IL-25-induced activation of nasal fibroblast and its association with the remodeling of chronic rhinosinusitis with nasal polyposis
IL-25-induced activation of nasal fibroblast and its association with the remodeling of chronic rhinosinusitis with nasal polyposis
Soo-Kyoung Park 0 1 2
Yong-De Jin 0 2
Yeong-Kyu Park 0 1 2
Sun-Hee Yeon 0 1 2
Jun Xu 0 1 2
Rui- Ning Han 0 1 2
Ki-Sang Rha 0 1 2
Yong-Min Kim 0 1 2
☯ These authors contributed equally to this work. 0 2
0 fund of Chungnam National University 2016 and the national research fund , fund number: NRF- 2016R1D1A3B03934918
1 Department of Otorhinolaryngology-Head and Neck Surgery, Research Institute for Medical Science, Chungnam National University School of Medicine , Daejeon , Korea , 2 Department of Otorhinolaryngology- Head and Neck Surgery, Yanbian University Hospital , Yanji , China
2 Editor: Zheng Liu, Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology , CHINA
Background and objective Interleukin (IL)-25 has been shown to play an important role in the pathogenesis of chronic rhinosinusitis with nasal polyps. Nasal polyps are associated with chronic inflammation of the mucous membranes in the paranasal sinuses and are involved in extracellular matrix (ECM) accumulation. The aim of this study is to evaluate the effects of IL-25 on myofibroblast differentiation, ECM production and the expression of matrix metalloproteinases in nasal polyp derived fibroblasts (NPDFs) and to determine the molecular mechanism underlying these processes.
Data Availability Statement; All relevant data are within the paper
Materials and methods
A total of 40 patients were enrolled in this study for Immunofluorescence studies.
Expression of IL17 receptor B was evaluated by real time reverse transcription polymerase chain
reaction (PCR) in NPDFs. NPDFs were stimulated with IL-25 for 48 h in the presence or
absence of mitogen-activated protein kinase (MAPK) and NF-κB inhibitors or small
interfering RNAs (siRNA). The protein levels of fibrosis active mediators were examined using
western blotting. Fibroblast migration was evaluated with a scratch assay. The total collagen
amount was analyzed with the Sircol collagen assay.
IL-25 induced α-SMA, fibronectin, and MMP-1 and -13, which were dependent on IL-17RB.
IL-25 also induced activation of NF-κB and mitogen-activated protein kinase (MAPKs). By
using the specific inhibitor of ERK, p38, JNK and NF-κB (U, SB, SP and Bay), we found that
IL-25-induced expressions of α-SMA, fibronectin, and MMPs was regulated by the signaling
pathways of MAPKs and NF-κB. IL-25 also induces α-SMA, fibronectin, and MMPs
expression through IL-17RB-dependent pathways in NPDFs. The increased migration ability
induced by IL-25 was suppressed by the specific inhibitors of MAPKs and NF-κB.
Our data indicate that IL-25 induced myofibroblast differentiation, fibronectin production,
and MMP-1 and -13 expressions through the signaling pathways of MAPKs and NF-κB. in
NPDFs and increased expression of IL-25 were also involved in the pathogenesis of nasal polyposis by affecting nasal fibroblasts in chronic rhinosinusitis with nasal polyps.
Chronic rhinosinusitis with nasal polyposis (CRSwNP) is a chronic inflammatory disease of
the paranasal sinuses whose underlying etiology is multifactorial in nature [
]. Nasal polyposis
is histologically characterized by persistent inflammation and irreversible structural changes
that lead to remodeling in the sinonasal mucosa [
]. We have previously shown that nasal
polyps are composed of various cell types, including epithelial cells, fibroblasts/vascular
endothelial cells, eosinophils, CD4+ T cells, CD8+ T cells, B cells, macrophages, mast cells, and
dendritic cells. We previously reported that epithelial cells and fibroblasts, are two of the major
components of the nasal polyp derived cells [
] and fibroblasts confer mechanical strength by
providing a supporting framework for the extracellular matrix (ECM). Interleukin (IL) - 25 is
mainly produced from epithelial cells, and fibroblasts are a cellular source of ECM protein. [
The components of the ECM play essential roles in inflammatory reactions and can also be the
sites of numerous structural changes from fibrosis to extreme edema of the lamina propria [
]. Myofibroblasts that express alpha-smooth muscle actin (α-SMA) comprise an activated cell
phenotype of fibroblasts with a high capacity for ECM protein secretion and play an important
role in ECM remodeling of many pathologic conditions of the human airway, including
asthma, chronic rhinosinusitis, and nasal polyps [
A member of the IL-17 cytokine family and mainly produced by the epithelium, IL-25
noticeably promotes Th2 cell-mediated inflammatory responses and can promote the
recruitment of eosinophils, innate lymphoid cells, and mast cells to the inflammation site [
Epithelial-derived IL-25 can also induce the epithelium to produce more IL-25 and other potent
innate cytokines, such as IL-33 and thymic stromal lymphopoietin, thus intensifying the
allergic inflammation . In addition to driving Th2 inflammation, IL-25 expression is known to
be involved in airway remodeling by mediating pulmonary collagen deposition,
neovascularisation, peribronchial smooth muscle hyperplasia and airway hyperreactivity following allergen
]. According to a recent study, IL-25 protein levels were significantly increased in
NP tissue homogenates from patients with CRSwNPs, and further analysis has shown that
IL25 secreted from the sinonasal epithelia and infiltrating mast cells plays a crucial role in the
pathogenesis of CRSwNPs in Asian patients [
IL-25 binds a receptor complex composed of IL-17RB (also known as IL-25R), which
partners with IL-17RA [
]. IL-17rb-/- and IL-17ra-/- mice fail to respond to IL-25, and both
knockout strains are refractory to pulmonary inflammation induced by intranasal application
of IL-25 [
]. Recent studies have demonstrated that Act1 and STAT5 in the epithelium
and in T cells play critical roles in IL-25-dependent type 2 responses for allergic lung
]. Aside from signaling through Act1 or STAT5, IL-25 has also been shown to
activate MAPKs such as P38 and JNK as well as NF-kB . Although the role of IL-25 in
inducing type 2 allergic inflammatory responses and the components of its signaling cascade
are well recognized, the effects of IL-25 on fibroblast activation and the mechanism underlying
NPDFs has not yet been determined. Because epithelial cells and fibroblasts are the main
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components of nasal polyps, and epithelial-derived IL-25 expression has been shown to be
significantly higher in nasal polyp tissues than in control nasal tissues, an investigation into the
effects of IL-25 on NPDFs would be highly relevant to this field. This study aims to evaluate
the effects of IL-25 on fibroblast activation and differentiation, ECM production and MMP
expression in NPDFs and to determine the underlying molecular mechanism of these
Materials and methods
Human recombinant IL-25 was purchased from R&D System (Minneapolis, MN) and was
dissolved in sterile 4 mM HCl containing at least 0.1% bovine serum albumin. U0126 (a specific
inhibitor of ERK), SB203580 (a specific inhibitor of p38) and SP600125 (a specific inhibitor of
JNK inhibitor for JNK1, JNK2 and JNK3) were obtained from Calibiochem (Billerica, MA).
STAT5 inhibitor was purchased from Merck Millipore (Nottingham, United Kingdom). Bay
11±7082 (NF-κB inhibitor) and BX-795 (IRF3 inhibitor) were purchased from Sigma (St.
Louis, MO). All inhibitors were dissolved in dimethyl sulfoxide. Sircol collagen assay kits were
acquired from Biocolor Ltd (Belfast, N. Ireland, UK).
Patients and tissue preparation
A total of 40 patients were enrolled in this study for immunofluorescence studies. Forty
patients who visited the Department of Otorhinolaryngology of Chungnam National University
Hospital in Korea between January 2016 and November 2016 were included in the study. Of
the 40 patients, 20 had CRSwNP, 10 had CRS without NP (CRSsNP), and 10 subjects
underwent other rhinologic surgeries, such as dacriocystostomy, or endoscopic orbital
decompression surgery were enrolled as control subjects. Uncinate tissues (UT) were obtained from the
10 patients with CRSwNP, 10 patients with CRSsNP, and 10 control subjects. Polyp tissues
were obtained from the NP of patients with CRSwNP.
A sinus disease diagnosis was based on patient history, clinical examination, nasal
endoscopy, and computed tomography of the paranasal sinuses, as detailed by the guidelines
contained in ``EPOS 2012: European position paper on rhinosinusitis and NPs 2012º [
who used oral or nasal corticosteroids or other medications (e.g., antibiotics or
antileukotrienes) for 4 weeks before sample collection; those with recent upper respiratory tract
infections; and patients undergoing surgical revision were excluded from the study.
Details of the patients' characteristics are shown in Table 1. The median age of the patients
was in the CRSwNP group than those in the control and CRSsNP groups. The 3 groups did
not differ significantly with respect to gender, comorbity of bronchial asthma, atopy or aspirin
intolerance. NP tissues from 5 patients with CRSwNP were used for the NPDF culture. A
written informed consent was obtained from each patient and control subject before enrollment
into the study. The study was approved by the Institutional Review Board of the Chungnam
National University Hospital.
NPDFs were isolated from the surgical tissues by cutting them into small pieces with a
sanitized scissor, followed by collagenase (500 U/ml, Sigma) digestion. The cells were cultured in
Dulbecco's Modified Eagle Medium containing penicillin G (10,000 U/ml), streptomycin
(10,000 μg/mL), amphotericin B (25 μg/mL) and 10% fetal bovine serum (FBS) (Invitrogen,
Carlsbad, CA, USA).
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The NPDFs were seeded in culture plates for 7 days, after which the unattached cells were
eliminated. The purity of the obtained fibroblasts was confirmed by characteristic spindle and
bipolar or multipolar morphology as well as flow cytometry. The fourth cell passage provided
every cell used for this research. NPDF isolate from 5 patients were used for in vitro
experiment; including fluorescence activated cell sorting (FACS) analysis, siRNA, cell migration
assay, real-time polymerase chain reaction (RT-PCR) and western blotting.
The paraffin-embedded tissue samples were soaked first in xylene to remove the paraffin wax
and then sequentially in solutions of 100%, 95%, and 70% ethanol for rehydration. Antigen
retrieval was performed by heating the slides in a Decloaking Chamber (Biocare Medical,
Concord, CA) to 120ÊC. A protein block [10% normal chicken serum (Vector, Burlingame,
CA, USA) in phosphate-buffered saline (PBS) and 0.3% Triton X-100 (Biosesang, Seongnam,
Republic of Korea) for 1 hour at room temperature] was then applied to the tissue to prevent
non-specific protein binding. Endogenous peroxidase activity was blocked by incubating the
sections in 1% hydrogen peroxide solution (Sigma-Aldrich, St. Louis, MO, USA) in PBS with
0.3% Triton X-100 for 30 minutes at room temperature. Mouse anti-Vimentin antibody
(Invitrogen, Carlsbad, CA, USA), rabbit anti-alpha SMA (Abcam, Cambridge, MA, USA), and
rabbit anti-IL-25 (Abcam, Cambridge, MA, USA), at a concentration of 10μg/mL, were used as
the primary antibody which was incubated with the tissue at 4ÊC overnight. The sections were
rinsed 3 times with PBS and then incubated for 2 hours at room temperature with Alexa
Fluor@ 488-conjugated goat anti-mouse IgG and Alexa Fluor@ 594-conjugated goat
anti-rabbit (Life Technologies, Carlsbad, CA, USA) as a secondary antibody. After rinsing with PBS,
4',6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) was used at a
concentration of 300 nM for nuclear counterstaining. After final rinsing with PBS, the samples were
mounted using Fluoro-Gel with Tris Buffer (Electron Microscopy Science, Hatfield, PA,
USA). The slides were subsequently observed on a fluorescence microscope (Olympus, Tokyo,
Japan). Vimentin/alpha-SMA and IL-25/ alpha-SMA double-positive cells were counted per
high-powered field (HPF, × 400) at 3 different randomly chosen sites in the tissue, and the
mean values with range were calculated.
The NPDFs were stained with Vimentin (eBioscience, Ireland, United Kingdom) and
alphaSMA (eBioscience, Ireland, United Kingdom). 7-AAD and 7-amino-actinomycin D
(SigmaAldrich, St. ILouis, MO, USA) were used for staining the nonviable cells in our flow cytometric
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analysis. In brief, the NPDFs were washed in PBS buffer, and the staining dyes Vimentin
(eBioscience, Ireland, United Kingdom) and 7-AAD (Sigma-Aldrich, St. ILouis, MO, USA)
were then added and incubated in the dark for 20 minutes on ice. The NPDFs were washed in
PBS and then fixed with 4% paraformaldehyde. Through this process, more than 4 × 105 cells
were prepared and subjected to FACS analysis.
The tissue samples were prepared through fixation in 4% buffered formalin, followed by
embedding in paraffin and sectioning of the obtained paraffin block at every 4 μm thickness.
Immunohistochemistry was performed using a modified streptavidin- biotinylated peroxidase
technique with mouse anti-human vimentin (Affinity Bioreagents; Golden, CO), rabbit
antihuman alpha-SMA (Abcam, Cambridge, MA, USA) and mouse anti-human IL-25 (Abcam,
Cambridge, MA, USA).
The slides were prepared manually with pre-treatment by boiling them in sodium citrate
buffer (pH 10) for 60 minutes, at 90ÊC. Nonspecific protein staining was blocked by goat
serum for 30 min at room temperature. The sections were incubated with primary antibody at
4ÊC overnight. The sections were rinsed with PBS and incubated with biotinylated secondary
antibodies for 60 min. They were then washed and treated with 0.3% hydrogen peroxide in
methanol for 30 min to inhibit the activity of any endogenous peroxide. The slides were
washed, incubated with streptavidin-biotin-peroxidase complex for 30 min, and developed
according to the manufacturer's protocol. The immunoreaction was subsequently visualized
with 3, 3'-diaminobenzidine (DAB). After a final rinsing with PBS, the samples were mounted
using Fluoro-Gel with Tris Buffer (Electron Microscopy Science, Hatfield, PA, USA).
The slides were subsequently observed on a fluorescence microscope (Olympus, Tokyo,
Japan). Vimentin, alpha-SMA and IL-25 positive cells were counted per high-powered field
(HPF, × 400) using the tissues from the three different patients, and the mean values with
range were calculated.
Application of Short Interfering RNA (siRNA)
A duplex siRNA designed to target human IL-17RB (NCBI: NM_018725) was purchased from
Bioneer (Daejeon, Republic of Korea). A nonsilencing siRNA duplex targeting was used as the
control siRNA (random siRNA duplex). The NPDFs were seeded in six-well plates at a density
of 5 × 105 cells per well to obtain 70 to 80% confluence. Transfection of the siRNA was
performed with Lipofectamine1 RNAiMAX (Cat 13778075; Life Technologies, Carlsbad, CA,
USA) in culture medium (without FBS and antibiotics) according to the manufacturer's
instructions. Forty-eight hours after transfection of siRNA (at a concentration of 100 pmol/
μL), RT-PCR and western blot analysis were performed to examine the gene silencing effect.
Cell cytotoxicity and viability of the IL-25 protein were measured by MTT
(3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, Sigma-Aldrich, St Louis, MO,
USA). The NPDFs were seeded at a density of 5×104 cells in 96-well plates. After incubation at
37ÊC in the presence of 5% CO2 in a constant temperature and humidity incubator for 1day,
the NPDFs were treated with different doses of IL-25. After 72 h, 10μl of the MTT solution (5
mg/mL) was added 4 h before the end of the incubation duration, and the reaction was
terminated by the addition of 100 μL of dimethylsulfoxide (DMSO). Another MTT assay was
performed to exclude the possibility of specific inhibitors namely U (a specific inhibitor of ERK),
SB (a specific inhibitor of p-38), SP (a specific inhibitor of JNK) and Bay (a specific inhibitor of
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NF-κB)-induced cellular cytotoxicity of the NPDFs. The optical density (OD) or absorbance
was read at 570 nm. The experiments were performed at least 3 times in triplicate.
Cell migration assay
To study the effects of IL-25 and inhibitor treatment on cell migration, a monolayer wound
healing assay was performed. The NPDFs were seeded at a density of 1x104 cells/well in a
35mm culture-insert 4 well μ-Dish (ibidi, Germany). Upon reaching complete confluence,
the inner well of the μ-Dish was removed, and the detached cells and debris were washed 3
times with PBS. The cells were incubated in a serum free DMEM medium with IL-25 and/or
inhibitor of ERK, p38, JNK (U0126 10μm, SB203580 10μm, SP600125 10μm) and NF-κB (Bay
11±7082, 1μm). Photomicrographs were taken at time 0 (immediately following the scratch
wound), 24 and 48 hours under a phase-contrast microscope. For statistical analysis, 3
randomized microscopic fields containing the scratch in the center of the fields were captured for
each culture dish. The number of fibroblasts located in the scratches was counted and the
averages of the count numbers of the 3 randomized microscopic fields for each culture dish were
mRNA expression of IL-17 receptor B (IL-17RB) was evaluated using RT-PCR. Total RNA
was isolated from the NPDFs, CRSwNP (NP), control (UT) and HeLa cell (negative control)
using a TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's
instructions. For cDNA synthesis, 1 μg of total RNA was transcribed with AccuPower™ RT
PreMix (Bioneer, Daejeon, Republic of Korea), according to the manufacturer's instructions.
PCR was performed for cDNA synthesis using a T100TM Thermal Cycler (Bio-Rad
Laboratories, Hercules, CA, USA). The mRNA expression was analyzed using a CFX Connect TM
RealTime PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with PowerUp TM
SYBR TM Green Master Mix (Applied Biosystems, Carlsbad, CA, USA). PCR was performed
using the following primers: human IL-25R (IL-17BR) (sense sequence: 50-AACAGGCGTCC
CTTTCCCTCTGGA-30 and antisence sequence: 50-TTCTTGATCCTTTCGTGCCTCCAC-30);
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sense sequence: 5’-GTG GAT ATT
GTT GCC ATC AAT GAC C-3’ and antisense sequence: 5’-GCC CCA GCC TTC TTC ATG
All PCR assays were performed in triplicate. For each sample, the differences in threshold
cycles between the target molecules and GAPDH (ΔCttarget gene, ΔCtreference gene) were
determined; a calibrated delta Ct value (ΔΔCt, ΔCttarget gene -ΔCtreference gene) was
calculated; and the relative quantitation (RQ) values were then calculated using the following
equation: RQ = 2-ΔΔCt.
Sircol collagen assay
The Sircol Collagen Assay kit (Biocolor Ltd, UK) is a quantitative dye-binding method
designed to analyze acid soluble collagens extracted from mammalian tissues and collagens
released into culture medium by mammalian cells during in vitro culture. The assay was
performed according to the manufacturer's recommendations. The optical density of sirius red
was read against a blank at a wavelength of 540 nm (OD540), and the results were expressed as
collagen concentration (mg/mL). In some experiments, the cells were pretreated with U0126
(a MEK/ERK signaling inhibitor) or SB431542 (a ALK5/Smad2/3 signaling inhibitor) or
SP600125 (an MEK/JNK inhibitor) or Bay 11±7082 (a NF-κB inhibitor) for 2 hours, after
which human recombinant IL-25 was added and co-incubated for 3 days.
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Western blot analysis
NPDFs were treated with IL-25 and inhibitors for 48 h. A total of 5×105 cells were lysed in a
RIFA buffer (Cell Signaling Technology, USA) that contained protease inhibitors (iNtRON
Biotechnology, Korea) and phosphatase inhibitors (iNtRON Biotechnology, Korea) and
centrifuged at 12,000 × g for 20 min. The total protein concentration was determined using the
Bradford assay (Bio-Rad, USA). The samples were resolved in 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.45 μm Polyvinyl Difluoride
(PVDF) membranes and analyzed separately. After blocking with 5% skim milk at room
temperature for 60 min, the blots were surveyed with primary antibodies against rabbit
antimouse, rat and human fibronectin (1:1000; Santa Cruz, CA), rabbit anti-human α-SMA
(1:1000; Abcam), rabbit anti-human, mouse MMP1 (1:1000; Mybiosource), rabbit
antihuman, Rat MMP13 (1:1000; Mybiosource), rabbit anti-human p-IκBα (1:1000; Cell Signaling
Technology), rabbit anti-human p-ERK (1:1000; Cell Signaling Technology), rabbit
antihuman-p-38 (1:1000; Cell Signaling Technology), rabbit anti-human p-JNK (1:1000; Cell
Signaling Technology), anti-human p-STAT5 (1:1000; Cell Signaling Technology), anti-human
p-IRF3 (1:1000; Cell Signaling Technolog) and rabbit anti-human GAPDH (1:3000; Cell
Signal), at 4ÊC for overnight. Both the NF-κB and MAPKs (which comprise ERK, p38 and JNK)
families were activated by IL-25. After serum-starvation for 24 h, the cells were stimulated
with IL-25 (100 ng/ml) for 2 h. To evaluate the signaling pathways involved in the induction of
α-SMA, fibronectin, MMP-1, and MMP-13 by IL-25 stimulation in the NPDFs, the cells were
pretreated for 1 h with U0126 (a specific inhibitor of ERK), SB203580 (a specific inhibitor of
p38), SP600125 (a specific inhibitor of JNK inhibitor for JNK1, JNK2 and JNK3), and Bay 11±
7082 (NF-κB inhibitor). The cells were then stimulated in the presence of these inhibitors with
100ng/ml of IL-25 for 48 h, and the expression levels of α-SMA, fibronectin, MMP-1, and
MMP-13 were determined by western blot assay. The membranes were washed 3 times with
TBST buffer (20 mmol/L Tris-buffered saline and 0.1% Tween 20) for 1 h. Peroxidase
conjugated anti rabbit-IgG were used as secondary antibodies. Chemiluminescence was performed
with the Amersham ECL plus western blotting detection system (GE Healthcare, USA).
The sample size was chosen based on previous studies [
]. We conducted a power analysis
that indicated that with 10 patients in both the CRS w NP and the control group, using the
standard deviation in the number of double positive cells (Vimentin+ α-SMA+) and a
twosided .05 level of significance, the power was determined to be 0.99.
Statistical analyses were performed using SPSS 22 (version 18.104.22.168, International Business
Machines, Armonk, NY, USA) and GraphPad Prism 6 (version 6.01, GraphPad Software, La
Jolla, CA, USA). For continuous variables, the results are expressed as mean with standard
error of mean (SEM). D'Agostino-Pearson omnibus normality test, Mann-Whitney U test,
Kruskal-Wallis tests (2-tailed), and two-way ANOVA were used for the normality test,
comparisons of two groups, and multiple testing, respectively. A p value of less than 0.05 was
considered statistically significant. The fourth cell passage provided all the cells used for this
research. Also, biological and technical triple tests were performed on all of the experiments.
Number of myofibroblasts was significantly higher in NP tissues
Double immunofluorescent staining (Vimentin/ α-SMA or IL-25/ α-SMA) was conducted to
investigate whether myofibroblasts (active form of fibroblast) were involved in pathogenesis of
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nasal polyp of the CRSwNP group. α-SMA and vimentin expression was observed in cytosol
and IL-25 expression was observed in membrane. The number of double positive cells
(Vimentin+ α-SMA+ cells) was significantly higher in the NP tissues of the CRSwNP group
than in the other groups (p = 0.012) (Fig 1A and 1B). As shown in the above experiment, our
FACS analysis using Vimentin (FITC) and α-SMA (APC) divided into four groups confirmed
that vimentin was most abundant in the CRSwNP (NP) group (S1 Fig). This result suggests
that activation of fibroblasts is associated with nasal polypogenesis in patients with CRSwNP.
We then examined the correlation between the IL-25 level and the activated NPDFs in the
polyp tissues. Compared to the other group, the number of double positive cells (IL-25+
αSMA+) in the NP tissues of the CRSwNP group significantly increased (p = 0.016). To show
the IL-25 positive cell distribution in the image of the histological sections, we conducted
immunohistochemical staining for IL-25, α-SMA and vimentin. Vimentin, α-SMA and IL-25
levels in the patients' tissues were higher in the CRSwNP (NP) group than in the other 3
groups (S2 Fig). Taken together, these data show that IL-25 induced activation of nasal
fibroblast was associated with CRSwNP (Fig 2A and 2B).
Expression of IL-25 receptor on NPDFs was confirmed by RT-PCR
Microscopic examination revealed that NPDFs were morphologically bipolar or multipolar,
with elongated shapes, and were growing attached to a substrate (Fig 3A). Approximately 80%
of the cells in the cultured NPDFs tested positive for vimentin, which was used as a fibroblast
marker (Fig 3B). To determine whether the IL-25 receptor was involved in the IL-25-induced
ECM and MMP expressions in the NPDFs, mRNA expression of IL-17 receptor B (IL-17RB)
was evaluated using RT-PCR. IL-17RB mRNA levels were also measured in HeLa cells
(negative control), CRSwNP(NP) and control (UT). We found that IL-17RB mRNA was expressed
on the NPDFs. In addition, IL-17RB mRNA was expressed more in NPDFs than in HeLa cells,
and it was found to be less than in CRSwNP (NP) and control (UT) tissues. (Fig 3C). We
measured IL-17RB mRNA and IL-17RB protein expression levels with IL-17RB siRNA (p < 0.001)
and confirmed that IL-17RB mRNA and IL-17RB protein expression was suppressed by
approximately 60% and 80%, respectively, by IL-17RB siRNA (Fig 3D).
IL-25 induced myofibroblast differentiation (α-SMA), fibronectin
production and MMP-1 and -13 expressions in NPDFs
We found that IL-25 did not directly influence the survival of the NPDFs at doses ranging
between 10 and 1500 ng/ml for 72 hours (Fig 4A). U (specific inhibitor of ERK), SB(specific
inhibitor of p38), SP (specific inhibitor of JNK) and Bay(specific inhibitor of NF-κB) also did
not affect cell viability until the concentration reached 10μg/mL for 72hours (Fig 4B). The
expression levels of α-SMA, fibronectin and MMP-13 protein were determined by western
blot assays which revealed that the expression of these proteins was significantly increased
after treatment with various concentrations (10-1500ng/mL) of IL-25 (p < 0.0001) (Fig 4C,
4D, 4E and 4G). The expression of MMP-1 was increased at 10 and 100 ng/ml of IL-25
stimulation, but reduced at 1000 and 1500 ng/ml of IL-25 stimulation (p < 0.0001) (Fig 4C and 4F).
These results indicate a dose dependent manner in which IL-25 induced myofibroblast
differentiation (α-SMA) ECM production, and MMP-13 expression occur.
IL-25 induced activation of MAPKs and NF-κB in NPDFs
Activations of NF-κB and MAPKs has been shown to play essential roles in IL-25R-mediated
cellular activation and gene expression in a number of different cell types [19, 21±23]. As
representative MAPKs, phosphorylation of ERK, JNK, and p38 MAPK were also induced by
IL8 / 23
Fig 1. Double±immunofluorescent staining of myofibroblasts in the tissues of the four groups. Double-immunofluorescence staining was
undertaken to colocalize the cells with Vimentin (green color)/ α-SMA (red color) among the groups (A). The number of double positive cells
(Vimentin+ α-SMA+) was considerably higher in the NP tissues of the CRSwNP group compared to the other groups (*p < 0.05) (B). Vimentin: white
arrow head, α-SMA: yellow arrow head, double positive cells: red arrow. Triple tests were performed on all of the experiments.
25 treatment, and the use of U (specific inhibitor of ERK), SB (specific inhibitor of p38) and
SP (specific inhibitor of JNK) showed a markedly reduced expression of p-ERK, p-p38, and
JNK. (p-p38 reduced expression p value is 0.0028; the others are p < 0.001) (Fig 5A±5C). IL-25
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Fig 2. Double±immunofluorescent staining of the correlation between IL-25 level and myofibroblast in the tissues of the four
groups. Double-immunofluorescence staining was undertaken to colocalize the cells with IL-25 (green color)/ α-SMA (red color) among the
groups (A). The number of double positive cells (IL-25+ α-SMA+) was considerably higher in the NP tissues of CRSwNP group compared to
the other groups. Vimentin and IL-25: white arrow head, α-SMA: yellow arrow head, double positive cells: red arrow (*p < 0.05). Triple tests
were performed on all of the experiments.
also induced phosphorylation of IκBα, a frequently used marker for the activation of the
NFκB pathway, which significantly decreased when pretreated with Bay (p < 0.0001) (Fig 5D).
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Fig 3. Microscopic morphology of NPDFs and expression of IL-25 receptor on NPDFs. Microscopic morphology of
NPDFs (A). Approximately 80% of the cells in the cultured NPDFs were positive for vimentin (B). The mRNA expression of
IL17 receptor B (IL-17RB) was evaluated using RT-PCR (C), the IL-17RB mRNA and IL-17RB protein expression levels were
significantly suppressed by IL-17RB siRNA compared with the control siRNA (*p < 0.05) (D). Triple tests were performed on all
of the experiments.
Taken together, our data indicates that IL-25 induces activation of NF- κB and MAPKs in
IL-25-induced α-SMA, fibronectin and MMPs expressions are
differentially regulated through various signaling pathway in NPDFs
Treatment with the specific inhibitors significantly inhibited IL-25-induced α-SMA, MMP-1,
and MMP-13 protein expressions. Treatment with the significantly inhibited IL-25-induced
αSMA protein expressions (p < 0.0001), (Fig 6A, 6B, 6D and 6E). IL-25-induced fibronectin
expression was inhibited by pretreatment with the NF-κB, JNK, and p38 pathway inhibitors
(p < 0.0001), but not by the ERK inhibitor (p = 0.02) (Fig 6A and 6C). However, with
pretreatment of the ERK inhibitor, the IL-25-induced fibronectin mRNA expression was significantly
inhibited (p < 0.0001) (Fig 6F). Similar results were observed for soluble total collagen levels,
and collagen production induced by IL-25 was inhibited by all the inhibitors (p < 0.0001) (Fig
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Fig 4. IL-25 induced myofibroblast differentiation (α-SMA), fibronectin production and MMPs
expression in NPDFs. An MTT assay was performed to exclude the possibility of IL-25-induced and specific
inhibitors-induced cellular cytotoxicity of the NPDFs (A). The specific inhibitors did not affect cell viability (B)
The expression levels of α-SMA, fibronectin, MMP-1 and MMP-13 protein were determined by western blot
assays (*p < 0.05) (C-G). Triple tests were performed on all of the experiments.
6G). These results indicate that IL-25-induced myofibroblast differentiation, ECM production
and MMP expression are related to the signaling pathways of MAPKs, and NF-κB, although to
differing degrees. We can further report that NF-κB is the main signaling pathway in
IL25-induced myofibroblast differentiation, ECM production and MMP expression.
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Fig 5. IL-25 induced activation of MAPKs and NF-κB in NPDF. Effects of IL-25 on the activation of mitogen-activated protein
kinases (ERK, p38, JNK) and NF-κB evaluated by Western blotting. Phosphorylation of representative MAPKs, ERK (A), JNK
(B), and p38 MAPK (C) was induced by IL-25 treatment, after which they became inhibited by their specific inhibitors, including
U, SP, and SB, respectively. (p < 0.05). IL-25 induced phosphorylation of IκBα; p-IκBα expression was inhibited by Bay
(p < 0.05) (D). Values are expressed as means ± standard errors of independent experiments. (*p < 0.05 vs control). ²p <0.05
vs IL-25 alone. Triple tests were performed on all of the experiments. U = specific inhibitor of ERK, SB = specific inhibitor of p38,
SP = specific inhibitor of JNK, Bay = specific inhibitor of U = specific inhibitor of NF-κB.
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Fig 6. Regulation of IL-25-induced α-SMA, fibronectin and MMPs expression through various
signaling pathways in NPDFs. (A) The expression levels of α-SMA, fibronectin, MMP-1, and MMP-13 were
determined by western blot assay or RT-PCR. Treatments with the specific inhibitors significantly inhibited
IL25-induced α-SMA (B), MMP-1 (D), and MMP-13 (E) protein expression (p < 0.05). IL-25-induced fibronectin
(C) expression was inhibited by pretreatment with NF-κB, JNK, and p38 pathway inhibitors, but not by the
ERK inhibitor. IL-25-induced fibronectin mRNA expression level was measured by RT- PCR (F). The total
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soluble collagen level was measured by collagen assay (G) Values are expressed as means ± standard errors
of independent experiments. *P<0.05 vs control; ²p <0.05 vs IL-25 alone. Triple tests were performed on all of
the experiments. U = specific inhibitor of ERK, SB = specific inhibitor of p38, SP = specific inhibitor of JNK,
Bay = specific inhibitor of U = specific inhibitor of NF-κB.
IL-25 induced α-SMA, fibronectin and MMPs expressions through IL
17RB-dependent pathways in NPDFs
To assess whether IL-25-induced α-SMA, fibronectin and MMPs expressions depend on the
IL-25 receptor signal pathway, we suppressed IL-25R expression by IL-17RB siRNA (Fig 3D).
Knock down of IL-17RB decreased IL-25-induced expression of α-SMA, fibronectin and
MMP in the NPDFs (p = 0.0292, 0.0071, 0.00421 and 0.0007 respectively), and the expression
levels were determined by western blot assay (Fig 7A±7E). These data indicate that IL-25 the
expression of induces α-SMA, fibronectin, and MMPs proteins through IL-17RB-dependent
pathways in NPDFs, and the expression levels were determined by western blot assay.
IL-25 facilitated migration of NPDFs
To examine the IL-25-induced influence of migration in NPDFs, cell migration assays were
performed. The number of fibroblasts was counted in a ªcell-free gapº (500μm in width) after
24 and 48hours. Compared to the controls, the number of fibroblasts that had migrated into
the cell-free gap was significantly higher in the cells treated with IL-25. Pretreatment with the
specific inhibitors for signaling molecules significantly decreased the number of migrated cells
(p < 0.05) (Fig 8).
Chronic rhinosinusitis (CRS) is associated with severe inflammation, and CRSwNP may
overwhelmingly lead to severe edematous change, while CRSsNP usually exhibits fibrotic
remodeling of the lamina propria [
]. Although CRSwNP and CRSsNP present predominant
edematous and fibrotic patterns, respectively, a recent study demonstrated that many aspects of
the changes in tissue composition are not specific to the diseases and can present a wide range
of severity [
]. The precise molecular factors mediating this differential remodeling pattern are
not fully understood, but a recent study has suggested that TGF-β1 may play a key role in this
process; this study reported that low levels of TGF-β1 were observed in CRSwNP while high
levels were observed in CRSsNP [
]. Another study, however, reported higher expressions of
TGF-β1 in the nasal secretions of CRSwNP, than in the control or CRSsNP patients [
Despite differing reports regarding the expression levels of TGF-β1 or patterns of
connective tissue composition in CRSwNP, it is generally understood that the expression of IL-25 is
significantly higher in the nasal polyps of CRSwNP patients than in CRSsNP patients or
control subjects [
]. As IL-25 is generally considered to be the dominant innate cytokine with a
critical role in the pathogenesis of polyp formation in CRSwNP, [
] and because expression
of IL-25 is known to be involved in airway remodeling,  in the present study, we examined
whether IL-25 influences tissue remodeling in CRSwNP. We also evaluated the effects of IL-25
on the activations of fibroblasts, one of the main components of nasal polyps and a main
determiner of tissue composition. We demonstrated that IL-25 induced an increased production of
collagen and fibronectin as well as expression of MMPs through the NF-κB and MAPKs
signaling pathway in the NPDFs.
IL-25, also known as IL-17E, is mainly produced by mucosal epithelial cells.
Over-expression of IL-25 is associated with increasing eosinophilia and a TH2 dominant immune
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Fig 7. IL-25 induced α-SMA, fibronectin and MMPs expressions via IL-17RB dependent pathways in
NPDFs. Knock down of IL-17RB by IL-17RB siRNA decreased IL-25-induced a-SMA, fibronectin and MMPs
expression in NPDFs (A, B, C, D, E). Values are expressed as means ± standard errors of independent
experiments. *P<0.05 vs control. Triple tests were performed on all of the experiments.
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Fig 8. Migration of NPDFs facilitated by IL-25 facilitated the migration of NPDFs. Migration cells were
examined by a cell migration assay. Photos of our microscopic observations of cell migration assays showing
nasal fibroblast migration by IL-25 and pretreatment with specific inhibitors after 24h (A) and 48h (BC). The
number of fibroblasts was counted in a `cell-free gap' (500μm in width) after 24h and 48h (C). Values are
expressed as means ± standard errors of independent experiments. *P<0.05 vs control; ²p <0.05 vs IL-25
alone; rp <0.05 vs 24hr. Scale bar = 500 μm; Triple tests were performed on all of the experiments.
U = specific inhibitor of ERK, SB = specific inhibitor of p38, SP = specific inhibitor of JNK, Bay = specific
inhibitor of U = specific inhibitor of NF-κB.
response. Although it has been demonstrated that IL-25 receptor (IL-25R) is composed of
IL17RB and IL-17RA and forms a functional complex, the specific contribution of each subunit
to downstream signaling remains unclear [
]. Ongoing studies have indicated that IL-17RB is
expressed by various cell types such as epithelial cells, T cells, monocytes and innate lymphoid
cells, [28±30] but studies of IL-25 have been focused only on their immunologic functions on
T cells or various inflammatory granulocytes, and the expression of IL-17RB on fibroblasts is
yet to be investigated. We have demonstrated that IL-17BR is also expressed on NPDFs and
induces activation of the fibroblasts, as well as ECMs and MMPs production. To our
knowledge, our study is the first to report that IL-25 activates nasal fibroblasts and thereby induces
myofibroblast differentiation and ECMs and MMPs production in nasal polyps.
IL-17RB encodes a TRAF6-binding motif in its cytoplasmic tail. Antibody-mediated
crosslinking of the receptor activates NF-κB, which can be blocked by a dominant-negative form of
]. The IL-17RB cytoplasmic tail contains a SEFIR domain, and was recently
shown to bind ACT1 in a SEFIR-dependent manner . Apart from signaling through Act1
and TRAF6, IL-25 has been shown to activate MAPKs such as p38 and JNK as well as a NF-κB
in eosinophils [
The MAPK pathways consist of many phosphorylation cascades, each of which modulates
different signaling event, either alone or in combination. The MAPK pathways have been
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reported as Smad-independent TGF-β signaling pathways [
], ERK, JNK, and p38 are
known to participate in TGF-β signaling cascades [
]. MAPK activation regulates
phosphorylation of the down-stream transcription factor NF-κB, which leads to the activation of
the NF-κB signaling pathway [
]. We used the inhibitors U0126 (a specific inhibitor of
ERK, 10μM), SB203580 (a specific inhibitor of p38, 10μM), SP600125 (a specific inhibitor of
JNK, 10μM), and Bay 11±7082 (NF-κB inhibitor, 1μM) to elucidate the intracellular signaling
mechanisms regulating the induction of the fibroblast activation. Based on previous
publications and toxicity results we used the optimal concentrations of U0126 (a specific inhibitor of
ERK, 10μM), SB203580 (a specific inhibitor of p38, 10μM), SP600125 (a specific inhibitor of
JNK, 10μM), and Bay 11±7082 (NF-κB inhibitor, 1μM) with the highest inhibitory effect
without any cell toxicity . Our inhibition experiments have demonstrated that IL-25-induced
fibroblast activation and ECM and MMPs production were mediated by NF-κB, p38, and JNK
activities. In other words, IL-25 induced the migration ability of the fibroblast, which was
mediated by the NF-κB, p38, and JNK signal pathways. In addition, the NF-κB pathway was
the transcription factor most involved in the IL-25-induced activation of the NPDFs. In our
study, the production of fibronectin and collagen was not significantly inhibited by the ERK
inhibitor, which indicates that ERK is not involved in the production of fibronectin and
collagen. However, the production of α-SMA, MMP-1, and -13, and migration ability was
associated with the ERK signaling pathway.
Current studies in this field have demonstrated that MMPs and TIMPs play an essential
role in tissue remodeling, [34±36] as described in detail previously [
], this is because
increased expression of MMPs or reduced TIMP-1 could induce ECM rupture and promote
deposits of substances such as water and albumin. This ECM remodeling could result in
edema and trigger a local inflammatory process, [
]. Therefore, it would be reasonable to
hypothesize that MMPs expression could be a factor in a more pronounced edema and
consequently larger disease extension. Indeed MMPs and TIMP expression levels iffer between
CRSwNP and CRSsNP patients, suggesting that MMPs could be pivotal in worsening wound
healing and promoting tissue edema in nasal polyps [
]. Myofibroblasts are known to play a
crucial role in MMPs production in the tissues, and evidence to support the hypothesis that
fibroblasts perform a crucial role by differentiating into myofibroblasts has been reported [
Myofibroblasts, which express α-SMA, produce a large quantity of ECM components. This
process results in the accumulation of ECM and has an important impact on the structural
modification of nasal polyps [
]. Our results indicate that IL-25, one of the dominant
cytokines in Asian nasal polyps, also induce the expression of α-SMA in NPDFs in a
It is widely known that various substance, such as cell adhesive molecules and ECM-like
type IV collagen, type VII collagen, laminin, fibronectin, and heparin sulfate comprise the
mucosal basement membrane, which could be destroyed by various MMPs [
addition, a thickening of the layer below the basal lamina consisting of collagens type I, III, and V
plus fibronectin is a hallmark of remodeling in asthma . At least 23 MMP family members
have been characterized [
]. In particular, MMP-13 is thought to play key roles in tissue
remodeling and repair through the degradation of type IV collagen, a major component of
the basement membrane zone . Malinsky et al. reported a higher expression of MMP-1
mRNA in patients with nasal polyps than in the controls, suggesting that MMP-1 (also known
as collagenase 1 and known to initiate degradation of type I and III fibrillar collagen) could
play an important role in CRSwNP [
]. MMP-13 (precursor of collagenase 3) degrades
fibrillar collagen found in extensible connective tissues such as the skin, lungs, and vascular system,
frequently in association with type I collagen [
]. Molet et al. demonstrated that collagen
types I, III, and V were increased in all nasal polyp tissues, with a predominance of types III
18 / 23
and V . Muro et al. [
] and Huang et al. [
] reported that in diseases such as allergic
rhinitis and sinusitis, histological evidence of ECM remodeling includes basement membrane
thickening, subepithelial fibrosis, with increased deposition of collagen type I and III along
with other matrix products. Therefore, we evaluated IL-25-induced expressions of MMP-1
and MMP-13 on NPDFs and found that IL-25 increased the MMP-1 and MMP-13 expressions
and that this process was mediated by intracellular MAPKs and NF-kB activation. De Borja
et al. [
] reported the expression of MMPs and TIMPs in different tissue structures and
cells from NP and nasal mucosa, and included a variety of MMPs and TIMP in their
experiments. The present study has several limitations. We did not conduct any studies on TIMPs
that regulate MMPs, and only a few types of MMPs related to nasal polyps are evaluated in this
study. In addition, other cytokines secreted in association with IL-25 are not included.
Inclusion of other cytokines would help to clarify the IL-25 associated signaling.
This is the first study on the activation of NPDFs for the release of ECM proteins and MMPs
by IL-25. Our results suggest that IL-25-induced release of α-SMA, fibronectin, collagen,
MMP-1 and MMP-13 from NPDFs is mediated by the combined activation of the MAPK and
NF-kB pathways, thereby providing new clues for fibroblast-mediated inflammation by
changing the ECM composition in nasal polyps. Further investigations are required for other
potential intracellular signaling pathways for the regulation of the release of ECM and MMPs.
S1 Fig. A flow cytometric analysis of double positive cells between Vimentin and α-SMA.
S2 Fig. Immunohistochemistry staining pictures for IL25, a-SMA and vimentin.
We thank the staff of the Flow Cytometry Facility of Chungnam National University for their
excellent assistance with the FACS analysis. We also offer our thanks to the study participants,
all joint research workers, and Dong-Woon Kim from the Department of Anatomy for his
assistance in the double immunofluorescence study. Finally, we thank the patients who
provided samples for this study.
Conceptualization: Soo-Kyoung Park, Ki-Sang Rha, Yong-Min Kim.
Data curation: Soo-Kyoung Park, Yong-De Jin, Yeong-Kyu Park, Sun-Hee Yeon, Yong-Min
Formal analysis: Soo-Kyoung Park, Yong-De Jin, Sun-Hee Yeon.
Investigation: Sun-Hee Yeon, Ki-Sang Rha, Yong-Min Kim.
Methodology: Sun-Hee Yeon, Jun Xu.
Project administration: Soo-Kyoung Park, Yong-De Jin, Yeong-Kyu Park, Ki-Sang Rha,
Resources: Sun-Hee Yeon, Jun Xu, Rui-Ning Han.
19 / 23
Software: Jun Xu, Rui-Ning Han.
Supervision: Jun Xu, Rui-Ning Han, Yong-Min Kim.
Validation: Yong-De Jin, Yeong-Kyu Park, Jun Xu, Ki-Sang Rha.
Visualization: Soo-Kyoung Park, Yong-De Jin, Yeong-Kyu Park, Jun Xu, Ki-Sang Rha.
Writing ± original draft: Soo-Kyoung Park, Yong-De Jin.
Writing ± review & editing: Soo-Kyoung Park.
20 / 23
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