Expression of E-prostanoid receptors in nasal polyp tissues of smoking and nonsmoking patients with chronic rhinosinusitis
Expression of E-prostanoid receptors in nasal polyp tissues of smoking and nonsmoking patients with chronic rhinosinusitis
Li Xie 0 2
Ai-Guo Liu 0 2
Li-Yan Peng 0 2
Su-Jie Wang 2
Yin-Ping Zhang 1 2
Xian-Song Wang 1 2
0 Department of Otolaryngology-Head and Neck Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan , P.R. China , 2 Department of Otolaryngology-Head and Neck Surgery, The Fifth Affiliated Hospital of Zheng Zhou University , Zhengzhou, Henan , P.R. China
1 Department of Pathology, Tongji Medical College, Huazhong University of Science and Technology , Wuhan , P.R. China
2 Editor: Takuma Kato , Mie Daigaku , JAPAN
Different inflammatory reactions have been observed in the polyp tissues of nonsmokers and smokers with chronic rhinosinusitis (CRS). E-prostanoid (EP) receptors play a role in the inflammatory processes. Cigarette smoke (CS) exposure regulates EP-receptor expression levels promoting inflammatory mediator release from various inflammatory cells. In this study, we characterize the EP-receptor expression profiles in the polyps of nonsmoking and smoking CRS patients to explore the possible role of CS in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). Polyp biopsies were obtained from 28 non-smoking and 21 smoking CRSwNP patients. Histopathological characteristics were observed under a light microscope. The prostaglandin E2 (PGE2), TNF-?, and IL-8 contents in polyp tissues were detected using enzyme-linked immunosorbent assay. Immunostaining was used to locate EP receptors in polyps. Messenger RNA and protein expression of EP receptors were examined using quantitative real-time polymerase chain reaction and Western blot, respectively. More severe inflammatory reactions occurred in polyp tissues of smoking CRSwNP patients. The PGE2, TNF-?, and IL-8 in tissue homogenate levels were significantly higher in smoking CRSwNP patients than those in nonsmoking CRSwNP patients. Moreover, the distribution of each EP receptor subtype was similar in both groups. Compared with the EPreceptor expression in nonsmokers, messenger RNA and protein of EP2 and EP4 receptor were significantly down-expressed in smoking patients, but EP1 and EP3 receptors did not show significant differences.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Competing interests: The authors have declared
that no competing interests exist.
CS exposure downregulates the expression levels of EP2 and EP4 receptors and stimulates
the production of PGE2 and the proinflammatory cytokine IL-8 and TNF-? in polyp tissues of
CRS patients. The down-expressed EP2 and EP4 receptors might be associated with
severe inflammatory reactions in smoking CRSwNP patients.
Chronic rhinosinusitis (CRS) is characterized by chronic and persistent inflammation of nasal
and paranasal sinus mucosa. Epidemiological analyses indicate that 20% of CRS patients
exhibit nasal polyps [
]. Clinically, CRS with nasal polyps (CRSwNP) is more recalcitrant
and refractory than CRS without nasal polyps. Thus, its mechanism has become a significant
interest among many clinicians and researchers over the last decade . Current studies
suggest that the development of CRSwNP might involve a complex of intrinsic and exogenous
factors, including infections, allergy, environmental exposure, and genetic predisposition [
Cigarette smoke (CS) exposure is an important environmental factor. A higher CRSwNP
prevalence and a less favorable response to sinus surgery have been reported in cigarette
smokers compared with nonsmokers [5?7]. Moreover, chronic exposure to CS aggravates
eosinophilic inflammation, which favors nasal polyp formation [
]. These results suggest that CS
might play a role in CRSwNP pathogenesis. In the lower airway, CS exposure can damage
epithelial cells and promote the release of various proinflammatory mediators and inflammatory
cell recruitments, thereby leading to numerous inflammatory diseases, such as asthma,
emphysema, and chronic obstructive pulmonary diseases [
]. Although these proinflammatory
mediators and infiltrating cells are also associated with the development of nasal polyps in
CRS patients , relatively little is understood whether smoking also promotes cell
recruitment or causes a change in relevant mediator profiles in nasal polyps to participate in the
formation of nasal polyps or CRSwNP.
Prostaglandin E2 (PGE2) is a one of the most important arachidonic acid metabolites. In
inflammatory processes, PGE2 is usually produced in high amounts and promotes or inhibits
inflammatory responses by regulating the activities of various inflammatory cells or
modulating immune cell functions [
]. The contradictory actions of PGE2 are mediated by binding
four diverse E-prostanoid (EP) receptors, namely, EP1, EP2, EP3, and EP4 receptors. EP1/EP3
receptors mediate the proinflammatory effects, whereas EP2/EP4 receptors mediate the
antiinflammatory effects [
]. Several studies showed that CS influences the expression of EP
receptors in inflammatory cells to generate proinflammatory effects [
]. Whether CS
exposure can change EP receptor expression levels in nasal polyp tissues of CRSwNP patients to
affect the effects of PGE2 remains to be clarified. In this study, we compared the characteristics
of EP receptor expression in polyp tissues between the nonsmoking and smoking CRSwNP
patients to explore the possible role of cigarette smoking in the pathogenesis of CRSwNP.
A total of 49 patients with CRSwNP were enrolled from March 2016 to January 2017 at the
Department of Otolaryngology-Head and Neck Surgery, Tongji Hospital (Wuhan, China).
CRSwNP was diagnosed using the typical symptoms (nasal obstruction, nasal discharge, facial
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pain, reduction, and loss of smell) that persisted beyond 12 weeks, endoscopic signs of nasal
polyps, and a positive computed tomography scan change according to the current European
Position Paper on Rhinosinusitis and Nasal Polyps 2012. CRSwNP was classified as
eosinophilic when percent tissue eosinophils exceeded 10% of total inflammatory cells [
]. Up to 28
subjects were nonsmoking patients with CRSwNP who neither smoke nor live or work in a
smoky environment. A total of 21 patients were currently active smokers with CRSwNP. Each
smoking patient smoked more than 20 cigarettes per day for the last five years or more [
According to the criteria, smoking volume was defined in pack-years (number of packs
smoked per day times the number of years of smoking) [
]. Subjective symptoms were scored
on a visual analog scale (VAS) as mentioned elsewhere [
]. This focused on five major
symptoms including rhinorrhea, nasal obstruction, decreased sense of smell, facial pain or fullness,
and headache. A total symptom score was calculated based on the sum of these five VAS
symptom domains. Endoscopy findings were recorded using Lund-Kennedy scoring system [
Sinus computed tomography (CT) findings were staged according to the Lund-Mackay system
]. Clinical information of subjects is described in Table 1.
All subjects were excluded from this study if they had a diagnosis of atopy, aspirin
hypersensitivity, asthma, antrochoanal polyps, cystic fibrosis, or primary ciliary dyskinesia. The use
of any topical or systematic medications, including steroids, nonsteroidal anti-inflammatory
drugs, and anti-leukotrienes that may affect the research measurements, was prohibited in all
patients for at least four weeks before surgery. This study was approved by the ethics
committee of Tongji Hospital of Huazhong University of Science and Technology and conducted with
written informed consent from all patients.
Tissues from the apical region of polyps were obtained during functional endoscopic sinus
surgery. Fresh nasal biopsies were divided into at least two parts. One part was fixed in 10%
formaldehyde and embedded in paraffin for hematoxylin?eosin (HE) and immunohistochemical
staining. The other parts were snap frozen in liquid nitrogen and then stored at ?80 ?C for
RNA and protein preparation.
Paraffin sections were stained with HE and observed with a Nikon microscope. The number of
inflammatory cells (eosinophils, neutrophils, and mononuclear cells) and mucosal glands in
lamina propria was counted at 10 random fields of high-power (HP) magnification (?400).
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The results were expressed as cell or gland counts per HP field. Basement membrane thickness
(BMT) and sub-epithelial edema were scored based on degree (0, none; 1, mild; 2, moderate;
or 3, marked) at a magnification of ?100, as described in a previous study [
deposition was evaluated by Masson staining. The percentage of total collagen content was
quantified using Image pro-plus 6.0 (Media Cybernetics, Inc., MD, USA) [
]. According to the
criteria similar to that of Gao et al [
], squamous metaplasia was identified from specimens,
in which the pseudostratified columnar epithelium was replaced by stratified squamous
epithelium with total loss of cilia. Goblet cells were stained with Alcian blue and counted per
millimeter of intact epithelium. Histological analysis was conducted by two pathologists who were
blind to clinical data, as previously described [
Measurements of PGE2, TNF-?, and IL-8 in polyp tissues
Each 0.1 g of tissue specimens was homogenized in 1 mL of buffer (0.1 M phosphate, pH 7.4,
containing 1 mM EDTA). The homogenates were sonicated and centrifuged at 3000 rpm for
10 min at 4 ?C. The contents of PGE2, TNF-?, and IL-8 in supernatants were measured using
enzyme-linked immunosorbent assay kits according to manufacturer's protocols. PGE2 kits
were from Cayman, Michigan, USA. TNF-? and IL-8 kits were from R&D Systems, MN, USA.
The detection limit of the assay was 15 pg/ml for PGE2, 4 pg/ml for IL-8 and TNF-?.
Paraffin sections were used for immunohistochemical staining as described previously [
Briefly, sections were incubated with 3% hydrogen peroxidase to block endogenous peroxidase
activity, with 5% bovine serum albumin to block nonspecific binding sites. Sections were
incubated with polyclonal rabbit antihuman EP receptors (1:400 dilution for EP1, EP2, or EP3 and
1:200 for EP4, Cayman Chemicals, Michigan, USA) or polyclonal rabbit antihuman
myeloperoxidase (MPO) (1:100 dilution, ZSGB-BIO, Beijing, China) overnight at 4 ?C. The primary
antibodies were detected with the StreptAvidin Biotin-peroxidase Complex kit (Boster
Biotechnology, Wuhan, China). The immunoreaction was visualized using
3,3-diaminobenzidine-tetrahydrochloride, which causes brown staining of positive cells. Negative controls were
performed by omitting primary antibodies and using non-immune sera of the same species.
Quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA from tissues was extracted and reverse transcribed to cDNA. Quantitative RT-PCR
was performed using StepOnePlus system (ABI, Foster City, USA) and SYBR Premix Ex Taq
kit (TaKaRa Biotechnology, Dalian, China) as described previously [
]. The PCR
amplification consisted of one cycle at 95 ?C for 2 min, followed by 40 cycles of denaturation at 95 ?C
for 10 s, annealing at 60 ?C for 10 s, and then extension at 72 ?C for 15 s. At the end of each
PCR run, a melting curve analysis was used to confirm production specificity. Glyceraldehyde
phosphate dehydrogenase (GAPDH) gene expression was used for normalization. Relative
gene expression was calculated using comparative cycle threshold (2???Ct) method. No PCR
product was amplified in the negative control. The following primers for PCR were adopted:
EP1, forward 5'-GGTGTCGTGCATCTGCTGGA-3' and reverse 5'- CAAGA
GGCGAAGCAGTTGGC-3' (187 bp); EP2, forward 5'-AGACGGACCAC CTCA
TTCTC-3' and reverse 5'-GATGGCAAAGACCCAAGG-3' (176 bp); EP3,
forward 5'-CCCGCCTCAACCACTCCTA-3' and reverse 5'-CACCGATCCGCAAT
CCTC-3' (107 bp); EP4, forward 5'-AACTTGATG GCTGCGAAGACCTAC-3'
and reverse 5'-TTCTAATATCTGGGCCTCTGCTGTG-3' (128 bp); GAPDH,
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forward 5'- AGAAGGCTGGGGCTCATTTG -3' and reverse 5'-AGGGGCCAT
CCACAGTCT TC -3' (258 bp).
Total protein was extracted by homogenizing tissues in radioimmunoprecipitation assay/
sodium lauryl sulfate and protease inhibitors as described previously [
]. Denatured total
proteins (50 ?g) were separated by sulfate-polyacrylamide gel electrophoresis followed by
transferring into polyvinylidenedifluoride membranes. The membranes were incubated with block
solution containing Tween-20 and 5% fat-free milk powder for 2 h at room temperature, and
then incubated with polyclonal rabbit antihuman EP receptors (EP1, 1:1000 dilution; EP2,
1:1200 dilution; EP3, 1:1500 dilution and EP4, 1:500 dilution) overnight at 4 ?C. Negative
controls were performed in the absence of primary antibody or including an isotype control
antibody. Membranes were incubated with horseradish-peroxidase conjugated goat anti-rabbit
antibody (Sigma) for 1 h at room temperature. The immunoreactions were detected using a
chemiluminescent method (Supersignal West Pico Chemiluminescent Substrate, Thermo,
MA, USA) and exposed to GeneGnome HR Model (Synoptics Ltd., Cambridge, UK). The
protein levels were semiquantified as ratios to the GAPDH band intensities using GeneTools.
For continuous variables, the results were presented as medians and interquartile ranges or in
box and whisker plots representing the medians and interquartile ranges. The nonparametric
Kruskal-Wallis H test was used for data analysis within different groups. The Mann-Whitney
U-test was applied for between-group comparisons. Difference in proportions was tested
using the chi-square test or Fisher's exact test. Significance was set to p < 0.05.
In the present study, CRSwNP smokers and CRSwNP nonsmokers were comparable in terms
of sex, age distribution and the proportions of eosinophilic CRSwNP. No significant difference
in VAS symptom scores between the two groups was found. In comparison with non-smoking
patients, smoker patients had significantly higher preoperative CT scores, and endoscopic
scores (Table 1).
Epithelium cell and goblet cell hyperplasia, basement membrane thickening, stromal edema,
and inflammatory cell infiltration can be observed in smoking and nonsmoking CRSwNP
patients (Fig 1). The quantitative analysis for the histopathological changes in polyp tissues
from both patient groups is summarized in Table 2. Neutrophils (MPO+) were the prominent
infiltrating inflammatory cell in nasal polyps of smokers and nonsmokers. The amounts of
total inflammatory cells, neutrophils, and goblet cells were significantly higher in smoking
CRSwNP patients than those in nonsmoking CRSwNP patients. Squamous metaplasia was
found in 7/21 (33.3%) of nasal polyps in smoking CRSwNP patients and 2/28 (7.1%) in
nonsmoking CRSwNP patients. The rate of squamous metaplasia was significantly higher in
smoking CRSwNP patients compared with that in nonsmoking CRSwNP patients. No statistical
differences in stromal edema, collagen deposition, BMT, and the count of eosinophils,
mononuclear cells, and submucosal glands between two groups were observed.
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Fig 1. Representative photomicrographs of HE, Alcian blue or immunohistochemistry staining for polyp tissues. (A)
Epithelium hyperplasia (HE, ?200). (B) Epithelial squamous metaplasia (HE, ?200). (C) Epithelial goblet cells in the nonsmoking
CRSwNP patients (Alcian blue, ?400). (D) Goblet cell hyperplasia in the smoking CRSwNP patients (Alcian blue, ?400). E-F:
Immunohistochemistry stainings for myeloperoxidase (MPO) in the CRSwNP nonsmokers (E) and CRSwNP smokers (F)
(Original magnification, ?400).
Levels of PGE2, TNF-?, and IL-8
As shown in Fig 2, the values of PGE2, TNF-?, and IL-8 in the polyp tissues were significantly
higher in smoking CRSwNP patients than those in nonsmoking CRSwNP patients.
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Immunohistochemical staining for EP receptor subtypes
Immunohistochemical staining for EP1, EP2, EP3, and EP4 receptors in nasal polyp tissues is
shown in Fig 3 (CRSwNP smokers) and S1 Fig (CRSwNP nonsmokers). The cellular location
of each EP receptor subtype was similar in smoking and nonsmoking CRSwNP patients. EP1
was expressed primarily on infiltrating inflammatory cells in sub-epithelial regions. Both EP2
and EP3 were expressed in inflammatory cells, mucosal epithelium, and glands; however, EP3
was expressed in blood vessels as well. Moreover, EP4 was expressed on the epithelium and
mRNA expression of EP receptor subtypes
Comparing the mRNA expression of the EP receptor subtype between the nonsmoking and
smoking groups, as illustrated in Fig 4, EP2 or EP4 receptor was significantly downregulated
in the smoking group compared with the nonsmoking group (p < 0.05). No significant
differences were observed in the mRNA expression of EP1 or EP3 receptor between the two groups.
Protein expression of EP receptor subtypes
The expression levels of the EP receptor proteins in polyp tissues from nonsmoking and
smoking CRSwNP patients are illustrated in Fig 5. The EP2 and EP4 receptors were significantly
downregulated in smokers compared with nonsmokers (p < 0.05). However, the protein levels
of EP1 and EP3 receptors between the two groups showed no significant differences.
The reports about the clinical impact of smoking on CRSwNP patients have been discrepant.
Zeynep found that there was no correlation between smoking and polyp size, CT scores, or
subjective symptom scores [
]. Erbek [
] and Kilty et al [
] reported that smoking was not
associated with polyp stage, CT score, but related to higher symptom scores. The present study
showed that there were higher preoperative CT scores and endoscopic scores in smoking
CRSwNP patients, which was consistent with those reported by Uhliarova et al [
scores of CT and endoscopy imply more extensive inflammatory lesions present in smoking
CRSwNP patients. Our study also demonstrated that CS exposure is associated with an
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Fig 2. The contents of PGE2, TNF-?, and IL-8 in polyp tissue homogenates of the smoking CRSwNP and nonsmoking CRSwNP.
Values are reported as medians and interquartile ranges. Mann-Whitney U test was applied for between-group comparisons. N-NP,
nonsmoking CRSwNP; S-NP, smoking CRSwNP. P < 0.05.
increased CRS prevalence. The results suggest that CS exposure might be involved in the
degree of nasal damage in CRS patients.
The histopathological changes of nasal mucosa in smokers have been reported to be
different from those in nonsmokers [
]. Few studies indicated that CS, which is considered a
potent proinflammatory stimulator in the lower airway, contributes to nasal polyp
]. However, relevant reports about the effects of smoking on polyp histopathology
of CRSwNP patients were controversial [
22, 24, 27
]. In this study, we found that the
inflammatory reactions, including cellular infiltrates, edematous stroma and epithelial remodeling,
which have been reported in previous studies [
], were present in the polyp tissues of
smoking and nonsmoking CRSwNP patients. Our finding that the epithelial squamous
metaplasia and goblet cell hyperplasia were more prominent in smoking patients than in
nonsmoking patients corresponds with the study of Gao et al [
] in Chinese patients, as well as in other
studies performed in Asian populations [
]. The increased number of total inflammatory
cells and neutrophils in the polyp tissues of smokers corresponds with the report that CS
inhalation causes an increased numbers of macrophages, neutrophils, and T lymphocytes in
bronchoalveolar lavage [
]. The results suggest that CS exposure might be in favor of
furthering the inflammatory reactions in the nasal polyp tissues.
Various proinflammatory factors, including cytokines, chemokines, and adhesion
molecules, have been implicated in the development of nasal polyps [
]. IL-8, which is produced
primarily from epithelial cells and macrophages, is increased in nasal polyps versus normal
control mucosa [
]. IL-8 is a chemoattractant for neutrophils and elevated IL-8 level attracts
more neutrophils to the nasal mucosa or polyp tissues. TNF-? is produced by macrophages,
neutrophils, T-cells, mast cells, and epithelial cells. Moreover, TNF-? mRNA and protein
expression levels are increased in nasal polyps versus inferior turbinate tissues [
]. TNF-? has
been correlated with increased IL-8 expression and enhances neutrophil chemotaxis and
]. TNF-? stimulates adhesion molecule expression in endothelial cells and
upregulates CC chemokine ligand 2 in fibroblast cultures derived from nasal polyps, thereby
facilitating the recruitment of inflammatory cells [
]. Studies showed that inflammatory cytokines,
such as IL-1?, IL-6, IL-8, RANTES, and TNF-?, are significantly increased in the
bronchoalveolar lavage of smokers [
]. In this study, the concentrations of IL-8 and TNF-? in polyp
tissues from smoking CRSwNP patients were significantly higher compared with those in
nonsmoking CRSwNP patients. The results suggest that CS might induce inflammatory
cytokine release in polyps as it does in the lower airways. Moreover, high levels of IL-8 and TNF-?
might account for the increased number of total inflammatory cells or neutrophils
accumulating in the nasal polyps of smoking CRSwNP patients. Overproduction of IL-8 and TNF-?
attracts more inflammatory cells into nasal polyp tissues. Conversely, the infiltrating cells
release more mediators, including IL-8 and TNF-? after activation. Both of which lead to
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Fig 3. Representative immunostainings for EP receptors in CRSwNP smokers (Magnification, ?400). EP1 is expressed primarily on
infiltrating inflammatory cells in lamina propria. Both EP2 and EP3 are expressed in inflammatory cells, mucosal epithelium, and glands. EP3
is also present in blood vessels. EP4 is located in the epithelium and glands.
PLOS ONE | https://doi.org/10.1371/journal.pone.0200989
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Fig 4. Comparison of EP receptor mRNA expression between the nonsmoking CRSwNP and smoking CRSwNP groups. N-NP,
nonsmoking CRSwNP; S-NP, smoking CRSwNP. P < 0.05.
amplification of the inflammatory response and cause severe inflammatory injuries in the
polyps of smoking CRSwNP patients.
PGE2 is one of the most abundant arachidonic acid metabolites in the body and is primarily
produced by epithelial cells, endothelial cells, smooth muscles, and monocytes/macrophages
in the airways, as well as by infiltrating cells, such as neutrophils, eosinophils, and mast cells, in
inflammatory tissues [
]. Various proinflammatory and mitogenic stimuli, such as
lipopolysaccharides, proinflammatory cytokines (TNF-? and IL-1?), or growth factors, have been
proved to enhance of PGE2 synthesis and release; thus the PGE2 level is usually increased
largely at inflammation sites [
]. Several in vitro studies have reported that CS extract can
induce PGE2 production by activating or upregulating the expression of three key enzymes,
namely, phospholipase A2, cyclooxygenase-2, and membrane-bound prostaglandin E
synthase-1, for catalyzing the PGE2 biosynthesis from arachidonic acid [
13, 36, 37
]. In this
study, we found that PGE2 concentration in the nasal polyp tissues was statistically higher in
smoking patients than in nonsmoking patients. The result suggests that CS might be a
potential stimulator for PGE2 production. However, it is unclear whether the increased PGE2
production in smoker was attributed to the smoking-induced inflammatory response or the direct
promotion of eicosanoid metabolism by enhancing the activities of key enzymes, or other
unknown mechanism yet.
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Fig 5. Comparison of EP receptor protein expression between the nonsmoking CRSwNP and smoking CRSwNP
groups. Representative immunoblots are shown. N-NP, nonsmoking CRSwNP. S-NP, smoking CRSwNP. P < 0.05.
PGE2 is a known potent proinflammatory mediator involved in the initiation and
progression of inflammation [
]. In addition to directly causing inflammatory response, PGE2 also
plays a critical role in regulating the inflammatory process by modulating various immune
]. PGE2 promotes the activation of TH17 cells to produce IL-17 or stimulate human T
lymphocytes to produce IL-8 [
]. IL-17 or IL-8 recruits more monocytes and/or
neutrophils to the inflammation site and sustains inflammation. However, PGE2 also exerts an
antiinflammatory action by inhibiting the activities of various cells, such as neutrophils,
monocytes, and epithelial cells . The opposing effects of PGE2 in the inflammatory processes are
performed by EP1, EP2, EP3, or EP4 receptors [
]. In general, EP2 and EP4 receptors mediate
anti-inflammatory actions by activating adenylate cyclase to increase intracellular cAMP
concentration [42?46]. By contrast, EP1 and EP3 receptors increase intracellular calcium (EP1) or
decrease intracellular cAMP contents (EP3) to mediate proinflammatory effects [47?49].
Aside from the differential EP receptor subtype, the altered expression or coexpression of EP
receptor in tissues causes diverse or contrasting PGE2 effects [
]. In the present study, EP2
and EP4 receptor expression levels were significantly lower in the smoking group compared
with those in the nonsmoking group. This result suggests that smoking exposure might
downregulate the expression levels of EP2 and EP4 receptors in nasal polyps. Moreover, the
decreased expression levels of EP2 and EP4 receptors will weaken the anti-inflammatory
effects of PGE2, thereby relatively enhancing the EP1 and EP3 receptor-mediated
proinflammatory actions. Since both EP1 and EP3 receptors were primarily expressed in the infiltrating
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inflammatory cells, we speculate that the increased PGE2 level would promote the activities of
inflammatory cells to release more inflammatory mediators by activating EP1/EP3 receptors,
thereby leading to more serious inflammatory reactions in polyp tissues from the smoking
CRS patients. The more intense inflammatory responses might contribute to the increased CT
scores and endoscopic scores observed in smoking CRSwNP patients.
In this study, we found that the cellular distribution of the four EP receptors was similar in the
polyp tissues of smoking and nonsmoking CRSwNP patients. CS exposure enhanced PGE2
production and the proinflammatory cytokines IL-8 and TNF-? and downregulated the
expression levels of EP2 and EP4 receptors in nasal polyps of CRS patients. The
downexpressed EP2 and EP4 receptors might be associated with severe inflammatory reactions in
smoking CRSwNP patients. These results suggested that CS exposure might play a role in the
pathogenesis of CRSwNP by affecting EP receptor expression.
S1 Fig. Representative immunostainings for EP receptors in CRSwNP nonsmokers
S1 Dataset. Original data of this work.
S1 File. Original immunoblots of EP receptor protein expression.
Conceptualization: Li Xie, Xian-Song Wang.
Data curation: Xian-Song Wang.
Formal analysis: Xian-Song Wang.
Funding acquisition: Ai-Guo Liu, Xian-Song Wang.
Investigation: Ai-Guo Liu, Xian-Song Wang.
Methodology: Li Xie, Li-Yan Peng, Su-Jie Wang, Yin-Ping Zhang.
Project administration: Li Xie, Ai-Guo Liu, Xian-Song Wang.
Resources: Ai-Guo Liu.
Software: Li Xie, Li-Yan Peng.
Supervision: Ai-Guo Liu, Li-Yan Peng.
Validation: Ai-Guo Liu.
Writing ? original draft: Li Xie.
Writing ? review & editing: Ai-Guo Liu, Xian-Song Wang.
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