Keywords
thymic stromal lymphopoietin - Th2 cytokine - eosinophilic chronic rhinosinusitis
Introduction
Chronic rhinosinusitis (CRS), characterized by nasal discharge, postnasal drip, and
nasal congestion, is commonly encountered in the otorhinolaryngological practice.[1] Since 2012, CRS has been classified into two subsets: CRS without nasal polyps (CRSsNP)
and CRS with NPs (CRSwNP).[1] Patients with CRSsNP respond well to treatment; however, the prognosis for most
patients with CRSwNP remains poor.[1] Infiltration of eosinophils is typically observed in the NPs of patients with CRSwNP
in Western countries; however, immune cells other than eosinophils, such as neutrophils
and lymphocytes, often infiltrate the NPs of patients with CRSwNP in East Asia.[2]
[3]
[4]
[5] Therefore, ethnicity may influence the CRS phenotype. To distinguish between cases
of CRS with a poor or good response to treatment in Japan, an East Asian country,
eosinophilic CRS (ECRS) has been proposed as a refractory type based on diagnostic
criteria, including computed tomography (CT) findings, presence of NPs, and percentage
of eosinophils in the peripheral blood.[6] Although the etiology of CRS remains largely unknown, the categorization of CRS
into ECRS and non-ECRS for outcome prediction is considered a reasonable approach
to select CRS treatment in Japan.[6] Recently, even in Europe, the type-2 immune response has been emphasized, as well
as the phenotype that is the presence or absence of polyps.[7] The concept of “one airway, one disease,” which was proposed in 1997[27], is adopted to several cases in the otorhinolaryngological practice. For example,
the severity of allergic rhinitis and the proportion of patients with a poor prognosis
of asthma are correlated in Japan.[8] Thus, it is possible that eosinophils are involved in ECRS pathogenesis, as is the
case with airway epithelial cells in patients with asthma. Therefore, we hypothesized
that allergic airway diseases share a common etiology.
In addition, epithelial cells and mucosa in the airway are commonly stimulated through
pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), upon viral
infection, which exacerbates asthma.[9]
[10]
[11] Most of the viruses that cause respiratory-tract infections are RNA viruses. Once
viral infection occurs, double-stranded (ds) RNAs (dsRNAs) are generated as replication
intermediates of the viral genome.[12] These dsRNAs, as well as polyinosinic:polycytidylic acid, or poly(I:C), in experimental
settings, are recognized by TLR-3, a member of the TLR family of PRRs.[12]
[13] Epithelial cells activated by PRRs secrete cytokines that promote the activation
of dendritic cells such as thymic stromal lymphopoietin (TSLP), which play an important
role in allergic inflammation.[14] Dendritic cells activated by TSLP promote the differentiation of Th2 cells from
naïve CD4+ T cells.[15] Thymic stromal lymphopoietin also plays a role in maintaining a Th2-dominant environment
because memory Th2 cells can proliferate in response to TSLP.[14] The expression of the Th2 cytokine interleukin-13 (IL-13) is associated with the
recruitment of eosinophils to the airway in response to allergen challenge in patients
with asthma.[16]
[17] Another Th2 cytokine, IL-4, plays a fundamental role in allergic responses by inducing
Th2 cells and initiating the production of immunoglobulin E (IgE) in B cells.[18]
[19]
[20] However, it remains unclear whether and, if so, how Th2 cytokines are involved in
ECRS pathogenesis. To address this issue, using sinus surgical specimens, we investigated
the level of TSLP production in cultured epithelial cells from the paranasal sinus
mucosa of patients with ECRS with varying severities in the presence and absence of
Th2 cytokines and PRR activation.
Methods
Patients
All the patients included provided written informed consent, and the study was approved
by the institutional Ethics Committee. The present study included 18 patients with
ECRS who underwent surgery at our facility, who were classified according to three
degrees of severity based on the JESREC score criteria.[6] As shown in [Figure 1], 4 patients had mild, 11 had moderate, and 3 had severe ECRS. Four healthy control
samples were obtained from patients with orbital plate fractures and from those who
underwent dacryocystorhinostomy. Patients with other infections (such as HIV, syphilis,
and hepatitis B and C), who underwent reoperation, those taking preoperative systemic
steroids, those with fungal sinusitis, or subjects with allergic fungal rhinosinusitis
were excluded. In total, 22 patients with or without ECRS were enrolled (16 men and
6 women, with a mean age of 56.55 ± 16.744 years) between January 2016 and August
2017.
Fig. 1 Diagram of eosinophilic chronic rhinosinusitis (ECRS) severity. This classification
was adopted from Tokunaga et al.6 Factor A comprises > 5% of eosinophils in the peripheral blood and an ethmoid-dominant
shadow on computed tomography, whereas factor B is comorbid (bronchial asthma, aspirin
intolerance, intolerance to non-steroidal anti-inflammatory drugs). Factor A2: both factors are applied (0 or 1); at least one factor is not applied. Factor B
(1≦): at least one factor is applied, (0): none of three factors are applied. The
numbers presented under the ECRS severity indicate the number of specimens used.
The preparation of paranasal sinus epithelial cells and stimulation was as follows:
the uncinate process of the ethmoid bone was used as a specimen to obtain paranasal
sinus epithelial cells. The paranasal sinus mucosa collected during surgery was separated
from the bone tissue and fragmented. These fragments were cultured in a collagen I-coated
culture dish using bronchial epithelial cell growth medium (BEGM BulletKit [CC-3170],
Lonza Group AG) containing penicillin-streptomycin. The paranasal sinus epithelial
cells were then stimulated with 25 μg/mL of poly(I:C)) (Sigma-Aldrich) in the presence
or absence of 100 ng/mL of recombinant human IL-4 (PeproTech) or 100 ng/mL of recombinant
human IL-13 (PeproTech). After 24 hours, the culture supernatant was collected and
analyzed for TSLP concentration using the human TSLP enzyme-linked immunosorbent assay
(ELISA) Ready-SET-Go! kit (Affymetrix eBioscience). The detection limit was determined
to be 8 pg/mL.
Statistical Analysis:
The IBM SPSS Statistics for Windows software, version 25.0 (IBM Corp.) was used for
all analyses. The Wilcoxon signed-rank test was used to examine changes due to differences
in stimulation among individuals. The Shapiro-Wilk test was performed to compare patient
groups. If a normal distribution was observed, a t-test was performed. The correlation analysis was performed using Pearson's product-moment
correlation coefficient. Statistical significance was set at p < 0.05.
Results
We classified the patients with ECRS into 3 groups, as shown in [Figure 1], based on the clinical scoring system from the Japanese Epidemiological Survey of
Refractory Eosinophilic Chronic Rhinosinusitis (JESREC).[6] Kim et al.[21] reported the upregulation of Th2 mediators such as Th2 cytokines and TSLP in the
epithelial cells of patients with moderate or severe ECRS. Based on these observations,
we hypothesized that TSLP is involved in the pathogenesis and progression of ECRS.
To clarify whether TSLP is induced in virus-infected paranasal sinus epithelial cells,
we obtained primary nasal epithelial cells from the paranasal sinus mucosa of patients
with ECRS and healthy controls. To mimic viral infection, we used poly(I:C), which
can activate innate immunity via TLR3. We cultured these cells in the presence or
absence of poly(I:C) and found that the paranasal sinus epithelial cells of both patients
with ECRS and healthy controls produced TSLP in the presence of poly(I:C) ([Fig. 2]). Although these cells did not produce TSLP in the culture with IL-4 or IL-13, these
Th2 cytokines enhanced TSLP production from the paranasal sinus epithelial cells in
the stimulation with poly(I:C) ([Fig. 2]). Therefore, viral infection appears to be the primary inducer of TSLP production
in paranasal sinus epithelial cells. In addition, the poly(I:C)-stimulated paranasal
sinus epithelial cells produced increased the levels of TSLP in the presence of either
IL-4 ([Fig. 2A]) or IL-13 ([Fig. 2B]).
Fig. 2 Production of thymic stromal lymphopoietin (TSLP) by paranasal sinus epithelial cells
in the presence of polyinosinic: polycytidylic acid, or poly(I:C). Paranasal sinus
epithelial cells, classified according to disease severity using the flowchart in
[Figure 1], were stimulated with 25 mg/mL of poly(I:C) in the presence or absence of 100 ng/mL
recombinant human interleukin-4 (rhIL-4) (A) or 100 ng/mL of rhIL-13 (B) for 24 hours. The TSLP protein in the culture supernatant was analyzed through enzyme-linked
immunosorbent assay.
To investigate the relationship between TSLP production and ECRS severity, we compared
the ratio of increase in TSLP production in paranasal sinus epithelial cells stimulated
with poly(I:C) alone or with poly(I:C) combined with IL-4 or IL-13. As shown in [Figure 3], the increase in TSLP production in all patients with varying degrees of ECRS severity
was greater than among the controls. Particularly, the increase in TSLP production
by paranasal sinus epithelial cells from patients with severe ECRS stimulated with
poly(I:C) combined with IL-13 was significantly greater than that of the controls
([Fig. 3A]; t-test; p = 0.042). Although the culture with IL-4 showed a trend similar to that observed
in the culture with IL-13, the difference in TSLP production ratio with IL-4 was not
statistically significant ([Fig. 3B]). However, a correlation was observed between the TSLP production ratios after IL-4
and IL-13 stimulation ([Fig. 4]). Accordingly, both IL-4 and IL-13 can enhance poly(I:C)-induced TSLP production
by paranasal sinus epithelial cells in patients with severe ECRS more than in those
with mild or moderate ECRS. Therefore, we propose that Th2 cytokine-rich paranasal
conditions exacerbate ECRS following viral infection, especially in patients with
severe ECRS.
Fig. 3 Increase in TSLP production by paranasal sinus epithelial cells in the presence of
poly(I:C) with IL-4 or IL-13 compared with poly(I:C) stimulation alone. The rate of
increase in TSLP production by paranasal sinus epithelial cells stimulated with poly(I:C)
alone or with poly(I:C) combined with IL-13 (A) or IL-4 (B) in control, mild ECRS, moderate ECRS, or severe ECRS groups (*p < 0.05).
Fig. 4 Correlation between the ratio of TSLP production by paranasal sinus epithelial cells
in the presence of poly(I:C) and IL-4 or IL-13, compared with stimulation with poly(I:C)
alone. The ratio of TSLP production by paranasal sinus epithelial cells stimulated
with poly(I:C) combined with IL-4 was compared with the ratio of TSLP production in
paranasal sinus epithelial cells stimulated with poly(I:C) combined with IL-13 (r = 0.904;
p < 0.001 by the Pearson's correlation coefficient).
Discussion
Various cell types can respond to TSLP, resulting in the development of Th2-dominant
responses that may lead to allergic diseases.[21] Therefore, understanding how TSLP production is regulated is crucial to elucidate
the pathogenicity of allergic diseases. In the present study, we showed that TSLP
was produced by epithelial cells derived from the paranasal sinus mucosa in response
to poly(I:C). The secretion of TLSP induced by poly(I:C) was enhanced in the presence
of Th2-type cytokines IL-4 or IL-13. Importantly, the epithelial cells from patients
with severe ECRS responded to Th2 cytokines with a greater increase in TSLP production
than those from patients with moderate or mild ECRS. Therefore, more severe TSLP-Th2
exacerbating cycles may be initiated in patients with severe ECRS following viral
infection.
Bronchial asthma and allergies to nonsteroidal anti-inflammatory drugs influence the
severity of ECRS.[6] Therefore, an allergic or Th2-skewed background may affect ECRS pathogenesis. Since
TSLP is a key factor in initiating and maintaining Th2-skewed conditions,[22] the ability to produce abundant TSLP in epithelial cells in severe ECRS patients
seems reasonable. In addition, the frequency of patients with severe ECRS and a clinical
history of asthma is much higher than that of other patients.[6] Therefore, patients with severe ECRS may have Th2-dominant status. This may be a
result of epigenetic modifications of the genome in the epithelial cells of patients
with severe ECRS under Th2-skewed conditions. Another possibility is the presence
of a specific single nucleotide variation (SNV) that may affect TSLP gene expression. Several SNVs in the TSLP gene have been identified and suggested to be involved in asthma susceptibility across
multiple ethnic backgrounds.[23] From the perspective of the “one airway, one disease” concept, a shared genetic
factor possibly influences TSLP promoter activity, thereby leading to airway disorders
and ECRS.[24]
Similar to asthma, respiratory-tract infections in patients with severe ECRS may cause
symptoms of exacerbated sinusitis, based on the results of the current study. One
possible reason for this observation is that, in patients with severe ECRS, TSLP production
is markedly increased in the paranasal sinus mucosa, where Th2 predominance is likely
when combined with viral infection. In the current study, cultured epithelial cells
did not produce TSLP in the absence of stimulation. Therefore, in the ECRS-affected
area of the paranasal sinus, constant stimuli that induce TSLP production likely exists
in epithelial cells and potentially in other types of cells. Currently, dupilumab,
an anti-IL-4α/IL-13α receptor antibody,[25] is used for the treatment of CRSwNP in Japan. In addition, tezepelumab, an anti-TSLP
antibody,[26] is currently in phase-III trials for CRSwNP, which is part of the ECRS in the United
States. Accordingly, our results suggest that the combination of dupilumab and tezepelumab
might be more effective for the treatment of severe ECRS than the individual use of
each drug.
Limitations
Since the number of samples was limited in the present study, a trend towards a correlation
between poly(I:C)-induced TSLP production rate with or without Th2 cytokines and ECRS
severity was not statistically proven ([Fig. 3]). Therefore, although the ratio of TSLP production by paranasal sinus epithelial
cells stimulated with poly(I:C) in the presence or absence of IL-13 was statistically
different compared with the controls, the mild and moderate ECRS groups might exhibit
further distinctions if additional samples are analyzed.
Conclusion
In the sinus mucosa of patients with severer ECRS, TSLP production was increased when
poly(I:C) stimulation was combined with IL-4 or IL-13 stimulation. Thus, the Th2-skewed
condition of the sinus mucosa and enhanced immune responses due to viral infection
in patients with more severe ECRS may accelerate disease exacerbation.
Bibliographical Record
Akiko Inoue, Yuriko Tanaka, Hidehito Matsui, Akira Fukuo, Motonari Kondo, Kota Wada.
TSLP Production Induced by Poly(I:C) Stimulation Increased in the Presence of Th2
Cytokines in Patients with Severe ECRS. Int Arch Otorhinolaryngol 2025; 29: s00451809159.
DOI: 10.1055/s-0045-1809159
