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\begin{document}

\title{PVP-I reduces LPS-induced airway inflammation by blocking TLR4 signaling
in airway epithelial cells}



\author[1]{Seung Hoon Lee}%
\author[1]{Sun-Hee Yeon}%
\author[1]{Seung-Hyeon Choi}%
\author[1]{Soo-Kyoung Park}%
\author[1]{Mi Ra Choi}%
\author[1]{Yong-Min Kim}%
\affil[1]{Chungnam National University School of Medicine}%


\vspace{-1em}



  
  \date{May 28, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: Povidone-iodine (PVP-I) is an antiseptic and a disinfectant
with broad-spectrum antimicrobial activity against various pathogens.
However, it is unclear whether PVP-I nasal instillation can suppress
mucosal inflammation in non-eosinophilic chronic rhinosinusitis (CRS)
mice. Objective: This study aimed to explore the anti-inflammatory
effects and underlying molecular mechanism of PVP-I on
lipopolysaccharide-stimulated airway epithelial cells and investigate
whether nasal instillation of PVP-I can suppress mucosal inflammation in
non-eosinophilic CRS mice. Methods: Inflammation-related molecules in
the nasal epithelial cells and non-eosinophilic CRS mice were measured
by enzyme-linked immunosorbent assay, western blotting, quantitative
real-time polymerase chain reaction, immunoprecipitation, and
histopathological analysis (hematoxylin and eosin staining,
immunohistochemistry, and periodic acid-schiff staining). Results: PVP-I
blocked expressions of various inflammation-related molecules, such as
NLRP3, NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65, caspase-1, and IL-1\selectlanguage{greek}β; \selectlanguage{english}induced translocation of NF-\selectlanguage{greek}κ\selectlanguage{english}B
to the nucleus; and promoted assembly of NLRP3/ASC complexes in the
nasal epithelial cells and non-eosinophilic CRS mice. Notably, PVP-I
strongly blocked the receptor interaction of TLR4 and MyD88 in the
epithelial cells of nasal mucosa. Conclusion: We demonstrated that PVP-I
significantly attenuated inflammatory molecules and cytokines via
blocking the formation of TLR4 and MyD88 complexes during LPS-induced
mucosal inflammation in non-eosinophilic CRS.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Chronic rhinosinusitis (CRS) is a persistent inflammatory disease of the
nasal and paranasal sinuses mucosa.\textsuperscript{1} CRS is a
heterogeneous disorder with distinct pathophysiologic mechanisms, and
evidence from several studies has revealed different
endotypes.\textsuperscript{2--4} The selective expression of type 1, 2,
or 3 immune responses is associated with increasing CRS
heterogeneity.\textsuperscript{5-8} An endotype of CRS with prominent
type 2 immune responses is associated with eosinophilic infiltration
into sinonasal mucosa and can benefit from treatment with systemic
steroids and biologics, whereas non-eosinophilic CRS shows resistance to
these therapy.\textsuperscript{9--12}

Nucleotide-binding oligomerization domain-like receptor family pyrin
domain-containing 3 (NLRP3) could combine with the Apoptosis-associated
speck-like protein containing a caspase recruitment domain (ASC) and the
pro-caspase-1 to compose the NLRP3 inflammasomes \textsuperscript{13}.
NLRP3 inflammasomes play essential roles in the innate and adaptive
immune responses serving as a pattern-recognition receptor and a trigger
for caspase-1 activation and the maturation of IL-1\selectlanguage{greek}β \selectlanguage{english}and
IL-18.\textsuperscript{14,15}

The NLRP3 and its downstream IL-1\selectlanguage{greek}β \selectlanguage{english}were identified as potent inducers of
neutrophilic inflammation in various inflammatory
diseases.\textsuperscript{16,17} Previous studies showed that NLRP3 was
highly expressed in nasal polyps (NPs) from subjects with CRSwNP, and
significantly correlated with neutrophilic nasal
polyps.\textsuperscript{18,19} Wei et al. reported that macrophages and
epithelial cells were the primary sources of NLRP3 in the polyp
tissues.\textsuperscript{19}

Povidone-iodine (PVP-I) is an antiseptic and a disinfectant with
broad-spectrum antimicrobial activity. It has been used as a topical
treatment and surgical scrubs for several
decades.\textsuperscript{20--22} PVP-I consists of a complex of
povidone, hydrogen iodide, and elemental iodine.\textsuperscript{23}

PVP-I is effective in reducing edema or tissue inflammation and
promoting wound healing, as well as eradication of most
pathogens.\textsuperscript{24,25} Here, PVP-I was more effective in
non-eosinophilic CRS than its eosinophilic counterpart in terms of
decreasing sinus discharge and improving olfaction and up to a 17\%
reduction in serum inflammatory markers was measured post-PVP-I
rinsing.\textsuperscript{26}

Although non-eosinophilic CRS has been associated with various bacterial
infections, refractory CRS has been revealed to be closely associated
with Gram-negative bacterial infection, and the endotoxins and LPS
released by Gram-negative bacteria are considered as important
pathogenic mechanisms.\textsuperscript{27,28} While endotoxin may be a
noninfectious inflammatory factor, it can regulate the release of
inflammatory mediators, resulting in CRS.\textsuperscript{27,29}

Here, we explore the anti-inflammatory effects and the underlying
molecular mechanism of PVP-I on LPS-stimulated airway epithelial cells
and investigate whether nasal instillation of PVP-I can suppress mucosal
inflammation in non-eosinophilic CRS mice.

\textbf{METHODS}

\textbf{Chemicals}

Adenosine triphosphate (ATP, A2383), lipopolysaccharide (LPS, L2630),
and Poly(vinylpyrrolidone)--iodine complex (PVP-I, PVP1) were purchased
from Sigma (St. Louis, MO, USA).

\textbf{Cell cultures, human nasal airway epithelial primary cells}

A549 cells (human lung airway epithelial) and RPMI 2650 cells (airway
epithelial, human nasal septum) were cultured at 37\selectlanguage{ngerman}°C, and 5\%
CO\textsubscript{2} in RPMI medium (Gibco, Gaithersburg, MD USA)
supplemented with 10\% FBS (Gibco), 100 U/mL penicillin, and 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL
streptomycin (Gibco). For preparation, primary human nasal epithelial
cells (pHNECs, n=19) were derived from patients undergoing elective
endoscopic sinus surgery and were provided by the Department of
Otorhinolaryngology-Head and Neck Surgery, Chungnam National University
Hospital. The pHNECs were incubated overnight in 1\% Dispase II (Roche,
Belmont, CA, USA) at 4\selectlanguage{ngerman}°C, digested with 0.25\% Trypsin-EDTA (Gibco) for
15 min at 37°C, and neutralized with 10\% FBS. The digested tissue was
passed through a 70 \selectlanguage{greek}µ\selectlanguage{english}M cell strainer (SPL, Pocheon, Korea) to remove the
undigested tissue for collecting pHNECs and washed twice with RPMI
medium. Then, the tube was centrifuged at 1,300 rpm for 5 min. The
obtained pHNECs were cultured in airway epithelial cell growth media
(PromoCell, Heidelberg, Germany) supplemented with a mixture of
amphotericin B (25 \selectlanguage{greek}μ\selectlanguage{english}g/mL), penicillin G (10,000 U/mL), and streptomycin
(10,000 \selectlanguage{greek}μ\selectlanguage{english}g/mL). The cells were placed in a humidified chamber and
incubated at 37\selectlanguage{ngerman}°C, 5\% CO\textsubscript{2}, and 95\% air for two weeks.
The second cell passage provided all of the cells used in this study.
The cells were stimulated with LPS (1000 ng/mL) and ATP (5 mM) for 24 h,
and PVP-I (0.1\%) was additionally added to the cell culture for 1 h
after LPS or ATP stimulation or both.

\textbf{Animal study}

Animal experiments were conducted using seven-weeks-old male C57BL/6
mice with 18--20 g body weight (Orient Bio Inc., Seungnam, Republic of
Korea). This study protocol was approved by the Chungnam National
University's Institutional Animal Care and Use Committees (CNUH-20018).
The mice were maintained under specific-pathogen-free conditions,
consisting of a 12-h light/dark cycle, a temperature of 18°C--23°C, and
humidity of 50\%--60\%. Food and water were available \emph{ad libitum}
. After one week of acclimation in an animal facility, mice were divided
into four groups: control, LPS group, LPS + intraperitoneal (IP) of
dexamethasone (DEX) at 1 mg/kg, and LPS + intranasal (IN) of PVP-I at
0.1\%. LPS was dissolved in Dulbecco's phosphate-buffered saline (DPBS)
and injected intranasally (10 \selectlanguage{greek}µ\selectlanguage{english}g/kg) in 20-\selectlanguage{greek}μ\selectlanguage{english}L DPBS with a 10-\selectlanguage{greek}μ\selectlanguage{english}L pipette
three times a week for three consecutive months. Then, PVP-I was
dissolved in DPBS and administered intranasally once per day for seven
days, and DEX administered intraperitoneally three times per day for
seven days.

The animals were sacrificed seven days after sensitization with DEX and
PVP-I treatment (See Figure 5A for a timeline). After partial tracheal
resection under deep anesthesia, a micropipette was inserted into the
posterior choana through the tracheal opening in the upper airway
direction. Each nasal lavage fluid (NLF) was gently perfused with 200 \selectlanguage{greek}μ\selectlanguage{english}L
DPBS, and fluid from both nostrils was collected and centrifuged for
IL-1\selectlanguage{greek}β \selectlanguage{english}enzyme-linked immunosorbent assay (ELISA). The head of each mouse
was removed and fixed in 4\% paraformaldehyde. Serial 4 \selectlanguage{greek}μ\selectlanguage{english}M thick
sections were cut on a paraffin microtome designed for histological
analysis using H\&E, IHC, and PAS. The nasal mucosa tissue was
meticulously excised using a small curet and micro-forceps. The
harvested nasal mucosa was immediately soaked in T-PER Tissue lysis
buffer for immunoblot analysis.

\textbf{Cytotoxicity measures}

A549 and RPMI2650 cells were plated as stated above in clear 96-well
plates. After incubation time, the ATP, LPS, and PVP-I were treated in
varying concentrations and incubated for another 24 h. After completion,
the cell suspension was discarded, and 1 mg/mL MTT solution was added
and incubated for 4 h, and then DMSO was added. The absorbance was then
taken at 570 nM. The absorbance of each well was measured using a
spectrophotometer (Versamax microplate reader, Molecular Device,
Sunnyvale, CA, USA) at a wavelength of 570 nM, and expressed as a
percentage of the control.

\textbf{Immunoblot}

The protein samples from airway epithelial cells (A549 and RPMI2650
cells) were prepared in radio immunoprecipitation assay (RIPA) lysis
buffer (5 mM sodium fluoride, 10 \selectlanguage{greek}μ\selectlanguage{english}g/mL phenylmethylsulfonylfluoride, and
1 mM sodium vanadate) containing a protease inhibitor cocktail (Roche,
Mannheim, Germany). Mouse mucosa tissue protein samples were prepared in
T-PER Tissue lysis buffer (78510, Thermo Fisher Scientific, T-PER Tissue
lysis Protein Extraction Reagent). All samples were separated on 12\%
SDS-polyacrylamide gels (SDS-PAGE) and then transferred to PVDF
membranes, blocked with 5\% w/v nonfat dry milk in PBS containing 0.5\%
Tween-20. The primary antibodies used were actin (CS-8457P, Cell
Signaling, Danvers, MA, USA), caspase-1 (MA5-32909, Thermo Fisher
Scientific, Waltham, MA, USA), IL-1\selectlanguage{greek}β \selectlanguage{english}(GTX74034, genetex, USA), I\selectlanguage{greek}κ\selectlanguage{english}B-\selectlanguage{greek}α
\selectlanguage{english}(4814P, Cell Signaling), lamin B1 (AB65986, abcam, United Kingdom),
NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65 (8242S, Cell Signaling), NLRP3 (AG-20B-0014, Adipogen, SA,
Switzerland), and tubulin (T8023, Sigma).

\textbf{Histopathology}

Cells were fixed by incubation with 4\% paraformaldehyde at room
temperature for 30 min. Fixed cells were rinsed in PBS, treated with
0.5\% bovine serum albumin for 30 min, and then incubated overnight at
4\selectlanguage{ngerman}°C with rabbit anti-NLRP3, ASC, NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65, and - IL-1\selectlanguage{greek}β \selectlanguage{english}antibodies (each
diluted 1:500). They were then incubated for 2 h with Alexa 488 and 594
fluor-conjugated secondary antibody (dilution 1:2000). The cells were
finally washed in PBS and mounted using Vectashield mounting-medium
containing 4'6-diamidino-2-phenylindole. Immunofluorescent images were
captured using a microscope (Olympus Microscope System BX51; Olympus,
Tokyo, Japan).

For H\&E analysis, each mouse's head was removed and fixed in 4\%
paraformaldehyde. Serial 4 \selectlanguage{greek}μ\selectlanguage{english}M thick sections were cut on a paraffin
microtome designed for histological analysis using H\&E.

For immunohistochemistry, paraffin sections were dewaxed, and incubated
with 3\% hydrogen peroxide at room temperature for 10 min to inactivate
endogenous peroxidase. The sections were placed into an antigen
retrieval solution (0.01 M citrate), repaired using a pressure cooker at
100\selectlanguage{ngerman}°C for 5 min and cooled to room temperature before washing five times
with PBS for 5 min each. Sections were subsequently blocked with 1\%
bovine serum albumin at room temperature for 60 min and incubated
overnight at 4°C with anti\selectlanguage{english}-mouse TLR4 monoclonal antibody at a dilution
of 1:100. After washing, the sections were incubated with horse-radish
peroxidase (HRP) at 37degC in a humidified chamber for 60 min and
subsequently washed five times with PBS for 5 min each. The sections
were developed with 3'-Diaminobenzidine (DAB) and washed immediately
with running water when brown particles appeared in the cytoplasm to
terminate the reaction. The sections were secondarily stained with
hematoxylin for 1 min.

For periodic acid\selectlanguage{english}-schiff staining (PAS), at room temperature,
paraffin-embedded tissue sections were serially dehydrated in xylene and
ethanol. Then, sections were stained with PAS. Slides were incubated
with 0.5\% periodic acid solution for 5 min, then stained with Schiff's
reagent for 15 min, followed by counterstaining with hematoxylin
solution for 1 min.

\textbf{Immunoprecipitation}

Protein samples from airway epithelial cell extracts and mouse mucosa
tissue cell extracts (500\selectlanguage{english} \selectlanguage{greek}μ\selectlanguage{english}g protein) were immunoprecipitated by
incubation with anti-TLR4 or MyD88 antibody (25 \selectlanguage{greek}μ\selectlanguage{english}g) overnight at 4\selectlanguage{ngerman}°C,
followed by incubation with protein G-agarose bead (40\selectlanguage{english} \selectlanguage{greek}μ\selectlanguage{english}L of a 1:1
slurry) for 1 h at room temperature. Immune complexes were washed five
times with a RIPA buffer, boiled for 5 min in a Laemmli sample buffer,
and analyzed by SDS-PAGE and immunoblotting with the indicated
antibodies. The immunoprecipitation antibodies used were TLR4
(SC-293072, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and MyD88
(SC-74532, Santa Cruz Biotechnology).

\textbf{Proteome profiler antibody array analysis}

The protein from airway epithelial cells and pHNECs was extracted using
a RIPA lysis buffer. Proteome Profiler Human XL Cytokine Array Kit
(\#ARY022B) is membrane-based sandwich immunoassay. Following
manufacturer recommendations, each membrane was incubated with 100 \selectlanguage{greek}μ\selectlanguage{english}L of
pooled plasma overnight at 4\selectlanguage{ngerman}°C. Captured plasma proteins were detected
with biotinylated detection antibodies and afterward, visualized with
Streptavidin-HRP and chemiluminescent detection reagents. A signal was
produced at each capture spot, proportionally with the amount of the
protein-bound. The signal produced is proportional to the amount of
analyte bound. The bands' intensities were analyzed using Quick Spots
Image Analysis Software (Western Vision
Software,\emph{http://www.wvision.com/QuickSpots.html}).

\textbf{Real-time quantitative polymerase chain reactions}

According to the manufacturer's instructions, total RNAs from the cells
and mice nasal mucosa were extracted using TRIzol reagent (Thermo Fisher
Scientific). AccuPower RT PreMix (Bioneer, Daejeon, Korea) was used for
complementary DNA (cDNA) synthesis according to the manufacturer's
instructions. The obtained cDNA was amplified with specific primers
(Table 1). Polymerase chain reaction (PCR) was performed for cDNA
synthesis using a T100 Thermal Cycler (Bio-Rad Laboratories, USA). The
mRNA expression was analyzed using a CFX Connect Real-Time PCR Detection
System (Bio-Rad Laboratories, USA) with PowerUp SYBR Green Master Mix
(Applied Biosystems, USA). All PCR assays were performed in triplicate.
The relative gene expression was analyzed using the
2\selectlanguage{english}\textsuperscript{-[?][?]Ct} method. The details of the primers are shown in
Table 1.

\selectlanguage{greek}\textbf{IL-1β Enzyme-Linked Immunosorbent Assay}\selectlanguage{english}

The supernatants from cultured A549, RPMI2650, and pHNECs were
collected. Protein levels of IL-1\selectlanguage{greek}β \selectlanguage{english}in the supernatants were detected
using ELISA kits (SLB50, R \& R\&D Systems, Minneapolis, MN, USA). Assay
diluent was added to each well with the standard and samples, and the
wells were covered using an adhesive strip and incubated at room
temperature. Each well was aspirated and washed as the complete removal
of the liquid at each step is essential for best performance. Conjugate
was added to each well, and the above steps were repeated. Plates were
washed with a wash buffer, and substrate solution was added to each well
and incubated for 30 min at room temperature. Notably, the procedure
requires the samples to be protected from light. Once 1 mol/L sulfuric
acid was added, the substrate reaction is halted, and the extinction was
measured at a wavelength of 450 nM using a multi-plate ELISA reader.
According to the manufacturer's instructions, in mice NLF, IL-1\selectlanguage{greek}β \selectlanguage{english}levels
were detected using the ELISA kits (SMLB00C, R\&D Systems).

\textbf{Isolation of cytosolic and nuclear fractions}

Nuclear protein extracts from A549 and RPMI2650 cells were prepared
using the NE-PER kit (Thermo Fisher Scientific, USA) according to the
manufacturer's instructions. After that, nuclear proteins (the
supernatant) were used immediately or stored at -80\selectlanguage{ngerman}°C.

\textbf{Statistical analysis}

Data are expressed as mean ± standard error (SEM). The data were
analyzed via a one-way analysis of variance (ANOVA) and post-hoc
multiple variation comparisons (Tukey's HSD test). All data were
analyzed using GraphPad Prism v.5.10 software (GraphPad Software Inc.,
San Diego, CA, USA). Statistical comparisons between the different
treatments were performed using one-way ANOVA with the Tukey multiple
comparison post-test. \emph{P} -values of \textless{}.05 were considered
as statistically significant.

\textbf{RESULTS}

\selectlanguage{greek}\textbf{PVP-I suppress NLRP3- and NF-κB--dependent inflammatory pathways
in airway epithelial cells}\selectlanguage{english}

LPS, ATP, and PVP-I concentrations were selected up to 1000 ng/mL, 5 mM,
and 0.1\% for the following experiments, respectively (Fig. S1A).
Protein expression of NLRP3 were elevated in the LPS and ATP (LPS/ATP)
treated cells compared with cells treated with LPS or ATP alone (Fig.
S1B). Expressions of NLRP3 mRNA were also significantly elevated in
LPS/ATP treated cells compared with cells treated with each alone (Fig.
S1C).

Protein and mRNA expression of NLRP3 induced by LPS/ATP was
significantly decreased by PVP-I treatment in airway epithelial cells
(Fig. 1A and B). NLRP3 inflammasome-associated molecules were also
evaluated in these airway epithelial cells, and expression of NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65,
caspase-1, and IL-1\selectlanguage{greek}β \selectlanguage{english}were strongly inhibited by PVP-I treatment in both
protein and mRNA levels (Fig. 1C and D). Treatment of LPS/ATP allowed
NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65 translocation to the nucleus and I\selectlanguage{greek}κ\selectlanguage{english}B-\selectlanguage{greek}α \selectlanguage{english}degradation in the
cytosol, and these phenomena were reversed by treatment of PVP-I (Fig.
1E). Immunofluorescence analysis also showed that LPS/ATP-induced
NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65 translocation to the nucleus was inhibited by PVP-I treatment
in airway epithelial cells (Fig.1F). NLRP3/ASC assemblies in the
cytoplasm were also induced by LPS/ATP treatment, and were inhibited by
PVP-I treatment in airway epithelial cells (Fig. 1G).

LPS/ATP-induced expressions of NLRP3, NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65, caspase-1, and IL-1\selectlanguage{greek}β,
\selectlanguage{english}and NLRP3/ASC assemblies were suppressed by PVP-I treatment in pHNECs
(Fig. 2B--D). These results indicated that PVP-I treatment could inhibit
the activation of NLRP3 inflammasome and associated molecules in airway
epithelial cells.

\textbf{PVP-I inhibits LPS/ATP-induced cytokine production in airway
epithelial cells}

LPS/ATP-induced production of IL-1\selectlanguage{greek}β \selectlanguage{english}was markedly reduced by PVP-I
treatment (Fig. 3A). PVP-I treatment reduced the NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling related
cytokines (APoA1, Ang-1, BAFF, CD40L, EGF, Fas L, GM-CSF, GRO\selectlanguage{greek}α, \selectlanguage{english}CD54L,
IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-1\selectlanguage{greek}α, \selectlanguage{english}IL-1ra, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-23,
IP-10, MCP1, M-CSF, MMP9, TFF3, TNF-\selectlanguage{greek}α, \selectlanguage{english}VESF, and VCAM-1) induced by
LPS/ATP stimulation in pHNECs (Fig. 3B and S3). Also, LPS/ATP-induced
IL-1\selectlanguage{greek}β \selectlanguage{english}cytokine production (red box in Fig. 3B) was significantly
decreased by PVP-I treatment in pHNECs (Fig. 3B and C). These results
demonstrated that PVP-I strongly inhibits the production of
pro-inflammatory or inflammatory cytokines, which inflammatory mediator
induces.

\textbf{PVP-I suppresses LPS/ATP-induced inflammatory response by
blocking TLR4 signaling}

The binding of TLR4 and MyD88 are crucial for signaling inflammatory
response.\textsuperscript{41} The formation of TLR4 and MyD88 was
increased after LPS/ATP stimulation, and treatment of PVP-I inhibited
the intensity of the TLR4 band immunoprecipitated with an anti-MyD88
protein (Fig. 4A and B). The reverse immunoprecipitation of MyD88 with
TLR4 antibody also showed blocking of complex formation after PVP-I
treatment (Fig. 4A and B). This result indicated that PVP-I could
inhibit TLR4/MyD88 complex formation, which is induced by LPS/ATP
stimulation.

\textbf{PVP-I inhibits mucosal inflammation in mice with CRS}

Mucosal thickness (H\&E stain, Fig. 5B) and the number of goblet cells
(PAS stain, Fig. 5C) were significantly decreased in the groups of
dexamethasone (LPS + DEX) or PVP-I treatment (LPS + PVP-I) compared with
the PBS treatment group (LPS + PBS). TLR4 expression among groups was
significantly higher in the LPS + PBS group compared with other groups
(Fig. 5D). TLR4/MyD88 complex formation stimulated with LPS was
inhibited by PVP-I or dexamethasone treatment in the nasal mucosa (Fig.
5E).

NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65, caspase-1, and IL-1\selectlanguage{greek}β \selectlanguage{english}were significantly inhibited by PVP-I
treatment (LPS + PVP-I) or dexamethasone (LPS + DEX) (Fig. 6A and B). In
addition, IL-1\selectlanguage{greek}β \selectlanguage{english}production in NLF was significantly decreased in the
mice treated with dexamethasone or PVP-I (Fig. 6C). The mRNA expressions
of TNF-\selectlanguage{greek}α, \selectlanguage{english}IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-4, and IL-17 were significantly higher in
LPS-induced CRS mice (LPS + PBS) compared with control mice, and these
cytokines were significantly decreased by PVP-I (LPS + PVP-I) and
dexamethasone (LPS + DEX) treatment (Fig. 6D). These results indicated
that topical instillation of PVP-I in mice alleviates mucosal
inflammation of LPS-induced CRS mice.

\textbf{DISCUSSION}

Inflammatory responses exert their effect on cells mainly through the
NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling.\textsuperscript{18} We found that PVP-I inhibited
several molecules' expression in this signaling, such as NLRP3,
NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65, caspase-1, and IL-1\selectlanguage{greek}β \selectlanguage{english}in LPS/ATP-induced airway epithelial
cells and pHNECs. (Figs. 1 and 2). Previous studies have reported
anti-inflammatory mimic effects of PVP-I.\textsuperscript{23-26}.
However, the underlying molecular mechanisms of PVP-I on
anti-inflammatory effects were not revealed. To our knowledge, this is
the first study that evaluates the underlying mechanisms of PVP-I on
inflammation in airway epithelial cells.

We tried verifying the anti-inflammatory effect of PVP-I using the NLRP3
inflammasome signal model \emph{in vitro} . Recent studies have shown
that NLRP3 inflammasome plays a pivotal role in various diseases, such
as CRS, asthma, obstructive pulmonary disease, inflammatory bowel
disease, metabolic disorders, multiple sclerosis, and other auto-immune
and auto-inflammatory diseases. \textsuperscript{34--38} Our results
demonstrated that PVP-I effectively suppressed NLRP3
inflammasome-associated molecules and cytokines, activated by LPS/ATP
stimulation.

To inhibit the inflammatory signal inside airway epithelial cells, free
iodine may be able to enter the cytosol through the cell membrane. This
allowed the destruction of cellular components, including enzymes
essential to survive. Because 0.1\% of PVP-I did not show cellular
toxicity, other mechanisms may be involved in suppressing cellular
signaling molecules and inflammatory cytokines in this study. Our
findings suggest that PVP-I could inhibit the inflammatory response via
TLR4 and MyD88 complex formation. Free iodine released by PVP-I is a
strong oxidizing agent, and there is a possibility that free iodine
destroys some parts of TLR4 and disturbs joining with MyD88; thus,
blocking signal transmission. Indeed, TLR4 induced by LPS was suppressed
by PVP-I treatment in airway epithelial cells (Fig. 4A and B).

A study indicated that NLRP3 inflammasome is more associated with
non-eosinophilic CRS Because the PVP-I target was thought to be
TLR4\emph{in vitro} , expressions of TLR4 and TLR4-related signal
molecules were evaluated in mice, and these were decreased in PVP-I
treated mice. Interestingly, IL-4 expression in mice nasal mucosa was
also suppressed after PVP-I treatment. Future studies investigating
PVP-I-induced suppression of PRR and other surface receptors are
required.

In NPs of patients with CRS, several types of cytokine expression are
involved with inflammation.\textsuperscript{7}Our results showed that
PVP-I inhibited the inflammation-induced IL-1\selectlanguage{greek}β \selectlanguage{english}secretion, and 27
cytokines in airway epithelial cell lines and primary cultured cells
from nasal polyp patients (Fig. 3 and S3). In the pathogenesis of CRSwNP
in Asian patients, NF-\selectlanguage{greek}κ\selectlanguage{english}B-associated inflammatory cytokines are a key
factor and thought of as a therapeutic target.\textsuperscript{45} This
finding suggests that the PVP-I strongly inhibited inflammation
signaling through NF-\selectlanguage{greek}κ\selectlanguage{english}B related molecule, such as IL-1\selectlanguage{greek}β, \selectlanguage{english}and PVP-I may
be used as a possible therapeutic remedy in CRS, especially
non-eosinophilic CRS.

The therapeutic effects of PVP-I were similar to those of dexamethasone
in our study. Anti-inflammatory effects of dexamethasone have been well
described.\textsuperscript{50} Dexamethasone showed its effect by
inhibiting NF-\selectlanguage{greek}κ\selectlanguage{english}B and other inflammatory transcription factors and
promoting anti-inflammatory genes.\textsuperscript{51} Although the
effects of dexamethasone were similar to those of PVP-I in this study,
these drugs' target and route were quite different. Generally, the side
effect of PVP-I is less serious than those of dexamethasone even when
used long term.

Here, PVP-I was the first used to study an anti-inflammatory response by
inhibiting the goblet cells in the NPs mouse models' mucosa tissue. In
this study, PVP-I ameliorated goblet cell hyperplasia in neutrophilic
CRS mice. Although we did not conduct an animal study in the
eosinophilic mice model, PVP-I may effectively suppress mucosal
inflammation, including goblet cell hyperplasia because PVP-I blocked
the inflammation-related cytokines, such as TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}and IL-4 in
mucosa tissue of NPs mice models (Fig. 6A and B). Future studies are
required to verify these effects in the eosinophilic CRS model. A study
also demonstrated that diluted topical PVP-I rinses showed notable
symptom improvement, especially discharge score in recalcitrant CRS,
without affecting thyroid function, mucociliary clearance, or
olfaction.\textsuperscript{57}

In summary, we demonstrated that PVP-I significantly attenuated
inflammatory molecules and cytokines via blocking the formation of TLR4
and MyD88 complexes in the LPS-induced mucosal inflammation in
non-eosinophilic CRS. We believe PVP-I could modulate critical
inflammatory signals, and the need to study PVP-I-related signal
inhibition on the cell surface receptors or molecules, such as
pattern-recognition receptors, in the future. We also believe that ACE2
(angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane protease
serine subtype 2) could be a target for PVP-I rinses of the upper
airway.

\textbf{FIGURE LEGEND}

\textbf{Figure 1. LPS plus ATP activates NLRP3 inflammasome response in
airway epithelial cells.}

Treatment of airway epithelial cells with LPS and ATP with or without
the PVP-I inhibited the induction of NLRP3 inflammasome protein, as
shown \textbf{(A)} immunoblot and \textbf{(B)} qRT-PCR. Cells were
stimulated with LPS and ATP for 24 h, and PVP-I (0.1\%) was additionally
added to the cell culture for 1 h (to produce an inflammation
environment) after LPS and/or ATP stimulation. Statistical significance:
*P \textless{}.05 and **P \textless{}.01 compared with the control
group; \textsuperscript{\#}P \textless{}.05 and \textsuperscript{\#\#}P
\textless{}.01 compared with the LPS/ATP group. \textbf{(C and
D)}Protein and mRNA expression of NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65, caspase-1 and IL-1\selectlanguage{greek}β \selectlanguage{english}in
airway epithelial cells treated with LPS, ATP with or without PVP-I, as
shown by immune blot and qRT-PCR. Statistical
significance:\textsuperscript{*}P \textless{}.05 and
\textsuperscript{**}P \textless{}.01 compared with the control group;
\textsuperscript{\#}P \textless{}.05, \textsuperscript{\#\#}P
\textless{}.01 and\textsuperscript{\#\#\#}P \textless{}.01 compared with
the LPS/ATP group. \textbf{(E)} Cytosolic and nuclear extracts were
analyzed by immune blot with indicated antibodies for NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65 and
I\selectlanguage{greek}κ\selectlanguage{english}B-\selectlanguage{greek}α. \selectlanguage{english}Anti-Lamin B and anti-\selectlanguage{greek}α-\selectlanguage{english}tubulin antibodies were used as loading
controls for the nucleus and cytosol, respectively. \textbf{(F)}
Representative Immunofluorescence of NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65 translocation to nucleus
(green) and DAPI (blue) localization in airway epithelial cells. The
scale bar indicates 100 \selectlanguage{greek}μ\selectlanguage{english}m. \textbf{(G)} Immunofluorescence for NLRP3
(red) and ASC (green) co-localization in airway epithelial cells. The
scale bar indicates 100 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 2. Alteration of inflammation molecules in primary
cultured cells from nasal polyp patients.}

\textbf{(A} ) Optical microscope of cell viability in the human nasal
epithelial primary cells (pHNECs) from nasal polyp patients. Primary
cultured cells from nasal polyp patients were treated with LPS (1000
ng/mL), ATP (5 mM) and PVP-I (0.1\%) for 24 h. Representative
photomicrographs of cell morphology was measured using an optical
microscope. \textbf{(B and C)} mRNA and protein expression of NLRP3,
NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65, caspase-1 and IL-1\selectlanguage{greek}β \selectlanguage{english}in pHNECs treated with LPS, ATP with or
without PVP-I, as shown by qRT-PCR and immunoblot. Statistical
significance: \textsuperscript{*}P \textless{}.05, \textsuperscript{**}P
\textless{}.01 and \textsuperscript{***}P \textless{}.001 compared with
the control group; \textsuperscript{\#}P \textless{}.05 compared with
the LPS/ATP group. \textbf{(D} ) Immunofluorescence for NLRP3 (red) and
ASC (green) co-localization in pHNECs. The scale bar indicates 100 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 3. Profiling proteins by inflammation response in airway
epithelial cells.}

Inhibition of LPS and ATP-induced IL-1\selectlanguage{greek}β \selectlanguage{english}secretion by PVP-I. Airway
epithelial cells were treated with PVP-I (0.1\%) for 1 h after LPS (1000
ng/mL) and ATP (5 mM) treatment. \textbf{(A)} After 24 h-incubation, the
airway epithelial cell culture medium was assayed using an ELISA for
IL-1\selectlanguage{greek}β. \selectlanguage{english}Statistical significance: \textsuperscript{*}P \textless{}.05
and\textsuperscript{**}P \textless{}.01 compared with the control
group;\textsuperscript{\#\#}P \textless{}.01 compared with the LPS/ATP
group.\textbf{(B)} After 24 h-induction, the pHNECs were subjected to
cytokine antibody array assay. Each proteins represented by duplicate
spots in the respective membrane. IL-1\selectlanguage{greek}β \selectlanguage{english}protein is boxed in red.
\textbf{(C)}Data were analyzed using the Quick Spots Image Analysis
Software. Statistical significance: \textsuperscript{***}P
\textless{}.001 compared with the control group; \textsuperscript{\#\#}P
\textless{}.01 compared with the LPS/ATP group.

\textbf{Figure 4. PVP-I inhibits TLR4 and MyD88 interaction by
inflammation response}

Airway epithelial cells were pre-treated with LPS (1000 ng/mL) and ATP
(5 mM) for 60 min and then exposed to 0.1\% PVP-I for 24 h. Cells lysate
were immunoprecipitation with anti- MyD88 and TLR4 antibody, and then
immunoprecipitated proteins were detected using anti-TLR4 and anti-MyD88
antibodies. The pHNECs had the same experimental methods as the airway
epithelial cells. PVP-I was found to inhibit the interaction with TLR4
and MyD88 complex formation. Actin was used as a control for input.

\textbf{Figure 5. PVP-I inhibits inflammation in LPS-stimulated mice.}

\textbf{(A)} Nasal polyps mouse models protocol. Mice were assigned to 4
groups and administered saline or PVP-I at doses of 0.1\% intra-nasally
for on 7 days after LPS administration (intra-nasal) for 12 weeks.

Nasal polyps mouse models protocol. Mice were assigned to 4 groups and
administered PVP-I at doses of 0.1\% intra-nasally or DEX at doses of 1
mg/kg intraperitoneally for on 7 days after 10 \selectlanguage{greek}µ\selectlanguage{english}g LPS intra-nasally for
12 weeks. \textbf{(B)} Representative hematoxylin and eosin staining to
demonstrate nasal mucosa tissue. Statistical
significance:\textsuperscript{*}P \textless{}.05 compared with the
control group;\textsuperscript{\#}P \textless{}.05 compared with the
LPS+PBS group;\selectlanguage{ngerman}\textsuperscript{§}P \textless{}.05 compared with the
LPS+DEX group.\textbf{(C)} Goblet cells increased in the epithelium with
PAS staining upon LPS administration. In the epithelium, number of
goblet cells of the LPS administration group was more than in the CON
group, and was further inhibited in the PVP-I treatment group.
Statistical significance: \textsuperscript{**}P \textless{}.01 compared
with the control group; \textsuperscript{\#}P \textless{}.05 compared
with the LPS+PBS group; \textsuperscript{§}P \textless{}.05 compared
with the LPS+DEX group. \textbf{(D)} Immunohistochemistry showing the
TLR4 and MyD88 in the nasal mucosa of mice in each group. Statistical
significance: *P \textless{}.05 compared with the control
group;\textsuperscript{§}P \textless{}.05 compared with the LPS+DEX
group.\textbf{(E)} Cells lysate in murine nasal mucosa tissue were
immunoprecipitation with anti- MyD88 and TLR4 antibody, and then
immunoprecipitated proteins were detected using anti-TLR4 and anti-MyD88
antibodies. PVP-I was found to inhibit the TLR4 and MyD88 complex
formation. Actin was used as a control for input.

\textbf{Figure 6. PVP-I inhibits TLR4 and MyD88 complex formation in
nasal polyps mouse model.}

\textbf{(A)} Bar graphs represent the relative expression of cytokines.
Statistical significance: \textsuperscript{*}P
\textless{}.05,\textsuperscript{**}P \textless{}.01 and
\textsuperscript{***}P \textless{}.001 compared with the control group;
\textsuperscript{\#}P \textless{}.05, \textsuperscript{\#\#}P
\textless{}.01 and\textsuperscript{\#\#\#}P \textless{}.001 compared
with the LPS+PBS group; \textsuperscript{§}P \textless{}.05,
\textsuperscript{§§}P \textless{}.01 and \textsuperscript{§§§}P
\textless{}.001 compared with the LPS+DEX group. \textbf{(B)} The
expression of NLRP3, NF-\selectlanguage{greek}κ\selectlanguage{english}B-p65, caspase-1 and IL-1\selectlanguage{greek}β \selectlanguage{english}were measured using
an immunoblot in murine nasal mucosa tissues. Actin protein was used as
a loading control.\textbf{(C)} Production of IL-1\selectlanguage{greek}β \selectlanguage{english}in the nasal lavage
fluid (NLF) were measured using the ELISA assay. Statistical
significance:\textsuperscript{***}P \textless{}.001 compared with the
control group;\textsuperscript{\#}P \textless{}.05 compared with the
LPS+PBS group;\selectlanguage{ngerman}\textsuperscript{§}P \textless{}.05 compared with the
LPS+DEX group.

\textbf{Table 1.} Details of the oligonucleotide sequences for qRT-PCR\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Supplementary Figure Legends}

\textbf{Supplementary E1. Cell viability of LPS, ATP and PVP-I in airway
epithelial cells.}

Effect of LPS, ATP and PVP-I on airway epithelial cells (A549, RPMI2650
cells). \textbf{(A)} After serum starvation, cells were treated with
various concentration of LPS, ATP and PVP-I for 24 h. The viability of
the cells were measured by the MTT assay. \textbf{(B and C)} LPS/ATP
induced changes of NLRP3 protein and mRNA levels in airway epithelial
cells. Cells were treated with LPS 1000 ng/mL and ATP 5 mM for 24 h.
Statistical significance: \textsuperscript{*}P \textless{}.05 compared
with the control group; \textsuperscript{\#\#}P \textless{}.01 compared
with the LPS group;
\selectlanguage{ngerman}\textsuperscript{§§}\textless{}.01,\textsuperscript{§§§}\textless{}.001
compared with the ATP group.

\textbf{Supplementary E2. Localization of NLRP3 in airway epithelial
cells.}

Airway epithelial cells were treated with LPS (1000 ng/mL) and ATP (5
mM) for 60 min. \textbf{(A)} After 24 h-indubation, immunofluorescence
for NLRP3 (red) localization in A549 cells. \textbf{(B)} After 24
h-indubation, immunofluorescence for NLRP3 (red) localization in
RPMI2650 cells. The scale bar indicates 100 \selectlanguage{greek}μ\selectlanguage{english}m.

\selectlanguage{greek}\textbf{Supplementary E3. Inflammatory cytokines with NF-κB in pHNECs.}\selectlanguage{english}

pHNECs were treated with PVP-I (0.1\%) for 1 h after LPS (1000 ng/mL)
and ATP (5 mM) treatment. After 24 h-incubation, the pHNECs culture
medium was assayed using an ELISA for inflammaroty cytokines-related
NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling. Densitometric ratios of the array showed differences in
the inflammation cytokines.

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