Deprived TLR9 Expression in Apparently Healthy Nasal Mucosa Might Trigger Polyp-Growth in Chronic Rhinosinusitis Patients
Cardell L-O (2014) Deprived TLR9 Expression in Apparently Healthy Nasal Mucosa Might Trigger
Polyp-Growth in Chronic Rhinosinusitis Patients. PLoS ONE 9(8): e105618. doi:10.1371/journal.pone.0105618
Deprived TLR9 Expression in Apparently Healthy Nasal Mucosa Might Trigger Polyp-Growth in Chronic Rhinosinusitis Patients
Lotta Tengroth 0
Julia Arebro 0
Susanna Kumlien Geore n 0
Ola Winqvist 0
Lars-Olaf Cardell 0
Lena Alexopoulou, Centre d'Immunologie de Marseille-Luminy, CNRS-Inserm, France
0 1 Division of Ear, Nose and Throat Diseases, Department of Clinical Sciences, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden, 2 Department of Medicine, Unit of Translational Immunology, Karolinska Institute , Stockholm , Sweden
Background: The origin of nasal polyps in chronic rhinosinusitis is unknown, but the role of viral infections in polyp growth is clinically well established. Toll-like receptors (TLRs) have recently emerged as key players in our local airway defense against microbes. Among these, TLR9 has gained special interest in viral diseases. Many studies on chronic rhinosinusitis with nasal polyps (CRSwNP) compare polyp tissue with nasal mucosa from polyp-free individuals. Knowledge about changes in the turbinate tissue bordering the polyp tissue is limited. Objectives: To analyse the role of TLR9 mediated microbial defense in tissue bordering the polyp. Methods: Nasal polyps and turbinate tissue from 11 patients with CRSwNP and turbinate tissue from 11 healthy controls in total were used. Five biopsies from either group were analysed immediately with flow cytometry regarding receptor expression and 6 biopsies were used for in vitro stimulation with a TLR9 agonist, CpG. Cytokine release was analysed using Luminex. Eight patients with CRSwNP in total were intranasally challenged with CpG/placebo 24 hours before surgery and the biopsies were collected and analysed as above. Results: TLR9 expression was detected on turbinate epithelial cells from healthy controls and polyp epithelial cells from patients, whereas TLR9 was absent in turbinate epithelial cells from patients. CpG stimulation increased the percentage cells expressing TLR9 and decreased percentage cells expressing VEGFR2 in turbinate tissue from patients. After CpG stimulation the elevated levels of IL-6, G-CSF and MIP-1b in the turbinate tissue from patients were reduced towards the levels demonstrated in healthy controls. Conclusion: Defects in the TLR9 mediated microbial defense in the mucosa adjacent to the anatomic origin of the polyp might explain virus induced polyp growth. CpG stimulation decreased VEGFR2, suggesting a role for CpG in polyp formation. The focus on turbinate tissue in patients with CRSwNP opens new perspectives in CRSwNP-research.
Funding: Swedish Medical Research Council (Grant number: 2011-3357; http://www.vr.se/). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Chronic rhinosinusitis (CRS) constitutes a public health
problem with a negative impact on quality of life . According
to recent nomenclature, CRS can be subdivided into CRS with or
without nasal polyps (CRSwNP or CRSsNP) . The
pathogenesis of CRS, as well as the origin of polyp development, still
remains substantially unknown, even though numerous
inflammatory signs and markers that have been associated with the
disease . Several studies have shown abnormalities in the
immune responses in patients with CRSwNP [3,4]. Defects in the
host response to external pathogens including virus, bacteria and
fungi have been suggested to underlie the persistence of the
inflammatory state . Clinically, respiratory viral infections are
often implicated as triggers of CRS flare-ups and these infections
are also known to damage the function of nasal epithelial cells and
cilia [6,7]. Viral infections of the airway are also shown to be
followed by bacterial infections. For example, Herpes simplex
virus type 1 expressed in nasal polyps results in damaging effects to
the epithelium, facilitating invasion of Staphylococcus aureus .
Staphylococcus aureus has further been shown to play a role in
CRSwNP by releasing enterotoxins that acts as superantigens,
amplifying a T helper 2 (Th2)-biased immune response .
Pattern-recognition receptors (PRRs) on the nasal mucosa have
the ability to detect and bind conserved microbial components and
initiate an immune response. The most well-known members of
the PRR family are the Toll-like receptors (TLRs) . Mammals
express at least 10 TLRs that recognize specific pathogen
molecules . TLR9, a receptor of the TLR family, induces a T
helper 1 (Th1)-biased immune response that is known to suppress
Th2-related activities, associated with CRSwNP .
Cytosinephosphate-guanine oligodeoxynucleotide (CpG-ODN) is a TLR9
ligand found in bacterial and viral DNA. The ability for CpG to
induce Th1-polarization has made it an interesting novel target for
the treatment of allergy and infectious diseases [12,13].
We have previously demonstrated TLR9 expression in nasal
mucosa from healthy individuals . It appears to be most
prominently expressed in the epithelium, but it can also be found
on leukocytes scattered in the intraepithelial and subepithelial
layer . A decreased expression of TLR9 mRNA has been
found in sinus mucosa from patients with recurrent CRSwNP,
compared to patients with milder disease  and this might be
linked to the increased effects of locally produced Th2 cytokines in
CRSwNP [15,16]. However, many studies in the polyp field have
focused on comparing polyp tissue with turbinate tissue from
healthy, polyp-free, individuals. Knowledge about changes in the
apparently healthy turbinate tissue close to the anatomic origin of
the polyp is scarce. It is limited to only a few reports regarding
inflammatory cell counts and cellular proliferation activities
[17,18]. The aim of the present study is to uncover novel
approaches for CRSwNP research by focusing on the role of
TLR9 in apparently healthy turbinate tissue bordering the polyp
Materials and Methods
The study was approved by the Ethics Committees of
Karolinska Institutet, Stockholm, Sweden (2012/148231/1). All
participants, those biopsied with or without intranasal challenge
with CpG, gave their written informed consent, while all
procedures were conducted according to the principles expressed
in the Declaration of Helsinki.
Subjects and Study design
The patients with CRSwNP were defined by historical and
endoscopic criteria and/or CT changes (accordingly to the
European Position Paper on Rhinosinusitis and Nasal Polyps
guidelines ). In all patients, steroids were withheld during at
least 6 weeks (topically) and 12 weeks (systemically) prior to
functional endoscopic sinus surgery (FESS). Patients on daily
inhaled steroid medication, as well as those with more than four
episodes of FESS, were excluded from the study. None of the
patients had history of smoking, 6 out of 19 had history of asthma
and 9 out of 19 had a positive phadiatop test, with presence of
serum-specific IgE towards one or several allergens in the standard
panel (birch, mugwort, timothy, dog, cat, horse, mites and mold).
Biopsies from patients with CRSwNP were taken from the
polyp tissue and the apparently healthy, turbinate tissue, during
FESS. The area close to middle nasal meatus, from the middle
turbinate or the inferior turbinate, where the mucosa had healthy
appearance and bordered to the polyp tissue or to the tissue
showing polypoidal changes, was defined as the turbinate tissue.
The healthy controls had no history of polyp disease or smoking
history and had negative phadiatop test. Biopsies were obtained as
previously described .
Biopsies from 11 patients with CRSwNP (9 males and 2 females
with a mean age of 51, range 1884), and 11 healthy controls (5
males and 6 female with a mean age of 28, range 2048) were used
in total, 5 biopsies were analysed immediately with flow cytometry
and 6 biopsies used for in vitro stimulation experiments. Further, 8
additional patients with CRSwNP were included in the study with
intranasal challenge of CpG. These patients were randomized to
receive either CpG (n = 4; 2 males and 2 females with a mean age
of 44, range 3256) or placebo (n = 4; 3 males and 1 female with a
mean age of 62, range 5671).
In vitro stimulation with CpG
Biopsies used for in vitro stimulation were collected into sterile
DMEM/F-12 (1X) (Gibco, Paisley, UK) and separated into
equally small pieces of 0.05 g. The tissue pieces were incubated on
24-well culture plates at 37uC in a humidified 5% CO2 air
atmosphere in 1 mL of DMEM/F-12 with 10% fetal bovine
serum (FBS) (Gibco, Paisley, UK), penicillin (100 U/mL; Gibco,
Grand Island, NY, USA), streptomycin (100 U/mL; Gibco,
Grand Island, NY, USA) and Fungizone (0.25 mg/ml; Gibco,
Grand Island, NY, USA). The biopsies were stimulated with or
without modified CpG-ODN for 4 and 24 hours, respectively
using 0.1 mM; 0.3 mM and 1.0 mM of CpG-ODN.
Nasal administrated CpG
Eight patients with CRSwNP were intranasally challenged with
either CpG-ODN (n = 4) or placebo (n = 4). Sterile physiological
saline solution (100 ml) containing 50 mM CpG or sterile saline
solution (100 ml, placebo) was applied by intranasal spray to both
nostrils after exsufflation.
Cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODN 2006) were synthesized by DNA
Technology (Aarhus, Denmark). The following oligonucleotide
sequence was used: 59 tcgtcgttttgtcgttttgtcgtt 39. Biopsies from
polyp tissue and the turbinate tissue were taken during FESS,
24 hours after the CpG/placebo administration.
Flow cytometry analysis
The biopsies was placed through a 100 mm cell strainer (BD
Falcon), into DMEM/F-12 (1X) containing 10% FBS and
incubated at RT for 5 min. The cells were washed and centrifuged
after which the supernatant was aspirated and discarded.
The Vybrant Apoptosis Assay Kit #3 from Molecular Probes
(Eugene, OR) was used to assess the percentages of viable cells
(.80%). The epithelial cells, 20 00030 000/sample, were
subsequently gated based on their EpCAM expression. The cells
derived from the biopsies were ,50% epithelial cells. These cells
were analysed for further receptor expression on LRSFortessa
analyser (BD, San Jose, USA). The following mouse monoclonal
antibodies were used for staining: TLR3-PE (clone 40C1285.6),
TLR7-AF488 (clone IMG4G6), TLR8-FITC (clone 44C143),
TLR9-AF647 (clone 26C593.2) (Imgenex, San Diego, CA, USA),
EpCAM-PerCP-Cy5.5 (clone EBA-1) (Beckman Coulter,
Marseille, France), unlabeled VEGFR2 (clone KDR/EIC) (Abcam,
Cambridge, UK) were used for staining together with
phycoerythrin (R-PE) mouse IgG1 labeling kit (Molecular Probes).
Isotype controls relevant for each antibody were used for
background staining. IntraPrep Permeabilization Reagent kit
(Immunotech, Beckman Coulter, Marseille, France) was used to
detect the intracellular TLRs, according to instructions of the
manufacturer. For both extracellular and intracellular staining,
cells were incubated with antibodies or appropriate isotype
controls for 20 min at room temperature, thereafter washed and
resuspended in phosphate buffered saline (PBS) (Gibco, Paisley,
UK). Data were analysed with FlowJo Analysis Software ( Tree
Star, Inc, Ashland, USA).
Multiplex cytokine measurement
Cytokines in the supernatants from the in vitro stimulations
were measured using the Human Cytokine Standard 17-plex
(BioRad Laboratories, Inc, Corp., Hercules, USA) and quantified on
the Luminex200 system. The analysis was carried out according to
manufacturers instructions. Briefly, specific antibodies labeled
with spectrally encoded beads were applied to the samples and
standards. After incubation, the beads were washed and mixed
with specific biotinylated detector antibodies. Subsequently, excess
of biotinylated antibodies was removed by a washing step, followed
by addition of Streptavidin-R-phycoerythrin (R-PE), in order to
conjugate and label the detector antibodies. By monitoring the
spectral properties of the beads and the amount of associated
RPE fluorescence by the Bio-Plex System (Bio-Rad Laboratories,
Hercules, USA), the concentration of the cytokines could be
determined. The cytokines measured were: granulocyte-colony
stimulating factor (G-CSF; 1.7 pg/ml), granulocyte
monocytecolony stimulating factor (GM-CSF; 2.2 pg/ml),
interferon-gamma (IFN-c; 6.4 pg/ml), IL-1b (0.6 pg/ml), IL-2 (1.6 pg/ml), IL-4
(0.7 pg/ml), IL-5 (0.6 pg/ml), IL-6 (2.6 pg/ml), IL-7 (1.1 pg/ml),
IL-8 (1.0 pg/ml), IL-10 (0.3 pg/ml), IL-12p70 (3.5 pg/ml), IL-13
(0.7 pg/ml), IL-17 (3.3 pg/ml), monocyte chemotactic protein-1
(MCP-1: CCL2; 1.1 pg/ml), macrophage inflammatory protein
1b (MIP-1b: CCL4; 2.4 pg/ml) and tumor necrosis factor-a
(TNFa; 6.0 pg/ml).
Statistical analysis was performed using Graphpad Prism 5.01
(San Diego, Calif). Mean is represented by a horizontal line for
data presented as individual values. Remaining data is represented
as mean 6 SEM. N equals the number of independent donors.
For parametric data, statistical differences were determined using
unpaired t-test (for two sets of data) or one-way repeated measures
analysis of variance together with Dunnetts post test (for more
than two sets of paired data). Non-parametric data were analysed
with a Kruskal-Wallis test together with a Dunns multiple
comparisons post test (for more than two sets of paired data). A
P value of 0.05 or less was considered statistically significant.
Mucosal TLR expression
The first set of experiments compared the basal expression of
virus-recognizing TLRs in turbinate epithelial cells from patients
with CRSwNP, with the TLR expression found in turbinate
epithelial cells from healthy controls. TLR3, TLR7 and TLR8
were seen in equal amounts in both groups (data not shown). In
contrast, no TLR9-expressing epithelial cells were detected in the
turbinate tissue from patients, compared to the significantly higher
percentage of turbinate epithelial cells expressing TLR9 from
healthy controls (Figure 1A). The polyp tissue demonstrated an
increased percentage of epithelial cells expressing TLR9 compared
to turbinate tissue from the corresponding patients, although this
did not reach significance (Figure 1AD).
Effects of CpG stimulation on mucosal TLR9 expression
To investigate if CpG impacts the TLR9 expression on
epithelial cells, turbinate tissue from healthy controls and turbinate
tissue and polyp tissue from patients with CRSwNP were cultured
with increasing concentrations of CpG during 4 or 24 hours.
Initially, epithelial cells from the turbinate tissue from patients with
CRSwNP showed a significantly lower percentage of cells
expressing TLR9 compared to turbinate tissue from healthy
controls and polyp tissue from patients with CRSwNP (Figure 2A).
The percentage of epithelial cells from the turbinate tissue from
patients with CRSwNP expressing TLR9 significantly increased as
a result of the 4 hours CpG stimulation. Incubation with CpG did
not affect the percentage of epithelial cells expressing TLR9 in the
turbinate tissue from healthy controls nor did it affect percentage
of epithelial cells expressing TLR9 in the polyp tissue itself
(Figure 2A). After 24 hours, epithelial cells from the turbinate
tissue from patients with CRSwNP still showed a significantly
lower percentage of cells expressing TLR9 compared to turbinate
tissue from healthy controls (Figure 2B). Incubation with CpG for
24 hours did not significantly affect the TLR9 expression in any of
the tissues (Figure 2B).
Effects of CpG on cytokine release
To analyse the cytokine/chemokine release as well as the effects
of CpG stimulation on cytokine/chemokine release in turbinate
tissue from healthy controls and turbinate tissue as well as polyp
tissue from patients with CRSwNP, supernatants from all
experiments were studied. The release of IL-5 in polyp tissue
from patients with CRSwNP (6.961.2 pg/ml) were significantly
higher compared to the release in turbinate tissue from both
healthy controls (0.660.0 pg/ml, P,0.001) and patients with
CRSwNP (0.760.1 pg/ml, P,0.001). A similar cytokine pattern
was seen with IL-10, where the release of IL-10 in polyp tissue
from patients with CRSwNP (70.869.6 pg/ml) were significantly
higher compared to the release in turbinate tissue from both
healthy controls (2.360.4 pg/ml, P,0.001) and patients with
CRSwNP (6.963.7 pg/ml, P,0.001).
Further, 4 hours of CpG stimulation gave a small increase in
GM-CSF release although this effect was not
concentrationdependent and was only significant at the lowest dose (Figure 3A).
No significant increase in GM-CSF release was seen in healthy
turbinate tissue (Figure 3A). In the polyp tissue, no significant
GM-CSF alteration was seen after CpG stimulations (0 mM,
81.2614.8 pg/ml; 0.1 mM, 82.4625.2 pg/ml; 0.3 mM,
88.8616.3 pg/ml; 1.0 mM, 70.6624.8 pg/ml). The following
cytokines and chemokine analysed were unaffected by CpG
stimulation for 4 hours (all tissues), G-CSF, IFN-c, IL-1b, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, MCP-1,
MIP-1b and TNF-a (data not shown).
After 24 hours of incubation the levels of IL-6, G-CSF and
MIP-1b all tended to be higher under control condition in the
turbinate tissue from patients as compared to the levels
demonstrated in turbinate tissue from healthy controls
(Figure 3BD). CpG stimulation for 24 hours seemed to reduce them
all towards the levels demonstrated in healthy controls (Figure 3B
D). Incubation with CpG during culture of turbinate tissue from
healthy controls did not affect the release of IL-6, MIP-1b and
GCSF (Figure 3BD). In the polyp tissue, no significant alteration
was seen after CpG stimulations analysing IL-6 (0 mM,
22598610713 pg/ml; 0.1 mM, 1915765332 pg/ml; 0.3 mM,
2359265769 pg/ml; 1.0 mM, 2760069155 pg/ml), MIP-1b (0
mM, 392693 pg/ml; 0.1 mM, 343631 pg/ml; 0.3 mM,
4096119 pg/ml; 1.0 mM, 264695 pg/ml) or G-CSF (0 mM,
740462409 pg/ml; 0.1 mM, 465261039 pg/ml; 0.3 mM,
738861172 pg/ml; 1.0 mM, 793363819 pg/ml). The following
cytokines and chemokine analysed were unaffected by CpG
stimulation for 24 hours (all tissues), GM-CSF, IFN-c, IL-1b, IL-2,
IL-4, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, MCP-1 and
TNF-a (data not shown).
Effects of CpG stimulation on VEGFR2 expression
To explore whether CpG affected polyp development, vascular
endothelial growth factor receptor 2 (VEGFR2) expression was
studied on the epithelial cells. Both 4 and 24 hours CpG culture of
the turbinate tissue from patients with CRSwNP resulted in
reduction in the percentage of epithelial cells that express
VEGFR2. In contrast, the VEGFR2 expression in turbinate
epithelial cells from healthy controls as well as in epithelial cells
Figure 1. Epithelial TLR9 expression in turbinate tissue from healthy controls as well as turbinate tissue and polyp tissue from
patients with CRSwNP. Expression of TLR9 on turbinate epithelial cells from healthy controls compared to turbinate and polyp epithelial cells from
patients with CRSwNP, n = 5 (A). Intracellular staining for TLR9 (open histogram, black line) and isotype control (filled histogram) on turbinate
epithelial cells from a healthy control (B), turbinate epithelial cells (C) and polyp epithelial cells from a patient (D), analysed using flow cytometry.
Results are presented as mean 6 SEM, **P,0.01.
Effects of nasal challenge with CpG
Eight patients with CRSwNP in total were intranasally
challenged with CpG or placebo 24 hours prior to FESS. We
could demonstrate a high percentage of TLR9 expressing
epithelial cells in the turbinate tissue in the active CpG-treated
group. In the placebo group, almost none of the corresponding
epithelial cells expressed TLR9 (Figure 5A). Similar to the in vitro
experiments presented above, the active CpG treatment appeared
to some extent decrease the number of VEGFR2 positive
epithelial cells, this by 21 %, 24 hours after active CpG treatment,
although this did not reached statistical significance (P = 0.242)
(Figure 5B). No significant reduction was seen in the polyp tissue
(data not shown).
The present paper reveals several important differences between
the mucosa bordering the polyp and the polyp tissue itself. Of
Figure 2. Epithelial TLR9 expression after CpG stimulation. Epithelial TLR9 expression after 4 hours (A) and 24 hours (B) of culture with
vehicle/CpG (0.1 mM; 0.3 mM and 1.0 mM). Expression on turbinate epithelial cells from healthy controls, turbinate and polyp epithelial cells from
patients with CRSwNP, analysed using flow cytometry. Results are presented as mean 6 SEM, n = 6, **P,0.01 (unstimulated vs. CpG stimulated),
##P,0.01 (unstimulated turbinate tissue from patients vs. unstimulated turbinate tissue from healthy controls and polyp tissue from patients).
special interest is that TLR9 expression was detectable in CRS
polyps and in the nasal mucosal from healthy volunteers, but the
expression was almost absent in epithelial cells from the mucosa
close to the anatomical origin of the polyps. However, TLR9
expression could be reconstituted in the deficient mucosal
epithelium bordering the polyp following CpG stimulation, both
in vitro and in vivo. Stimulation also decreased the expression of
VEGFR2 on the epithelial cells in turbinate tissue from patients
with CRSwNP. Four virus-recognizing TLRs were measured;
TLR3, TLR7, TLR8 and TLR9, but only TLR9 was found to be
absent in the mucosa bordering the polyp. This highlights the
importance of TLR9 expression in the airway epithelium from
patients with CRSwNP.
TLR9 activation induces a Th1-biased immune response that is
known to suppress Th2-related activities associated with
CRSwNP. This response is initiated upon recognition of CpG in
the endosome after a non-specific uptake into the cell . Several
studies have demonstrated immunostimulatory properties of CpG
in vitro in humans [21,22] as well as in vivo in mice [23,24]. These
findings demonstrate the ability of CpG to strongly activate
different cell types to promote a Th1-biased immune response
[13,22]. An almost absent expression of TLR9 was seen in
epithelial cells from the mucosa bordering the polyp, but
stimulation with CpG, both in vitro and in vivo, upregulated this
expression. The discrepancies in TLR9 expression between freshly
analysed biopsies and in vitro unstimulated biopsies is probably
related to serum content in the media of the latter. However, a
similar TLR9 upregulation could not be induced in polyp derived
epithelial cells. The upregulation of TLR9 in epithelial cells from
the turbinate tissue bordering the polyp may have been due to the
low, but probably present, TLR9 expression responding to
CpGODN. However, the mechanism by which CpG-ODN may
upregulate TLR9 on epithelial cells needs to be further evaluated.
It is clinically well-established that viral infections aggravate
inflammation causing concomitant polyp growth . TLR9 is
known to respond to such microbial intrusion by initiating a host
response reaction . The lack of epithelial TLR9 activity at the
site of polyp growth might therefore contribute to the virus
Figure 3. Cytokine secretion from the nasal mucosa after CpG stimulation. CpG induced/reduced cytokine release in culture with turbinate
tissue from healthy controls and turbinate tissue from patients with CRSwNP. Samples analysed after culture of 4 hours (n = 4) or 24 hours (n = 58).
Levels of GM-CSF (A), IL-6 (B), G-CSF (C) and MIP-1b (D) were analysed with Luminex. Data presented as mean 6 SEM, *P,0.05; **P,0.01
(unstimulated vs. CpG stimulated), #P,0.05 (unstimulated turbinate tissue from healthy controls vs. unstimulated turbinate tissue from patients).
induced polyp development seen among patients with CRSwNP.
The restored and activated TLR9 receptor, as the result of the
CpG-ODN stimulation, may improve the immune defense of the
turbinate tissue that could lead to viral infection in smaller extent
causing polyp growth. Studies in mice have demonstrated that the
innate immune defenses activated by CpG-ODN, given by
injection, inhalation, or even by oral administration, can protect
against a wide range of viral, bacterial, and even some parasitic
pathogens, such as Listeria monocytogenes, Mycobacterium
tuberculosis, herpes simplex virus and respiratory syncytial virus .
The CpG-ODN treatment could, through the activated TLR9,
provide temporary protection against diverse pathogens. However,
interaction with other functions of the adaptive immunity resulting
in polyp growth inhibition cannot be ruled out. The small
upregulation of TLR9 expression due to serum in the media seen
in all the tissues were of the same magnitude in all tissues
specimens tested and it did not affect the CpG upregulated TLR9
expression. The increased percentage of cells that express TLR9 in
the turbinate tissue from patients with CRSwNP was also
confirmed in vivo after 24 hours. Kodama et al. have
demonstrated that weekly administrated CpG in the nasal cavity appears
unharmful to mice . Equivalently, we have previously
registered no apparent side effects in conjunction with nasal
CpG challenges of healthy volunteers . Our data along with
previous studies may impact the idea of CpG derived ligands as a
future therapeutic drug for patients with CRSwNP.
The nasal mucosa from patients with CRSwNP in the western
world is characterized by eosinophilic predominance and a Th2
cytokine profile . The present study demonstrates different
cytokine profiles, under control conditions, in the polyp tissue
compared to both the turbinate tissue from patients with
CRSwNP and from healthy control. IL-5 is an important cytokine
in patients with CRSwNP as it is responsible for delaying
eosinophilic apoptosis in nasal polyps . The anti-inflammatory
nature of IL-10 also contributes to polyp formation by enhancing
local Th2 inflammation and the associated tissue damage .
Both cytokines were higher in polyp tissue compared to the
turbinate tissue from patients with CRSwNP. Other groups have
demonstrated a higher release of IL-5 and IL-10 in polyp tissue
than in turbinate tissue from healthy controls [30,31]. The
development of an acute sinusitis is usually preceded and
accompanied by inflammation and cytokine secretion from the
nasal mucosa . Dysregulation of the innate antimicrobial
processes that normally protect the host can promote microbial
colonization or infection . Similarly, failure to limit the virus
induced innate immune responses can create a persistent
damaging inflammatory process. In the absence of TLR9 activity
and a robust Th1 inflammatory activation, an eosinophilic
Th2skewed inflammation can arise, in our study demonstrated by
upregulated IL-5, IL-6, IL-10, G-CSF and MIP-1b. This impaired
microbial defense could allow the microorganisms to become
permanently deposited in the nasal mucosa .
The Th2 cytokine profile is also known to be suppressed by
TLR9 induced Th1 activity . Stimulation of turbinate tissue
from patients with CRSwNP with CpG during 4 hours resulted in
a small increase in GM-CSF release. In line with this, GM-CSF
has been demonstrated to enhance neutrophilic responses to CpG
through a TLR9-dependent mechanism . After 24 hours of
CpG stimulation, the high levels of IL-6, G-CSF and MIP-1b of
the turbinate tissue from patients with CRSwNP had decreased,
Figure 4. Percentage of cells that express VEGFR2 after CpG culture. Epithelial VEGFR2 expression after 4 hours (A) and 24 hours (B) of
culture with vehicle/CpG (0.1 mM; 0.3 mM and 1.0 mM). Expression on turbinate epithelial cells from healthy controls, turbinate and polyp epithelial
cells from patients with CRSwNP, analysed using flow cytometry. Results are presented as mean 6 SEM, n = 5, *P,0.05, **P,0.01 (unstimulated vs.
CpG stimulated), ##P,0.01 (unstimulated turbinate tissue from patients vs. unstimulated turbinate tissue from healthy controls and polyp tissue
reaching levels corresponding to the release from turbinate tissue
from healthy volunteers. In line with this, Zhang et al. have
demonstrated an increased IL-6 release in polyp tissue from
patients with CRSwNP . It is possible that increased levels of
IL-6, seen in turbinate tissue from patients with CRSwNP, may be
responsible for the recruitment and retention of T cells seen
among patients with CRSwNP [35,36]. As the IL-6 levels
decreased after CpG stimulation, this could be an efficient way
of preventing this recruitment. Though TLR9 primarily is
expressed on B cells and plasmacytoid dendritic cells (pDCs), it
has also been detected on epithelial cells [37,38]. As the B cell
numbers are comparably low in the nasal mucosa we propose that
epithelial cells that are high in numbers, have an important role in
the cytokine secretion [19,39]. Hence, the reported cytokine
secretion could be emanating from any of these cells in the nasal
mucosa. Administration of a CpG-ODN activates pDCs to
promote Th1 adaptive immune responses. This is reflected in B
cells and pDCs showing increased expression of costimulatory
molecules, upregulation of the chemokine receptor, CCR7, and
secretion of Th1-promoting chemokines as well as cytokines, such
as MIP-1b and IFN-cinducible protein-10 . Although no
mouse model currently exists for CRSwNP, mice with upper and
lower allergic Th2 inflammation exhibit a decreased expression of
innate immune genes and reduced capacity to clear infection
[41,42]. It is important to recognize that, in contrast to what was
seen in epithelial cells from turbinate tissue, no cytokine reduction
was found in the polyp tissue as a result of the CpG stimulation.
The downregulation of cytokine release to the level seen in healthy
controls could further support the use of CpG for immunotherapy
against infectious disease.
The present finding of high VEGFR2 expression on epithelial
cells in turbinate tissue from patients with CRSwNP compared to
the low expression found in turbinate tissue from healthy controls,
after 24 h of culture, suggests that VEGFR2 is important in polyp
development. Nasal polyp growth requires epithelial proliferation
and accumulation of extracellular matrix [1,43,44]. For optimal
function these growth processes are in need of an increased
vascular supply, something that could be regulated by VEGFR
Figure 5. TLR9 and VEGFR2 expression after nasal challenge with CpG. Eight patients with CRSwNP were nasally challenged with either CpG
(n = 4) or placebo (n = 4). Biopsies from the turbinate tissue were obtained during FESS, 24 hours later. The nasal turbinate epithelial cells were stained
intracellularly for expression of TLR9 (A) and VEGFR2 (B) and analysed using flow cytometry. Results are presented as mean 6 SEM, *P,0.05.
located on epithelial cells . VEGFR participates in
angiogenesis by enhancing proliferation, migration and vascular
permeability . The VEGFR also regulates capillary and basal
membrane permeability in nasal polyps and increased VEGFR
expression can cause edema and polyp growth . Soluble
VEGF and VEGFR2 have been shown to be upregulated on
epithelial cells in nasal polyp tissue [48,49]. Bobic et al. show that
VEGFR2 not is upregulated in nasal polyps compared to turbinate
tissue from controls, but these polyps originate from patients that
had taken nasal corticosteroids . The patients in our study did
not take steroids for 6 (topically) and 12 weeks (systemically) prior
to FESS. This indicates that VEGFR2, compared to VEGFR1, is
affected by steroid treatment and could thereby be an important
player in polyp growth.
Further, CpG stimulation resulted in a clear downregulation of
VEGFR2 on epithelial cells in turbinate tissue from patients with
CRSwNP after both 4 and 24 h in vitro. Locally administrated
CpG reduced the VEGFR2 expression on turbinate epithelial cells
from patients with CRSwNP by 21 %, 24 hours after active CpG
treatment. No such reduction was seen in the polyp tissue. Viral
infections have been demonstrated to further upregulate the
VEGFR expression in epithelial cells . The finding of high
VEGFR2 expression on epithelial cells from turbinate tissue and a
downregulation following CpG stimulation further demonstrates
the function of CpG as a restrictor of polyp growth. Reduced
VEGFR expression decreases microvascular permeability, leading
to less plasma proteins accumulation in the extracellular matrix,
thereby contributing to polyp growth inhibition . The
VEGFR2 expression in healthy controls was unaffected by CpG
stimulation and the generally high level found in the turbinate
tissue from patients with CRSwNP further indicates the
importance of an inhibitory role of CpG in polyp growth.
To summarize, this paper investigates epithelial cells derived
from an area close to the anatomical origin of the polyps in
patients with CRS. A deprived expression of TLR9 was detected
in this area. This deficiency was seen neither in the polyp tissue
nor in the healthy turbinate tissue. CpG stimulation upregulated
the TLR9 expression and downregulated the VEGFR expression,
suggesting a correlation between TLR9 and VEGFR. It is
therefore tempting to assume a role for TLR9 activation in
restricting polyp growth. The demonstrated ability of CpG to
upregulate and activate TLR9 has to be considered important in
the struggle to reduce or inhibit return of polyps after surgery.
However, even more important for our future CRS research is the
finding that the polyp itself does not necessarily reflect the
inflammatory conditions at its nasal epithelial growth zone. Hence,
some of our previous conclusions based on polyp findings might
have to be supplemented and revised.
We thank Maria Axelsson, Carina Israelsson and the surgeons at the
ENTdepartment for logistic support and skillful assistance with the handling of
Conceived and designed the experiments: LT JA SKG L-OC. Performed
the experiments: LT. Analyzed the data: LT SKG L-OC. Contributed
reagents/materials/analysis tools: LT JA L-OC. Wrote the paper: LT JA
SKG OW L-OC.
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