 23 Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, Republic of
Korea
b
Medical Science Research Institute, Kyung Hee University Medical Center, Seoul, Republic of Korea
c
Department of Obstetrics and Gynecology, St. Vincent's Hospital, The Catholic University of Korea, Suwon, Republic of Korea
d
Medical Research Center for Bioreaction to Reactive Oxygen Species and Biomedical Science Institute, School of Medicine, Graduate School, Kyung Hee University, Seoul,
Republic of Korea

ARTICLE INFO ABSTRACT

Keywords: Objectives: The endoplasmic reticulum (ER) is an intracellular organelle involved in the synthesis and secretion
Endoplasmic reticulum stress of proteins. The ER stress response, which protects cells from cytotoxic proteins such as unfolded proteins, is
Otitis media with effusion related to several diseases including inflammation. In this study, we investigated the effect of ER stress on the
Pathophysiology pathophysiology of otitis media with effusion (OME).
Methods: Thirty-nine pediatric patients who were diagnosed with OME and underwent ventilation tube insertion
were enrolled in this study. Exudate from the middle ear cavity was collected through ventilation insertion, and
ER stress gene expression was analyzed via real-time polymerase chain reactions(PCR).
Results: There were no significant differences in ER stress-related mRNA expression between effusion culture-
positive and culture-negative groups (p > 0.05). Expression of the C/EBP-homologous protein (CHOP) was
higher in the otitis-prone group than in the non-otitis-prone group (p < 0.05). The most common type of fluid
was mucoid, and inositol-requiring enzyme 1α expression was higher in serous fluid than in mucoid, muco-
purulent, or purulent fluid (p < 0.05).
Conclusions: Endoplasmic reticulum stress-related responses are activated in pediatric OME patients, and specific
ER-stress related pathways are related to both the characteristics of fluid and the frequency of OME. Thus, ER
stress-related responses affect the pathophysiology of OME in pediatric OME patients.

1. Introduction
Otitis media with effusion (OME) is a disease in which exudative fluid
accumulates in the middle ear cavity without symptoms of acute infec-
tion. The pediatric patient is usually asymptomatic, but OME may cause
mild fluctuating hearing loss and therefore affect language development,
behavior, and school performance [1]. OME usually occurs after any
symptoms of acute otitis media have resolved. About 90% of children
will have OME before school age. The point prevalence is 7%–13%, with
a peak during the first year of life, and the per-year period prevalence is
15%–30% [2]. Although the etiology of OME is not fully understood,
Eustachian tube dysfunction is the primary etiology identified to date.
Eustachian tube dysfunction may also be caused by acute otitis media,
upper respiratory infection and gastroesophageal reflux disease, as well
as by craniofacial disorders such as Down's syndrome and cleft palate and
adenoid vegetation, all of which may be risk factors for OME [3–6]. The
middle ear cavity constantly secretes mucus to prevent bacteria from
entering from the nasopharynx. This mucus contains immunoglobulins,
lysozyme, lactoferrin, and other complementary components which
protect the middle ear cavity from infection. Therefore, if a problem
occurs in this defense mechanism, OME can develop after acute infection.
Bacteria, primarily Haemophilus influenza, Streptococcus pneumoniae, and
Moraxella catarrhalis, have been detected in 20–40% of OME exudates by
conventional bacterial culture and in nearly 80% by PCR methods [7,8].
Viscosity of the fluid varies depending on the concentrations of mucin
and glycoprotein produced in middle ear cavity [9]. The exact relation-
ship between the viscosity of the fluid and the presence of bacteria has
not yet been clarified; however, in effusions with high viscosity, bacteria

∗
Corresponding author. Department of Otorhinolaryngology-Head and Neck Surgery, Kyung Hee University School of Medicine, 23 Kyungheedae-ro,
Dongdaemun-gu, Seoul, 02447, South Korea.
E-mail addresses: yeo2park@gmail.com, yeo2park@khmc.or.kr (S.G. Yeo).

https://doi.org/10.1016/j.ijporl.2019.03.006
Received 1 January 2019; Received in revised form 4 March 2019; Accepted 6 March 2019
Available online 09 March 2019
0165-5876/ © 2019 Published by Elsevier B.V.
D.W. Kang, et al. International Journal of Pediatric Otorhinolaryngology 121 (2019) 109–113

is cultured more frequently than in serous effusions and detection under external auditory canal was done before ventilation tube insertion. After
the microscope is also higher. Therefore, a correlation between viscosity an incision was made in the anterior inferior quadrant of the tympanic
of the fluid and presence of bacteria does exist [10]. membrane with a myringotome, effusion fluid was collected through the
The endoplasmic reticulum (ER) is the first organ involved in the incision from the middle ear cavity using a collector (Xomed Trace
synthesis and secretion of almost all proteins produced by intracellular Products, Jacksonville, FL, USA). A tympanostomy tube was then inserted
organelles [11]. These proteins are recognized as normal proteins by and the procedure was ended if there was no active bleeding. The effusion
the ER chaperones [12,13]. Accumulated unfolded proteins or protein fluid sample was transferred to an Eppendorf tube and kept at −80 °C.
aggregates induced by stresses such as hypoxia and oxidative conditions
are toxic to cells [14]. This toxicity is mitigated by activation of cellular 2.3. Bacterial culture
defense mechanisms, including ER stress responses and unfolded pro-
tein responses (UPR) [15,16]. The aseptically-collected effusion fluid was placed in culture tubes
ER stress responses are important in various diseases. For example, cy- (Xomed Trace Products) and then cultured in blood agar medium
tokines produced by ER stress responses induce inflammatory responses, (Hangang, Kun-po, Korea). Bacteria were transferred to the medium,
including the expression of pro-inflammatory cytokines, which are asso- incubated at 37 °C for 3 days, and identified by gram stain, catalase test,
ciated with obesity, type 2 diabetes, cancer and intestinal bowel disease. and oxidase test.
Pro-inflammatory cytokines, such as TNF , IL-1 , and IL-8, are also found in
OME exudates and are involved in chronic OME by increasing fluid viscosity 2.4. RNA extraction and real-time PCR
through activation of mucin production [17–20]. ER-stress responses were
recently shown to increase mucin production in human nasal mucosa [21]. All RNAs were purified from the effusion fluid sample with TRIzol re-
In addition, IL-8, which has been detected in the fluid of 92–100% of OME agent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA,
patients, is involved in chronic inflammatory diseases, such as rheumatoid USA). The first-strand cDNA synthesis used 1 μg of total RNA according to
arthritis, inflammatory bowel disease, psoriasis, and palmoplantar pustu- the manufacturer's instructions using a reverse transcription system with
losis. IL-8 is also produced through ER-stress responses in patients infected random hexamers (Promega, Madison, WI, USA). Primer sequences are
with HBV, causing chronic inflammation of hepatocytes [22–24]. shown in Table 1. Real-time PCR was performed with the StepOnePlus real-
Few studies to date have analyzed ER stress responses in OME. time PCR system using the Power SYBR Green PCR Master Mix (Applied
Although OME can be caused by chronic inflammation of the middle Biosystems, Foster City, CA, USA). PCR was processed with 2 μl of cDNA
ear cavity induced by Eustachian tube dysfunction resulting from an placed in 20 μl of a reaction mixture containing 10 μl of Power SYBR Green
acute infection, such as acute otitis media, the overall pathophysiology PCR Master Mix, 2 μl of primer, and 7 μl of PCR-grade water. The amplifi-
of OME is not yet known. Therefore, the relationship between the pa- cation protocol consisted of an denaturation at 95 °C for 10 min, followed by
thophysiology of OME and ER stress should be investigated. This study 40 cycles at 95 °C for 15 s and annealing and extension at 60 °C for 1 min.
therefore assessed the expression of ER stress-related genes as a func- formula 2–(target gene–β-actin) was applied to the cross-point of cDNA expres-
tion of the presence or absence of bacteria, the characteristics of middle sion of target genes with β-actin, and the relative amounts were quantitated.
ear cavity fluid, and the frequency of occurrence.
2.5. Statistical analysis
2. Material & methods
All results are expressed as mean ± standard deviation or percen-
2.1. Study design tage values. Real-time PCR results were expressed using formula 2–(target
gene–β-actin)
, which reflects the cross-point of target genes with β-actin.
This study enrolled 39 pediatric patients who first visited the outpatient The Mann-Whitney U test was used to compare the expression levels of
clinic of Kyung Hee University Hospital between March 2014 and May ER stress-related genes between the two groups, which were classified
2018, and were diagnosed with OME. Medical history taking, physical ex- according to the characteristics of fluid, presence of bacteria, and fre-
amination, pure tone audiometry or speech audiometry and impedance quency of OME. The data were analyzed using SPSS version 20.0 sta-
audiometry was done to the all patients. The patient was diagnosed with tistical software (SPSS Inc., Chicago, IL), with p values less than 0.05
OME if they had amber-color or air-bubble sign on the tympanic membrane considered significant.
and B- or C-type tympanogram on impedance audiometry and simulta-
neously no sign of acute inflammation. Ventilation tubes were inserted in Table 1
patients who showed no improvement after wait-and-see for 3 months, Primers for real-time RT-PCR.
retraction of the tympanic membrane, and more than 40 dB of hearing
Name Direction Primer sequences Length (bp)
threshold on pure tone audiometry. We evaluated age, sex, period between
ventilation tube insertion and symptom onset, frequency of OME, presence β-actin Forward 5′-GCGAGAAGATGACCCAGATC-3′ 77
or absence of otorrhea, and number of ventilation tubes inserted. The fre- Reverse 5′-GGATAGCACAGCCTGGATAG-3′
CHOP Forward 5′-GTACCTATGTTTCACCTCCTGG-3′ 150
quency of otitis media was defined as otitis-prone for three or more occa-
Reverse 5′-TGGAATCTGGAGAGTGAGGG-3′
sions per 6 months and non-otitis-prone for 4 or more per a year. sXBP1 Forward 5′-TGGATTCTGGCGGTATTGAC-3′ 146
Patients were divided into two groups according to fluid char- Reverse 5′-TCCTTCTGGGTAGACCTCTG-3′
acteristics, presence or absence of bacteria in middle ear cavity fluid, ATF6 Forward 5′-CCTGTCCTACAAAGTACCATGAG-3′ 148
and frequency of OME. Expression of ER stress-related genes was then Reverse 5′-CCTTTAATCTCGCCTCTAACCC-3′
BiP Forward 5′eCCTGGGTGGCGGAACCTTCGATGTG-3′ 358
compared between the two groups. Children suspected of having AOM, Reverse 5′-CTGGACGGGCTTCATAGTAGACCGG-3′
head or neck anomalies, systemic diseases, or congenital or acquired IRE1α Forward 5′-GCGAACAGAATACACCATCAC-3′ 147
immunodeficiencies were excluded. The parents and guardians of each Reverse 5′-ACCAGCCCATCACCATTG-3′
subject provided written informed consent for the use of patient sam- PERK Forward 5′-GAACCAGACGATGAGACAGAG-3′ 150
Reverse 5′-GGATGACACCAAGGAACCG-3′
ples. The study protocol passed the review process of the Kyung Hee
University Clinical Research Ethics Committee. †Abbreviations: ER stress-related mRNA, endoplasmic reticulum stress-related
mRNA; CHOP, C/EBP-homologous protein; sXBP1, X-box binding protein 1;
2.2. Surgical procedure ATF6, activating transcription factor 6; BiP, immunoglobulin heavy chain-
binding protein; IRE1α, inositol-requiring enzyme 1α; PERK, endoplasmic re-
Removal of cerumen and irrigation with potadine solution on the ticulum kinase; OME, otitis media with effusion; SD, standard deviation.

110
D.W. Kang, et al. International Journal of Pediatric Otorhinolaryngology 121 (2019) 109–113

Table 2 Table 4
Demographic and clinical characteristics of pediatric patients with OME. mRNA expression in the Culture-positive and Culture-negative groups.
Patients (N = 39) ER stress-related mRNA Mean ± SD p-value

Age (year), mean ± SD 4.9 ± 2.8 (0.00–11.00) Culture-positive Culture-negative

Sex, male:female (N, %) 24:15 (61.5%:39.5%)
Duration of OME (months), mean ± SD 3.5 ± 1.4 (3.00–5.00) CHOP 0.229 ± 0.244 0.175 ± 0.244 0.400
Otitis-prone:non-otitis-prone (N, %) 29:10 (74.3%:25.7%) sXBP1 0.112 ± 0.094 0.078 ± 0.059 0.265
Otorrhea (N, %) 1 (2.5%) ATF6 0.008 ± 0.009 0.013 ± 0.014 0.298
Culture-positive:culture-negative (N, %) 14:58 (35.8%:64.2%) Bip2 0.048 ± 0.683 0.028 ± 0.026 0.561
Number of V-tube insertions, mean ± SD 1.6 ± 0.9 (1.00–4.00) IRE1 0.012 ± 0.013 0.010 ± 0.211 0.200
Middle ear fluid character (N, %) PERK 0.007 ± 0.007 0.004 ± 0.006 0.179
Serous 17 (43.6%)
Mucoid 19 (48.7%) †Abbreviations: ER stress-related mRNA, endoplasmic reticulum stress-related
Mucopurulent 2 (5.1%) mRNA; CHOP, C/EBP-homologous protein; sXBP1, X-box binding protein 1;
Purulent 1 (2.5%) ATF6, activating transcription factor 6; BiP, immunoglobulin heavy chain-
binding protein; IRE1α, inositol-requiring enzyme 1α; PERK, endoplasmic re-
†Abbreviations: ER stress-related mRNA, endoplasmic reticulum stress-related
ticulum kinase; OME, otitis media with effusion; SD, standard deviation.
mRNA; CHOP, C/EBP-homologous protein; sXBP1, X-box binding protein 1;
††Comparison on Mann–Whitney U test, *P < 0.05, and **P < 0.01.
ATF6, activating transcription factor 6; BiP, immunoglobulin heavy chain-
binding protein; IRE1α, inositol-requiring enzyme 1α; PERK, endoplasmic re-
ticulum kinase; OME, otitis media with effusion; SD, standard deviation. Table 5
††Duration of OME, period between ventilation tube insertion and symptom mRNA expression in the otitis-prone and non-otitis-prone groups.
onset; otorrhea, ear discharge symptom at the time of v-tube insertion; otitis- ER stress-related mRNA Mean ± SD p-value
prone, more than 3 episodes of acute otitis media during 6 months or 4 episodes
during 1 year; non-otitis-prone, not more than 3 episodes of acute otitis media Prone Non-prone
during 6 months or 4 episodes during 1 year.
CHOP 0.266 ± 0.247 0.113 ± 0.218 0.003**
sXBP1 0.084 ± 0.076 0.104 ± 0.063 0.087
3. Results ATF6 0.011 ± 0.011 0.010 ± 0.016 0.231
Bip2 0.031 ± 0.045 0.020 ± 0.019 0.064
A total of 39 patients participated in this study. The mean age of the IRE1 0.012 ± 0.020 0.008 ± 0.011 0.074
PERK 0.006 ± 0.007 0.005 ± 0.004 0.091
patients was 4.9 ± 2.8 years (range, 0 to 11, 24 boys [61.5%], 15 girls
[39.5%]). The duration from symptom onset of OME to the insertion of a
†Abbreviations: ER stress-related mRNA, endoplasmic reticulum stress-related
ventilation tube was 3.5 ± 1.4 months (range, 3.00 to 5.00). Of all pa- mRNA; CHOP, C/EBP-homologous protein; sXBP1, X-box binding protein 1;
tients, 29 patients (74.3%) were classified as otitis-prone and 10 patients ATF6, activating transcription factor 6; BiP, immunoglobulin heavy chain-
(25.7%) as non-otitis-prone. Bacteria was identified from fluid samples of binding protein; IRE1α, inositol-requiring enzyme 1α; PERK, endoplasmic re-
14 (35.8%) patients and 58 (64.2%) were not identified. The mean ticulum kinase; OME, otitis media with effusion; SD, standard deviation.
number of ventilation tube insertions in the patients was 1.6 ± 0.9 ††Otitis-prone: More than 3 episodes of acute otitis media during 6 months or 4
(range, 1.00–4.00). In terms of the characteristics of middle ear effusion, episodes during 1 year, Non-otitis-prone: Not more than 3 episodes of acute
17 (43.6%) had serous, 19 (48.7%) had mucoid, 2 (5.1%) had muco- otitis media during 6 months or 4 episodes during 1 year.
purulent, and 1 (2.5%) had purulent middle ear effusion (Table 2). †††Comparison on Mann–Whitney U test, *P < 0.05, and **P < 0.01.
The most common bacteria detected from the culture of middle ear
effusion were Staphylococcus aureus (5, 12.8%), followed by methicillin- 4. Discussion
resistant S. aureus (2, 5.1%), Streptococcus pneumoniae, Bacillus, and St.
mitis (1, 2.5%, respectively) (Table 3). OME is otitis media with fluid effusion in the middle ear cavity, but
Expression of the ER-stress related genes CHOP, sXBP1, ATF6, BiP, without acute symptoms such as otalgia and fever. Although chronic
IRE1α, and PERK did not differ significantly according to presence or inflammation of the middle ear cavity resulting from upper respiratory
absence of bacteria in the middle ear effusion (p > 0.05) (Table 4). tract infections is a major cause of OME, the pathophysiology of OME is
Expression levels of CHOP mRNA were significantly higher in the not clear. ER stress responses, which are observed in patients with
otitis-prone group than in the non-otitis-prone group (p < 0.05), but
there were no significant differences in sXBP1, ATF6, BiP, IRE1α, or
Table 6
PERK mRNA levels (p > 0.05) (Table 5).
mRNA expression in the fluid characteristic groups.
Expression levels of IRE1α mRNA were significantly higher in the
serous group than in the mucoid, mucopurulent or purulent groups ER stress mRNA Mean ± SD p-value
(p < 0.05), but no significant differences were found in CHOP, sXBP1,
Serous Non-serous
ATF6, BiP or PERK (p > 0.05) (Table 6).
CHOP 0.131 ± 0.180 0.246 ± 0.277 0.151
sXBP1 0.104 ± 0.084 0.077 ± 0.062 0.305
Table 3 ATF6 0.011 ± 0.012 0.011 ± 0.013 0.774
Bacteria detected during culture of effusion fluid samples. Bip2 0.025 ± 0.032 0.043 ± 0.045 0.175
IRE1 0.018 ± 0.024 0.004 ± 0.006 0.030*
Bacteriology Patient no. (%) PERK 0.009 ± 0.008 0.003 ± 0.004 0.053

No growth 25 (64.1%) †Abbreviations: ER stress-related mRNA, endoplasmic reticulum stress-related

Growth 14 (35.9%) mRNA; CHOP, C/EBP-homologous protein; sXBP1, X-box binding protein 1;
Staphylococcus aureus 5 (12.8%)
ATF6, activating transcription factor 6; BiP, immunoglobulin heavy chain-
Coagulase-negative Staphylococcus 4 (10.2%)
binding protein; IRE1α, inositol-requiring enzyme 1α; PERK, endoplasmic re-
Methicillin-resistant Staphylococcus aureus 2 (5.1%)
Streptococcus pneumoniae 1 (2.5%)
ticulum kinase; OME, otitis media with effusion; SD, standard deviation.
Bacillus 1 (2.5%) ††Non-pathologic: serous discharge; Pathologic: mucoid, mucopurulent, and
Streptococcus mitis 1 (2.5%) purulent discharge.
†††Comparisons by the Mann–Whitney U test, *P < 0.05, and **P < 0.01.

111
D.W. Kang, et al.
neurodegenerative diseases such as Alzheimer's disease and Parkinson's
disease, as well as in atherosclerosis and ischemic diseases, are also
involved in chronic inflammatory reactions caused by infections
[17,18]. The present study investigated the relationship between ER-
stress related responses and the pathophysiology of OME by measuring
the expression of genes involved in several ER-stress response path-
ways, as well as by assessing fluid positivity for bacteria, the frequency
of OME, and fluid characteristics related to the chronicity of OME.
Three major transmembrane proteins are involved in ER stress re-
sponses: ATF6 (activating transcription factor 6), IRE1 (inositol requiring
protein 1), and PERK (protein kinase RNA-like endoplasmic reticulum
kinase). ATF6, which is usually inhibited by BiP, an endoplasmic re-
ticulum chaperone, is activated when exposed to unfolded proteins. If the
Golgi-localization sequence (GLS) of activated ATF6 is exposed, the latter
is cleaved. Cleaved ATF6 enters the nucleus and acts as a transcription
factor. IRE1α has intrinsic kinase activity and can act as an en-
doribonuclease. Activated IRE1α induces splicing of the mRNA encoding
the transcription factor XBP1 (X box-binding protein 1), producing spliced
XBP1 (sXBP1). PERK, which is activated by unfolded proteins, activates
eukaryotic translation-initiation factor 2a (EIF2a), which stops almost all
protein synthesis and cell growth [11,25–27]. The present study in-
vestigated the levels of expression of ATF6, IRE1, and PERK as indicators
of three representative pathways of ER stress responses. We also studied
sXBP1 and CHOP, which are the signaling messengers of IRE1 and PERK,
respectively, and BiP as an indicator of inhibition of the ER stress response.
By comparing the levels of these mRNAs, we analyzed the relationship
among these three representative pathways of the ER stress response.
Of the middle ear fluid samples cultured, 35.9% were positive for
bacteria, with the most common bacteria being Staphylococcus aureus
(12.8%) CNS (10.2%) and MRSA (5.1%). Although the high percentage
of CNS indicates contamination during the sampling process, the pro-
portion of CNS did not affect the results of other strains, with the results
of our study being similar to those of other studies [28,29]. The rates of
detection of Streptococcus pneumoniae, H. influenzae, and M. catarrhalis,
the predominant bacteria in OME, were low, due to increases in vacci-
nation rates for S. pneumoniae and H. influenzae and the high percentage
of patients transferred from other facilities, who had already received
empirical antibiotics such as erythromycin and amoxicillin/clavulanate.
However, the high rates of bacterial detection by PCR suggest that as-
sessments of our patients using PCR may yield different results [7,8].
The present study found that the level of expression of each ER
stress-related gene was unaffected by the presence of bacteria in middle
ear effusion. This is different from the prediction that the expression
levels of ER stress mRNAs would be higher in fluid with than without
bacteria, because chronic inflammation due to infection induces ER
stress responses. The reasons for this discrepancy include (1) the high
proportion of bacteria-free effusion; (2) congenital or acquired immune
responses during the 3-month wait-and-see period, possibly affecting
responses in the middle ear cavity, and (3) the use of antibiotics by most
patients before visiting our hospitals. Several previous studies have
reported that injecting nonviable bacteria or bacterial components into
the middle ears of animals causes inflammatory responses and OME
[30,31], suggesting that our findings may have been due to the effects
of nonviable bacteria.
CHOP, a final end product of the PERK signal pathway, was regarded
as an indirect measure of signal pathway activation through PERK. The
level of CHOP mRNA was significantly higher in the otitis-prone than in
the non-otitis-prone group, suggesting that the PERK pathway of ER
stress response is associated with the frequency of OME. However, the
levels of expression of PERK mRNA did not differ significantly in these
two groups. Because CHOP is an end-product of the PERK pathway and
activates the ER stress response, CHOP, rather than PERK, is likely the
starting point of the PERK/CHOP pathway. XBP-1, CHOP, and ATF6 have
been reported to regulate the production of human nasal airway mucins
by expression of MUC5AC and MUC5B [21]. Because this study revealed
that CHOP upregulates the production of mucins, our results showed that
112
International Journal of Pediatric Otorhinolaryngology 121 (2019) 109–113
CHOP may play a role in increasing fluid viscosity through the produc-
tion of mucin in middle ear mucosa [32].
IRE1α is activated by the ER stress response and enhances expression
of sXBP1. IRE1α is associated with human cerebral ischemia and liver
cancer, and the transcription factor sXBP1 is involved in bipolar disorder
and plasma cell differentiation [33,34]. The present study found that the
expression of IRE1 was higher in serous fluid than in mucoid, muco-
purulent, or purulent fluid, but there was no difference among the groups
in the expression of sXBP1 mRNA. Activation of IRE1 by the ER stress
response may influence the characteristics of middle ear effusion. IRE1α
has both phosphorylase (kinase) and endonuclease activity, activating
XBP1 through RNA splicing, but prolonged ER stress is more likely to
enhance the phosphorylation of JNK1 (c-Jun N-terminal kinase) than
XBP1, which induces cell apoptosis. These two pathways are expressed
independently of each other [35]. This study therefore indicated that
JNK1, a by-product of IRE1α, may be involved in OME. Moreover,
IRE1 , another by-product of IRE1, may affect fluid characteristics due to
the production of mucins and glycoproteins and increased ciliary func-
tion, with IRE1 expression a related to excretion of mucins [36].
This study had several limitations. First, for ethical reasons, we
could not harvest the middle ear mucosa and thus performed experi-
ments on middle ear effusion. Second, we did not include any children
with early stage OME. Third, we could not test all signal transduction
genes involved in ER stress and only representative genes were tested.
Finally, this study investigated the effects of ER stress responses on
mRNA, but not protein, expression. Western blotting analysis is there-
fore needed to compare protein expression in these groups of patients.
5. Conclusions
In conclusion, our findings show that CHOP is more common in
otitis-prone rather than non-otitis prone patients. Also, serous effusion
is more associated with IRE1 than mucoid, mucopurulent, or purulent
effusion. Therefore, different ER stress response pathways are activated
according to the frequency of OME and characteristics of middle ear
effusion in OME.
Conflicts of interest
There were no conflicts of interest for any authors involved in the
study.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Acknowledgments
This work was supported by the National Research Foundation of Korea
grant funded by the Korea government (NRF 2017R1D1A1B3030021)
(NRF-2018R1A6A1A03025124).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ijporl.2019.03.006.
References
[1] C.N. Brouwer, A.R. Maille´, M.M. Rovers, D.E. Grobbee, E.A. Sanders, A.G. Schilder,
Health-related quality of life in children with otitis media, Int. J. Pediatr.
Otorhinolaryngol. 69 (2005) 1031–1041.
[2] R.M. Rosenfeld, J.J. Shin, S.R. Schwartz, R. Coggins, L. Gagnon, J.M. Hackell,
D. Hoelting, L.L. Hunter, A.W. Kummer, S.C. Payne, D.S. Poe, M. Veling, P.M. Vila,
S.A. Walsh, M.D. Corrigan, Clinical practice guideline: otitis media with effusion
executive summary (update), Otolaryngol. Head Neck Surg. 154 (2016) 201–214.
D.W. Kang, et al.
[3] Helen Atkinson, Sebastian Wallis, Andrew P. Coatesworth, Otitis media with effu-
sion, Postgrad. Med. 127 (2015) 381–385.
[4] David S. Hurst, Per venge, the impact of atopy on neutrophil activity in middle ear
effusion from children and adults with chronic otitis media, Arch. Otolaryngol.
Head Neck Surg. 128 (2002) 561–566.
[5] Mauricio Schreiner Miura, Miguel Mascaro, M. Richard, Rosenfeld, Association
between otitis media and gastroesophageal reflux, Otolaryngol. Head Neck Surg.
146 (2012) 345–352.
[6] H. Kubba, J.P. Pearson, J.P. Birchall, The aetiology of otitis media with effu-
sion:review, Clin. Otolaryngol. Allied Sci. 25 (2000) 181–194.
[7] C.D. Bluestone, J.S. Stephenson, L.M. Martin, Ten-year review of otitis media pa-
thogens, Pediatr. Infect. Dis. J. 11 (1992) S7–S11.
[8] J.C. Post, R.A. Preston, J.J. Aul, M. Larkins-Pettigrew, J. Rydquist-White,
K.W. Anderson, R.M. Wadowsky, D.R. Reagan, E.S. Walker, L.A. Kingsley,
A.E. Magit, G.D. Ehrlich, Molecular analysis of bacterial pathogens in otitis media
with effusion, J. Am. Med. Assoc. 273 (1995) 1598–1604.
[9] S. Carrie, D.A. Hutton, J.P. Birchall, G.G. Green, J.P. Pearson, Otitis media with
effusion: components which contribute to the viscous properties, Acta Otolaryngol.
112 (1992) 504–511.
[10] J.F. Stanievich, C.D. Bluestone, J.A. Lima, R.H. Michaels, D. Rohn, M.Z. Effron,
Microbiology of chronic and recurrent otitis media with effusion in young infants,
Int.J.Ped.Otorhinoloaryngol. 3 (1981) 137–143.
[11] H.O. Rashid, R.K. Yadav, H.R. Kim, H.J. Chae, ER stress: autophagy induction, in-
hibition and selection, Autophagy 11 (2015) 1956–1977.
[12] R.J. Kaufman, Stress signaling from the lumen of the endoplasmic reticulum: co-
ordination of gene transcriptional and translational controls, Genes Dev. 13 (1999)
1211–1233.
[13] A. Helenius, M. Aebi, Intracellular functions of N-linked glycans, Science 291
(2001) 2364–2369.
[14] O. Pluquet, A. Pourtier, C. Abbadie, The unfolded protein response and cellular
senescence. A review in the theme: cellular mechanisms of endoplasmic reticulum
stress signaling in health and disease, Am. J. Physiol. Cell Physiol. 308 (2015)
C415–C425.
[15] K.E. Gunn, N.M. Gifford, K. Mori, J.W. Brewer, A role for the unfolded protein
response in optimizing antibody secretion, Mol. Immunol. 41 (2004) 919–927.
[16] A.V. Cybulsky, T. Takano, J. Papillon, K. Bijian, Role of the endoplasmic reticulum
unfolded protein response in glomerular epithelial cell injury, J. Biol. Chem. 280
(2005) 24396–24403.
[17] Celli Jean, M. Renee, Tsolis, bacteria, the ER and the unfolded protein response:
friends or foes? Nat. Rev. Microbiol. 13 (2015) 71–82.
[18] Aimin Xu, A. Richard Bellamy, John A. Taylor, BiP (GRP78) and endoplasmin
(GRP94) are induced following rotavirus infection and bind transiently to an en-
doplasmic reticulum-localized virion component, J. Virol. 72 (1998) 9865–9872.
[19] H.S. Jin, H.W. Suh, S.J. Kim, E.K. Jo, Mitochondrial Control of Innate Immunity and
Inflammation Immune Network vol. 17, (2017), pp. 77–88.
[20] M. Hotomi, T. Samukawa, N. Yamanaka, Inter-leukin 8 in otitis media with effusion,
Acta Otolaryngol. 114 (1994) 406–409.
[21] M.H. Kim, C.H. Bae, Y.S. Choi, H.G. Na, S.Y. Song, Y.D. Kim, Endoplasmic reticulum
stress induces MUC5AC and MUC5B expression in human nasal airway epithelial
cells, Clin Exp Otorhinolaryngol (2018) [Epub ahead of print].
[22] L. Skov, F.J. Beurskens, C.O. Zachariae, S. Reitamo, J. Teeling, D. Satijn,
113
International Journal of Pediatric Otorhinolaryngology 121 (2019) 109–113
K.M. Knudsen, E.P. Boot, D. Hudson, O. Baadsgaard, P.W. Parren, J.G. van de
Winkel, IL-8 as antibody therapeutic target in inflammatory diseases: reduction of
clinical activity in palmoplantar pustulosis, J. Immunol. 181 (2008) 669–679.
[23] M. Tsuge, N. Hiraga, Y. Zhang, M. Yamashita, O. Sato, N. Oka, K. Shiraishi, Y. Izaki,
G.N. Makokha, T. Uchida, M. Kurihara, M. Nomura, K. Tsushima, T. Nakahara,
E. Murakami, H. Abe-Chayama, T. Kawaoka, D. Miki, M. Imamura, Y. Kawakami,
H. Aikata, H. Ochi, C.N. Hayes, T. Fujita, K. Chayama, Endoplasmic reticulum-
mediated induction of interleukin-8 occurs by hepatitis B virus infection and con-
tributes to suppression of interferon responsiveness in human hepatocytes, Virology
525 (2018) 48–61.
[24] D.H. Kim, J.H. Cheon, Pathogenesis of inflammatory bowel disease and recent
advances in biologic therapies, Immune Network 17 (2017) 25–40.
[25] S.N. Sou, K.M. Ilieva, K.M. Polizzi, Binding of human BiP to the ER stress trans-
ducers IRE1 and PERK requires ATP, Biochem. Biophys. Res. Commun. 420 (2012)
473–478.
[26] J. Maiuolo, S. Bulotta, C. Verderio, R. Benfante, N. Borgese, Selective activation of
the transcription factor ATF6 mediates endoplasmic reticulum proliferation trig-
gered by a membrane protein, Proc. Natl. Acad. Sci. U.S.A. 108 (2011) 7832–7837.
[27] M.D. Shoulders, L.M. Ryno, J.C. Genereux, J.J. Moresco, P.G. Tu, C. Wu, J.R. Yates,
A.I. Su, J.W. Kelly, R.L. Wiseman, Stress-independent activation of XBP1s and/or
ATF6 reveals three functionally diverse ER proteostasis environments, Cell Rep. 3
(2013) 1279–1292.
[28] J.S. Lee, M.G. Kim, S.M. Hong, S.Y. Na, J.Y. Byun, M.S. Park, S.G. Yeo, Changing
patterns of bacterial strains in adults and children with otitis media in Korean
tertiary care centers, Clin exp otorhinolaryngology 7 (2014) 79–86.
[29] H. Jung, S.K. Lee, S.H. Cha, J.Y. Byun, M.S. Park, S.G. Yeo, Current bacteriology of
chronic otitis media with effusion: high rate of nosocomial infection and decreased
antibiotic sensitivity, J. Infect. 59 (2009) 308–316.
[30] N. Nonomura, Y. Nakano, O. Fujioka, H. Niijima, M. Kawana, M. Fujita,
Experimentally induced otitis media with effusion following inoculation with the
outer cell wall of nontypable Haemophilus influenzae, Arch. Oto-Rhino-Laryngol.
244 (1987) 253–257.
[31] N. Okazaki, T.F. DeMaria, B.R. Briggs, D.J. Lim, Experimental otitis media with
effusion induced by nonviable Hemophilus influenzae: cytologic and histologic
study, Am. J. Otolaryngol. 5 (1984) 80–92.
[32] J. Lin, V. Tsuprun, H. Kawano, M.M. Paparella, Z. Zhang, R. Anway, S.B. Ho,
Characterization of mucins in human middle ear and Eustachian tube, Am. J.
Physiol. Lung Cell Mol. Physiol. 280 (2001) 1157–1167.
[33] C.D. Bown, J.F. Wang, B. Chen, L.T. Young, Regulation of ER stress proteins by
valproate: therapeutic implications, Bipolar Disord. 4 (2002) 145–151.
[34] B. Tirosh, N.N. Iwakoshi, L.H. Glimcher, H.L. Ploegh, XBP-1 specifically promotes
IgM synthesis and secretion, but is dispensable for degradation of glycoproteins in
primary B cells, J. Exp. Med. 202 (2005) 505–516.
[35] M.J. Jurczak, A.H. Lee, F.R. Jornayvaz, H.Y. Lee, A.L. Birkenfeld, B.A. Guigni,
M. Kahn, V.T. Samuel, L.H. Glimcher, G.I. Shulman, Dissociation of inositol-re-
quiring enzyme (IRE1alpha)-mediated c-Jun N-terminal kinase activation from
hepatic insulin resistance in conditional X-box-binding protein-1 (XBP1) knock-out
mice, J. Biol. Chem. 287 (2012) 2558–2567.
[36] M.B. Martino, L. Jones, B. Brighton, C. Ehre, L. Abdulah, C.W. Davis, D. Ron,
W.K. O'Neal, C.M. Ribeiro, The ER stress transducer IRE1β is required for airway
epithelial mucin production, Mucosal Immunol. 6 (2013) 639–654.