S100A8 enhances IL-1β production from nasal epithelial cells in eosinophilic chronic rhinosinusitis.

BACKGROUND
Chronic rhinosinusitis is classified into eosinophilic chronic rhinosinusitis (ECRS) and non-eosinophilic chronic rhinosinusitis (NECRS). ECRS is a refractory allergic disease involving a variety of immune and epithelial cells. S100A8 is a damage-associated molecular pattern that is closely related to allergic inflammation. However, the pathological implications of S100A8 in ECRS have not been clarified.


METHODS
We evaluated the role of S100A8 in the pathogenesis of ECRS. Gene expression profiles of nasal polyps obtained from patients with ECRS or NECRS were evaluated using RNA sequencing.


RESULTS
S100A8 was identified as a significantly upregulated gene in nasal polyps associated with ECRS. Immunohistochemistry consistently revealed intense S100A8 staining in nasal polyps from patients with ECRS. Human nasal epithelial cells expressed the receptor for advanced glycation end products and Toll-like receptor 4. Recombinant S100A8 protein induced interleukin-1β secretion in human nasal epithelial cells.


CONCLUSIONS
Our data demonstrate that S100A8 results in production of interleukin-1β in the nasal epithelium, which may be involved in the pathogenesis of ECRS.


Abstract
Chronic rhinosinusitis is classified into eosinophilic chronic rhinosinusitis (ECRS) and non-eosinophilic chronic rhinosinusitis (NECRS). ECRS is a refractory allergic disease involving a variety of immune and epithelial cells. S100A8 is a damage-associated molecular pattern that is closely related to allergic inflammation. However, the pathological implications of S100A8 in ECRS have not been clarified. We evaluated the role of S100A8 in the pathogenesis of ECRS. Gene expression profiles of nasal polyps obtained from patients with ECRS or NECRS were evaluated using RNA sequencing. S100A8 was identified as a significantly upregulated gene in nasal polyps associated with ECRS. Immunohistochemistry consistently revealed intense S100A8 staining in nasal polyps from patients with ECRS. Human nasal epithelial cells expressed the receptor for advanced glycation end products and Toll-like receptor 4. Recombinant S100A8 protein induced interleukin-1β secretion in human nasal epithelial cells. Our data demonstrate that S100A8 results in production of interleukin-1β in the nasal epithelium, which may be involved in the pathogenesis of ECRS.

Chronic rhinosinusitis (CRS) is an airway disease with paranasal sinus inflammation. In
East Asia, the phenotypes of CRS are heterogeneous, and CRS is subdivided in two groups according to Japanese clinical criteria: eosinophilic chronic rhinosinusitis (ECRS) and non-eosinophilic chronic rhinosinusitis (NECRS) 1 2 3 . ECRS is an allergic disease that causes olfactory disturbances, nasal obstruction, and nasal discharge. In addition, bronchial asthma is a common complication of ECRS, which is designated as an intractable disease in Japan. ECRS is a type 2 inflammatory disease involving various types of immune cells. Recent studies have shown that the most severe forms of eosinophilic inflammation, including ECRS, are caused by inflammation resulting from a combination of type 2 and type 3 cytokines 4 5 . The main treatments for ECRS are endoscopic sinus surgery (ESS) and systemic glucocorticoids. However, ESS has a high relapse rate, while long-term systemic glucocorticoid steroid therapy is associated with side effects. Therefore, molecularly targeted therapy based on an understanding of pathogenesis is an attractive alternative for treatment of ECRS.
Nasal epithelial cells are the interface between the host and the environment and act as the first defense mechanism. Nasal epithelial cells produce a variety of cytokines and matrix metalloproteinases that exacerbate allergic inflammation 6 7 . In particular, when cellular damage occurs, nasal epithelial cells release damage-associated molecular patterns (DAMPs). DAMPs contribute to the activation of various immune cells by type 2 and type 3 cytokines, and previous reports have suggested a relationship between DAMPs and allergic diseases. High mobility group box-1 (HMGB-1), a representative DAMP, enhances interleukin-33 production from nasal epithelial cells 8 . However, the pathological implications of other DAMPs in CRS have not been sufficiently clarified. S100 proteins are a group of small Ca 2+ -binding modulator proteins. Recent work has revealed that S100 proteins are involved in tumor metastasis and immune responses 9 . In particular, S100A8 protein binds to Toll-like receptor 4 (TLR4) or the receptor for advanced glycation end products (RAGE) and initiates the activation of various immune cells and the production of reactive oxygen species and nitric oxide, contributing to the exacerbation of chronic inflammation 10 11 12 13 .
In this study, we attempted to discover the genes involved in the pathogenesis of ECRS via RNA sequencing. We found that S100A8 gene expression is upregulated in nasal polyps obtained from patients with ECRS. Thus, we sought to clarify the mechanism of ECRS by characterizing the effects of S100A8 on the nasal epithelium.

Human subjects
Paranasal sinus tissue samples were collected from seven patients with chronic

Histological analysis
Positive cell numbers were assessed in three regions at random points with 500 highpower fields. The sum of the positive cell numbers was used as the score for each individual case.

Immunofluorescence
After deparaffinization of paraffin-fixed tissue, antigen retrieval was performed with autoclaving for 15 min at 125°C in an ethylenediaminetetraacetate buffer solution (pH 9). Sections were blocked with 1% BSA in PBS and were reacted with S100A8/MRP8 Antibody  All experiments were performed in accordance with relevant guidelines and regulations.

Statistical analysis
All statistical analyses were performed with Prism version 7 (GraphPad Software, San Diego, CA, USA). Data were expressed as means ± SD. Comparisons between two groups were performed using Student's t-test, and p < 0.05 was considered to indicate statistical significance.

Upregulated genes in nasal polyps from patients with ECRS
In order to investigate the factors involved in the pathogenesis of ECRS, we first performed RNA sequence analysis of nasal polyps obtained from patients with ECRS or NECRS. S100A8 was identified as a significantly upregulated gene in nasal polyps associated with ECRS (Table 1). The expression of other S100 protein-related genes is shown in Table 2. S100A9 was significantly upregulated in nasal polyps from patients with ECRS, while S100A1 and S100B were significantly upregulated in nasal polyps from patients with NECRS. Therefore, we hypothesized that S100A8 plays an important role in the pathogenesis of ECRS. We evaluated the expression of S100A8 in locally inflamed tissue of patients with ECRS.
Evaluation of S100A8 expression in nasal polyps associated with ECRS First, we confirmed that the relative expression of S100A8 measured by RT-qPCR was slightly higher in ECRS than in NECRS, but not significantly ( Figure 1A). In immunohistochemistry studies, anti-S100A8 antibody staining of eosinophils that had infiltrated nasal polyps was stronger in patients with ECRS than in those with NECRS ( Figure 1B-D). The number of cells stained with S100A8 was significantly higher in ECRS than in NECRS ( Figure 1E, F). These results indicate that the increase in S100A8 in nasal polyps associated with ECRS may be due to infiltrating eosinophils. We also confirmed the expression of S100A8 in peripheral blood eosinophils, neutrophils, and mononuclear cells by western blotting (Supplemental Figure 1).
Expression of the receptor for S100A8 in human nasal epithelial cells To further investigate how S100A8 functionally contributes to ECRS, we first confirmed the expression of TLR4 and RAGE in HNEpCs using western blotting. Both TLR4 and RAGE were expressed in HNEpCs in the steady state ( Figure 2A). Next, the expression of TLR4 and RAGE were compared by immunohistochemistry in ECRS and NECRS. Interestingly, the expression of TLR4 was significantly higher in ECRS than in NECRS ( Figure 2B). The expression of RAGE was nonsignificantly higher in ECRS than NECRS ( Figure 2C).

S100A8 protein pathophysiology in nasal epithelial cells
Previous reports suggested that various cells promote proinflammatory cytokine production via S100A8 stimulation. Thus, we examined cytokine production by nasal epithelial cells following stimulation with recombinant S100A8 protein for 24 or 48 h.
Interestingly, S100A8 induced interleukin-1 beta (IL-1β) production in nasal epithelial cells ( Figure 3A) in a dose-dependent manner ( Figure 3B). However, stimulation by S100A8 did not induce interferon-gamma production ( Figure 3C). Next, we examined cytokine production by nasal epithelial cells following stimulation with recombinant S100A1, S100A9, and S100A3 proteins as negative controls. S100A8 induced IL-1β production in nasal epithelial cells more strongly than S100A1 or S100A3. S100A9 induced IL-1β production as strongly as S100A8 ( Figure 3D). In immunohistochemistry, IL-1β was more highly expressed in ECRS than in NECRS, but not significantly (Supplemental Figure 2).

DISCUSSION
In this study, we demonstrated the clinical implications of S100A8 in ECRS, a refractory disease involving type 2 inflammation. Some previous reports suggested that DAMPs released by damaged tissue might contribute to the exacerbation of allergic diseases 8 . S100A8, a type of DAMP, is produced mainly by blood cells 15 16 17 . It has been reported that RAGE and TLR4 are receptors for S100A8, and S100A8 induces inflammation by binding to these receptors. Thus, S100A8 is closely related to allergic dermatitis and autoimmune diseases, but its role in ECRS has not been fully explored 18 19 .
RNA sequencing showed that S100A8 was more highly expressed in nasal polyps associated with ECRS than those associated with NECRS. Among other genes in the S100 protein family, S100A9 was also significantly upregulated in nasal polyps associated with ECRS. Previous studies used RNA sequencing to compare gene expression in nasal polyps associated with ECRS and NECRS 20 . In RT-qPCR in this study, there was no significant difference in S100A8 expression between ECRS and NECRS, but expression was nonsignificantly higher in ECRS. However, we believe that the lack of significant difference between ECRS and NECRS is due to the small number of cases examined by RT-qPCR.
However, there are no studies that evaluated the expression of S100 family proteins in local tissues from patients with ECRS. It has been reported that S100A8 is mainly secreted by neutrophils and monocytes 15 16 17 . Thus, increased S100A8 gene expression in nasal polyps associated with ECRS might be due to the infiltration of various immune cells. In this study, histological evaluation revealed S100A8 protein expression in tissue-infiltrating eosinophils. No previous reports have described the contribution of eosinophils to the production of S100A8; this finding is new. Further studies will be needed to elucidate the factors that induce the production of S100A8 in eosinophils.
We evaluated the role of S100A8 in the exacerbation of ECRS. Nasal epithelial cells were predicted as a target of S100A8 action. Therefore, we assessed the receptors for S100A8 in the nasal epithelium. Previous reports have shown the expression of RAGE and TLR4 in lower airway epithelial cells 18 19 . This study showed that both receptors are expressed in the upper airway as well. In addition, S100A8 has been reported to contribute to the production of various proinflammatory cytokines and increased production of MUC5AC 11 . Hence, we stimulated nasal epithelial cells with S100A8 and found increased production of IL-1β, an important factor for both type 2 and type 3 inflammation. These results are consistent with recent studies showing that severe eosinophilic inflammation is caused by inflammation mediated both by type 2 and type 3 cytokines 4 5 21 22 23 .
We also showed that the expression of TLR4 was higher in ECRS than in NECRS. This result suggests that S100A8 may be highly expressed in ECRS due to the higher expression of TLR4 in ECRS.
This study has some limitations. We did not investigate whether IL-1β production by nasal epithelial cells is mediated by TLR4 or RAGE receptors.
Additionally, in immunohistochemistry, the expression of IL-1β was nonsignificantly higher in ECRS than NECRS.
However, the study has three novel features. Comprehensive analysis revealed that S100A8 is upregulated in nasal tissues of patients with ECRS. S100A8 protein expression was upregulated in eosinophils infiltrating nasal polyps in ECRS and enhances IL-1β production in upper airway epithelial cells. S100A8 worsens allergic inflammation, implying that S100A8 blockade could promote recovery from ECRS.
Therefore, studies with mouse models, as well as clinical tests of S100A8-blocking therapies, should be pursued in the future.
In summary, we found increased expression of S100A8 gene expression in nasal polyps from patients with ECRS. S100A8 might be derived from eosinophils that have infiltrated nasal tissue. We also demonstrated that S100A8 enhances IL-1β production in nasal epithelial cells, which may be involved in the pathogenesis of ECRS.

Figure 1 S100A8 was expressed in eosinophils that have infiltrated nasal tissue.
A. Gene levels of S100A8 were measured by RT-qPCR and compared (n = 8 ECRS, n = 4 NECRS).
B, C. Hematoxylin and eosin staining of nasal tissues and immunohistochemical detection of S100A8. Images are of representative samples from patients with ECRS.
E. Expression of S100A8 in nasal polyps from patients with ECRS or NECRS.  A. HNEpCs were stimulated with recombinant S100A8 protein (10 µg/ml) with control for 24 or 48 h. IL-1β levels in culture supernatant were determined using CBA.
B. HNEpCs were stimulated with recombinant S100A8 protein (1 µg/ml or 10 µg/ml) with control for 24 h. IL-1β levels in culture supernatant were determined using CBA.
C. HNEpCs were stimulated with recombinant S100A8 protein (10 µg/ml) with control for 24 h. INF-γ levels in culture supernatant were determined using ELISA.
HNEpCs, human nasal epithelial cells; IL-1β, interleukin-1 beta; CBA, cytometric bead array; Ctrl, control.  Figure 1 S100A8 was expressed in eosinophils that have in ltrated nasal tissue. A. Gene levels of S100A8 were measured by RT-qPCR and compared (n = 8 ECRS, n = 4 NECRS). B, C. Hematoxylin and eosin staining of nasal tissues and immunohistochemical detection of S100A8. Images are of representative samples from patients with ECRS. Scale bars, 100 μm. D. Immuno uorescence staining for S100A8 (green), MBP (red), or DAPI (blue). Scale bars, 100 μm. E. Expression of S100A8 in nasal polyps from patients with ECRS or NECRS. F. Immunohistochemical staining for S100A8; positive cells were counted (n =15 ECRS, n Expression of S100A8 receptor on HNEpCs. A. Western blots showing that both TLR4 and RAGE are expressed in HNEpCs. Full length blots/gels are presented in Supplementary Figure 3. B. Immunohistochemical staining for TLR4; positive cells were counted (n = 5 ECRS, n = 5 NECRS). Left: representative images for ECRS and NECRS; right: number of positive cells. C. Immunohistochemical staining for RAGE; positive cells were counted (n = 5 ECRS, n = 5 NECRS). Left: representative images for