NP tissues were obtained from patients with CRSwNP during routine functional endoscopic sinus surgery. All subjects provided written informed consent for study participation, and the study was approved by the Institutional Review Board of the Seoul National University Hospital, Seoul Metropolitan Government-Seoul National University Boramae Medical Center (No. 10-2018-80). The diagnosis of CRS was based on personal history, physical examination, nasal endoscopy, and sinus computed tomography (CT) findings according to a 2012 European position paper on rhinosinusitis and nasal polyps (EPOS) guidelines.1 Exclusion criteria were as follows: 1) younger than 18 years of age, 2) prior treatment with antibiotics, systemic or topical corticosteroids, or other immune-modulating drugs for 4 weeks before surgery, and 3) unilateral rhinosinusitis, antrochoanal polyp, allergic fungal sinusitis, cystic fibrosis, or immotile ciliary disease. Each sample was divided into 2 parts; one was fixed in 10% formaldehyde and embedded in paraffin for histological analysis, and the other was submersed in 1 mL phosphate-buffered saline (PBS) supplemented with 0.05% Tween-20 (Sigma-Aldrich, St Louis, MO, USA) and 1% PIC (Sigma-Aldrich) per 0.1 g of tissue. The latter tissue was homogenized with a mechanical homogenizer at 1,000 rpm for 5 minutes on ice. After homogenization, the suspensions were centrifuged at 3,000 rpm for 10 minutes at 4°C, and the supernatants were separated and stored at −80°C for further analysis of cytokines and other inflammatory mediators. The atopic status of study subjects was evaluated using the ImmunoCAP^® assay (Phadia, Uppsala, Sweden) to detect immunoglobulin E (IgE) antibodies against 6 mixtures of common aeroallergens (house dust mites; molds; trees; weed and grass pollen; and animal dander). Subjects were considered atopic if the allergen-specific IgE level was greater than 0.35 IU/mL to any one or more of the allergens. The diagnosis of asthma was based on medical history and lung function analysis, including challenge tests by an allergist. Lund-Mackay CT scores and global osteitis scoring scale were calculated with the preoperative CT scan.18 Odor threshold was evaluated using a butanol threshold test (BTT), performed as previously described.19 According to National Institute on Alcohol Abuse and Alcoholism (NIAAA) standards, 14 g of alcohol was defined as one “standard drink”. One quarter bottle of soju (Korean distilled spirits), 350 mL beer, or 1 glass of wine contain the same amount of alcohol as one “standard drink”. Based on these guidelines, alcohol consumption of patients was calculated in grams. The disease control status (DCS) of individual patients was evaluated 1 year after surgery and was classified into controlled and refractory (partly controlled plus uncontrolled) groups according to the EPOS guidelines.1 In brief, CRS control status takes into account the presence and severity of the four major sinonasal symptoms, sleep disturbance and/or fatigue, nasal endoscopic evaluation and need for oral medication. “Controlled” was defined postoperatively when there were no symptoms and abnormal endoscopic findings without medication. “Refractory” was classified 1 year after surgery, when there were symptoms or abnormal findings on endoscope which needs systemic medication such as antibiotics and steroids. Prior study demonstrated that the cutoff value of 70 eosinophils/high power field (HPF) presented the most significant difference in NP recurrence. 20 Thus, we defined eosinophilic nasal polyp (ENP) as the presence of greater than 70 eosinophils/HPF, while the presence of fewer than 70 eosinophils/HPF was designated as non-eosinophilic nasal polyp (NENP).20 Additionally, another previous study for subclassification of CRS showed that patients with 20 neutrophils/HPF exhibited the minimum P values for NP recurrence.21 Thus, in this study, tissue neutrophilia (Neu^high) was defined as greater than 20 human neutrophil elastase (HNE)-positive cells/HPF, while less than 20 HNE-positive cells/HPF was designated as the absence of tissue neutrophilia (Neu^low).21

As previously described,222324 immunohistochemical staining was performed using the polink-2 plus polymerized horseradish peroxidase (HRP) broad DAB-Detection System (Golden Bridge International Labs., Bothell, WA, USA). Briefly, sections were incubated in 3% hydrogen peroxidase to inhibit endogenous peroxidases. Heat-induced epitope retrieval was then performed by microwaving samples in 10 mmol/L citrate buffer (pH 6.0). The sections were incubated with a 1:500 dilution of mouse anti-human neutrophil elastase primary antibody (R&D Systems, Minneapolis, MN, USA) for 60 minutes at room temperature. Sections were then incubated with a broad antibody enhancer and polymer-HRP before staining with the DAB Detection System. Finally, slides were counterstained with hematoxylin. Positive cells were counted in the ten densest visual fields (400×), and the average value from 2 independent observers was determined.

As previously described,1425 protein concentrations of tissue extracts were determined using the Pierce 660 nm Protein Assay Kit (Thermo Scientific Inc., Rochester, NY, USA), and samples were thawed and vortexed at room temperature to ensure a well-mixed sample. Tissue homogenates were then assayed for periostin, HNE and α-1 anti-trypsin using commercially available ELISA kits (R&D Systems). Multiple cytokine analysis kits (tumor necrosis factor [TNF]-α, interleukin [IL]-1α, IL-1β, IL-1Ra, IL-5, IL-6, IL-17A, IL-18, IL-22, IL-23, IL-36α, OSM, chemokine [C-X-C motif] ligand [CXCL]-1, CXCL-8, myeloperoxidase [MPO], matrix metallopeptidase (MMP)-9, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, and IL-5) were obtained from R&D systems (catalog No. LMSAHM), and data were collected using a Luminex 100 reader (Luminex, Austin, TX, USA). Data analysis was performed using MasterPlex QT version 2.0 (MiraiBio, Alameda, CA, USA), and total IgE was measured using the Human IgE ELISA kit (K3231066: KOMA, Seoul, Korea). All assays were run in duplicate according to the manufacturer protocol. All protein levels in the tissue homogenate were normalized to the concentration of total protein. The sensitivity of the ELISA assays is presented in Supplementary Table S1.

To verify the relationship between markers expressed on HNE or MPO-positive cells and IL36R, NP tissues were plated. Immunofluorescence was evaluated using primary antibodies directed against HNE (1:500, MAB91671; R&D Systems), MPO (1:300, Abcam, Cambridge, UK), and IL36R (1:150, PA5-38013; Invitrogen, Carlsbad, CA, USA). After 24 hours incubation with primary antibodies, tissues were incubated for 1 h with an Alexa Fluor^® 488-conjugated anti-rabbit IgG (1:1,000, ab150077; Abcam) or Cy3-conjugated anti-mouse IgG (1:500, ab97035; Abcam) secondary antibody. The nucleus was stained with 4′,6-Diamidino-2-phenylindole dihydrochloride (1:1,000, Sigma) for 2 min, and tissues were mounted with aqueous-based medium. Fluorescent images were obtained using a fluorescence microscope (CELENA^® S; Logos Biosystems, Annandale, VA, USA). HNE, MPO, and IL-36R-positive cells were counted and analyzed in three randomly selected fields, with non-specific signals excluded.

Statistical analyses were performed using IBM SPSS 21 (SPSS, Inc., Chicago, IL, USA), R version 3.4.2 software (R Foundation for Statistical Computing, Vienna, Austria), and GraphPad Prism software 7.0 (GraphPad Software Inc, La Jolla, CA, USA). A χ² test and 2-tailed Mann-Whitney U test were used for unpaired comparisons. Bonferroni correction was applied to multiple comparison analyses. Linear by linear associations were also performed to evaluate the trend in proportion of refractoriness of each subtype. Multiple variate analyses were performed to identify the risk factor for disease refractoriness. The parameters statistically significant in univariate analysis were involved in binary logistic regression analysis for multivariate analysis, after adjusting for confounding factors. The significance level was set at α = 0.05. Exploratory factor analysis using principal component analysis (PCA) was used to describe patterns of inflammatory mediators. Variables with loadings greater than 0.3 were selected.

Subjects were divided into 4 groups based on tissue eosinophilia and neutrophilia. ENP and NENP accounted for 40 (25%) and 120 (75%) subjects, respectively. Out of the patients with ENP, 7 (4.4%) had tissue neutrophilia (ENP-Neu^high) and 33 (20.6%) did not have tissue neutrophilia (ENP-Neu^low). For patients with NENP, 69 (43.1%) had tissue neutrophilia (NENP-Neu^high) and 51 (31.9%) did not have tissue neutrophilia (NENP-Neu^low). In addition, we observed that tissue neutrophilia was inversely correlated with tissue eosinophilia in NP (r = −0.2932, P = 0.0001; Fig. 1A).

Patients with ENP-Neu^high had an increased incidence of asthma and a higher likelihood of smoking, in addition to an increased amount of smoking (packs/year) and alcohol intake (g/month), compared with ENP-Neu^low patients. However, there were no significant differences in smoking and alcohol intake between NENP-Neu^low and NENP-Neu^high patients, except the proportion of asthma was lower in NENP-Neu^high patients (Table 1).

NENP-Neu^low patients showed the higher rate of disease control (35/44, 79%) than NENP-Neu^high (25/50, 50%) and ENP-Neu^high (1/7, 14%) patients (Fig. 1B). Linear by linear association was performed to show the trend in refractoriness from NENP-Neu^low to NENP-Neu^high or ENP-Neu^low to ENP-Neu^high. These results imply that tissue eosinophilia may be correlated with a worse disease outcome, which is consistent with previous studies,26272829 and tissue neutrophilia was also associated with a worse disease outcome in CRSwNP.

To further identify risk factors for disease refractoriness in Asians with CRSwNP, we performed binary multivariate analysis (Table 2). We found that tissue neutrophilia was the strongest risk factor for CRSwNP refractoriness (hazard ratio [HR], 4.38, 95% confidence interval [CI], 1.76-10.85). The Lund-Mackay score (HR, 1.14; 95% CI, 1.04–1.23), age (HR, 0.96; 95% CI, 0.93–0.99), and tissue eosinophilic count (HR, 1.01; 95% CI, 1.002–1.01) were also risk factors for CRSwNP refractoriness.

Next, to determine the influence of neutrophils-related biomarkers on disease refractoriness, we performed exploratory factor analysis using PCA, with a correlation matrix of 17 neutrophils-associated mediators. The PCA retained 6 components, accounting for 74.7% of all variance in the data (Supplementary Table S2). The first, second, third, and fourth components accounted for 33.6%, 14.5%, 8.6% and 6.8% of the variance, respectively (Fig. 2). We found that IL-18, IFN-γ, IL-1Ra, TNF-α, OSM and MPO were associated with good disease control status (Fig. 2A and B), whereas IL-36α and IL-1α were associated with refractory disease control (Fig. 2C and D). Therefore, we speculated that neutrophils-associated mediators have distinct phenotypes related to disease control status in CRSwNP.

To confirm this, we compared various neutrophil-associated inflammatory mediators in CRSwNP, according to disease control status (Table 3). Among those, we found that HNE-positive cells, HNE, and IL-36α expression were significantly up-regulated in the refractory group (P = 0.0132, P < 0.0001, and P = 0.0395, respectively), whereas the expression of MPO and IL-18 was higher in the controlled group (P = 0.0002 and P = 0.0009, respectively). Additionally, there was no difference in the expression of type 2 inflammatory markers such as IL-5 and total IgE between the controlled (IL-5: 0.1 [0–7.22] and total IgE: 20.71 [7.77–44.45]) and refractory (IL-5: 0.1 [0–2.99] and total IgE: 21.01 [7.99–36.74]) groups.

Interestingly, our findings imply that expression of a neutrophil marker such as HNE or MPO in Asian NPs may have a different clinical outcome. Thus, to identify a possible association between expression of HNE or MPO-positive cells in Asian NP tissues and disease control status, we performed immunofluorescence analysis. Among HNE-positive cells, IL36R⁺HNE⁺ double-positive cells were significantly increased in the refractory group compared to the controlled group, whereas IL-36R⁺MPO⁺ positive cells were infrequently observed in NP tissues (Fig. 3). Additionally, we found that the concentration of α1 anti-trypsin was lower and the ratio of HNE-positive cells to α1 anti-trypsin was higher in the refractory group than in the controlled group (Fig. 4). The α-1 anti-trypsin is known to be one of the potent inhibitors of neutrophil elastase.3031 Therefore, we suggest that an imbalance of HNE-positive neutrophils to α-1 anti-trypsin might have an influence on refractoriness in Asian CRSwNP patients.