Age-Related Decline of Neutrophilic Inflammation Is Associated with Better Postoperative Prognosis in Non-eosinophilic Nasal Polyps

Background Innate and adaptive immune responses change with increasing age and affect the course of diseases. Previous study investigated immunologic alteration in Western nasal polyps (NP) which is mostly eosinophilic. However, there are no reports regarding age-related immune changes of non-eosinophilic NP (NE-NP) which is a predominant subtype in Asian population. Methods A total of 153 subjects, including 20 with control, 63 with chronic rhinosinusitis (CRS) without NP (CRSsNP), and 70 with CRS with NP were enrolled. Age-related changes in computed tomography (CT), cytokines and clinical information were investigated. Tissue samples were analyzed for protein levels of IL-5, IL-17A, IL-23, interferon (IFN)-γ, CCL-11, and CXCL-8, using Luminex immunoassay and for mRNA expression levels of interleukin (IL)-5, IL-17A, IL-23p19, IFN-γ, CCL-11, CXCL-1, CXCL-2, CXCL-8, and CXCR2 by quantitative RT-PCR. Immunohistochemistry (IHC) was performed for the number of inflammatory cells. Results We observed that Lund-Mackay CT scores decreased with age in NE-NP. The number of human neutrophil elastase-positive cells and myeloperoxidase gene expression decreased in older patients with NE-NP, but not in control subjects, CRSsNP, and E-NP. Neutrophil-associated cytokines including IL-17A and IL-23, were negatively correlated with age in NE-NP at the protein and mRNA levels. Additionally, the expression of CXCR2, a receptor for CXCL-1 and CXCL-2, was decreased with age in NE-NP. However, there were no age-related changes in blood neutrophil count, and neutrophil-recruiting chemokines such as CXCL-1, CXCL-2, and CXCL-8. Elderly NE-NP patients showed better endoscopic scores at 12 months after surgery compared with the non-elderly. Conclusion Age-related decline in neutrophil inflammation may favorably affect postoperative results in elderly patients with NE-NP.


Introduction
Recent research using both animal models and human subjects suggests that there are several important changes in the innate and adaptive immune responses with increasing age [1]. Alterations of immune response with aging may affect the pathophysiology of airway inflammation including asthma [2]. We previously reported age-related changes in Western patients with nasal polyps (NP) [3,4]. In that study, there was a significant age-related decline of eosinophilic inflammation and innate immune barrier function in patients with chronic rhinisinusitis with NP (CRSwNP). Altered barrier function such as decreased S100A8/9 and increased soluble gp130 may be associated with disease extent or asthma comorbidity in eosinophilic NP (E-NP). However, several studies have shown that the inflammatory response in NP removed during surgery is usually eosinophilic in the US and Europe, although the incidence of E-NP is likely overestimated since the population of these study is based on tertiary referral hospital, while NP removed from patients in Asian countries (including China, Korea and Japan) and even from 2 nd generation Asians in the US, have inflammation that is much more often noneosinophilic [5][6][7]. These two subtypes of NP show different levels of inflammatory cell accumulation and remodeling pattern. Neutrophilic infiltration in non-eosinophilic NP (NE-NP) is often associated with glandular hypertrophy and subsequent fibrosis whereas eosinophils induce edematous changes [8]. NE-NP showed different immunologic characteristics and pathologic mechanism compared with E-NP [9][10][11][12] so that age-related immunologic changes would affect disease progression in a different manner. However, up to date, age-related immunologic changes and its clinical implication remained poorly understood in NE-NP. Therefore, the investigation of age-related differences in NE-NP may provide novel clinical implications to clinicians who are treating NP.

Materials and Methods Subjects
One hundred fifty-three study subjects were studied, including 20 controls who underwent sinonasal surgery for unrelated reasons (e.g., endoscopic skull base surgery) without a history of nasal diseases and 133 chronic rhinosinusitis (CRS) patients. Patients were enrolled based on medical chart review (Table 1). CRS diagnoses were based on personal medical history, physical examination, nasal endoscopy, and CT findings of the sinuses according to the European position paper on rhinosinusitis and nasal polyps (EPOS) 2012 guidelines [13]. The presence of NP was confirmed by endoscopic examination. The Lund-MacKay sinus CT scoring system was used as an objective measure of the severity of the disease [14]. The diagnosis of asthma and aspirin sensitivity was performed by an allergist based on history taking, lung function and challenge tests. Aspirin sensitivity was excluded from this study. We evaluated nasal tissue samples such as uncinate process (UP) tissues or NP tissues from patients with CRS without NP (CRSsNP) or CRSwNP, or control subjects. Patients who had taken oral or topical steroids and oral antibiotics within 4 weeks prior to sample collection were excluded from this study. NP were divided into E-NP and NE-NP depending on whether the tissue eosinophils exceeded 10% of the total inflammatory cells [7,15]. Twenty-eight subjects with E-NP and 20 patients with NE-NP were followed up to out-patient clinic at 12 months after surgery. They underwent endoscopic examination for evaluating Lund-Kennedy endoscopic scores [16]. All patients provided written informed consent, and this study was approved by the Institutional Review Board of Boramae Medical Center, Seoul, Korea.

Immunohistochemical staining (IHC)
Initially, the authors investigated the influence of age on inflammatory cell distribution in CRS subjects, using IHC. IHC staining was performed by using the polink-2 plus polymerized horseradish peroxidase (HRP) broad DAB Detection System (Golden Bridge International Labs, WA). Briefly, after deparaffinization, the sections were incubated in 3% hydrogen peroxide for endogenous peroxidase inhibition and microwave-treated in 10 mmol/L citrate buffer (pH 6.0) for heatinduced epitope retrieval. The sections were incubated for 60 min at room temperature with each primary antibody, which included mouse anti-human eosinophil major basic protein (EMBP; 1:100; Merck Millipore, Darmstadt, Germany), mouse anti-human mast-cell tryptase (1:500; Abcam, Cambridge, UK), and anti-human neutrophil elastase (HNE) (1:100; Abcam, Cambridge, UK). A proteinase treatment with 0.1% trypsin for 15 minutes in water was performed prior to blocking and antibody staining, particularly during EMBP staining. The sections were incubated in broad antibody enhancer and polymer-HRP for rabbit and mouse antibodies and then stained with the DAB Detection System. Finally, slides were counterstained with hematoxylin. The positive cells in epithelia, glands, and submucosa were counted in the densest five visual fields (400×) by two independent observers, and the average of the resulting scores was used.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA). In this study, the post hoc two-tailed Mann-Whitney U-test for unpaired comparisons was used. Test for normality was confirmed by Kolmogorov-Smirnov test. The Spearman correlation coefficient was utilized to determine variable relationship because the data were not normally distributed. Partial correlation analysis was also performed to measure the degree of association between postoperative endoscopic score and age when the effect of infiltrated neutrophils was eliminated. A P value of less than 0.05 was considered statistically significant.

Age-related decline of neutrophil-associated cytokines in noneosinophilic nasal polyps
Up-regulation of cytokines that neutrophils produce or response to [18], including IL-17A and IL-23, was negatively correlated with age in NE-NP at the protein or mRNA levels ( , whereas there was no correlation in E-NP and UP from controls (data not shown) and CRS subjects (Fig 1 and S2 Fig). However, protein levels of IL-17A did not show age-related decline (Fig 1G), which might be affected by small number of study population. Therefore, authors took UP tissue, which is the ethmoidal mucosa near the site at which polyps form, and NP tissue from the same patient, then, subtracted cytokine values of UP from those of NP (NP-UP) to examine the decline of IL-17A and IL-23 with age. Interestingly, these subtracted values were decreased to minus with aging in neutrophils and its associated cytokines ( . These findings imply that neutrophilic decline with aging was prominent especially in NE-NP tissues compared to other nasal tissues.

No age-related changes in blood neutrophil and neutrophil-recruiting chemokines
To elucidate the mechanism of the age-related decline of neutrophils in NE-NP, the number of neutrophils in circulating blood, neutrophil-attracting chemokines such as CXCL-1, CXCL-2 and CXCL-8, and CXCR-2, a receptor for CXCL-1 and CXCL-2 in NP tissue were analyzed. Interestingly, there were no age-related differences in peripheral blood neutrophil counts and chemokine levels in subjects with CRS with NP (Figs 2D and 3A-3E), but age-related difference exists in the expression of CXCR2 in NE-NP, but not in E-NP (Fig 3F and 3G).
Postoperative endoscopic score has negative correlations with age in NE-NP To investigate the clinical impact of the age-associated decline of neutrophils in NE-NP, the association of postoperative prognosis with age, using Lund-Kennedy endoscopic score, was assessed. Postoperative endoscopic score showed significant negative correlations with age ( Fig  4A, R = -0.5550, P<0.05). When each group was divided into young-aged (18-39), middleaged (40-59) and elderly (60-81) groups, postoperative endoscopic score was statistically significantly decreased in elderly patients with non-eosinophilic NPs (Fig 4B, P<0.01). Partial correlation analyses were performed with statistical adjustment of the number of HNE positive cells to investigate their correlation when the effect of neutrophils was eliminated. Postoperative endoscopic scores were no longer significantly related to age (data not shown). These findings suggest that the decline of neutrophils with age may influence on endoscopic scores related to postoperative prognosis.

Discussion
We recently reported that elderly CRS patients have higher CT scores and show higher prevalence of asthma in a group of patients in Chicago, which would be mainly eosinophilic NPs [3,4]. In contrast to NPs of Western countries, earlier studies reported that elderly NPs showed better objective surgical outcomes and lower epithelial proliferative ability, compared with younger age groups in Korean population [19,20]. However, previous study design had a limitation that the subjects were not divided into each subtype of CRS and they did not investigate age-associated immune changes. For the first time, this study showed that neutrophilic infiltration was decreased with aging in NE-NP. Considering that neutrophil numbers in the peripheral blood and neutrophil-recruiting chemokines in the polyp tissues did not vary with age, aging may affect neutrophil survival or responsiveness rather than recruitment. CXCL-8-induced activation of neutrophils causes delayed apoptosis and enhanced production of cytokines and reactive oxygen species [21]. However, CXCL-8-stimulated neutrophils from the elderly did not show enhanced MPO and elastase activity, meaning poor responsiveness to CXCL-8 in elderly neutrophils [22]. Additionally, neutrophils from the elderly subjects were less protected from apoptosis when stimulated with LPS, IL-2 and GM-CSF, than neutrophils from younger ones [23]. In line with previous reports, neutrophil-producing enzymes such as MPO and neutrophilassociated cytokines such as IL-17A and IL-23 were significantly decreased with aging, although there was no difference in the expression of CXCL-8 in this study. Functional defect of neutrophils in the elderly NE-NP may be associated with inflammatory involution, taking it into account that neutrophils are one of main inflammatory cells in NE-NP. Recent study using a large all-payer database showed that patients >65 were three times more likely to have a major complication of endoscopic sinus surgery such as skull base, orbital and hemorrhagic injuries, compared to younger adults [24]. Additionally, elderly patients usually have more complicated underlying diseases and need more time to restore to normal life after surgery. The optimal surgical management of nasal polyps has not yet been established [13]. The efficacy of procedures may well be dependent on a variety of factors including disease extent and immunologic characteristics. Therefore, the specific details of the procedures performed need be considered carefully. Complete "full house" surgery can be recommended for nasal polyposis if the elderly patients have no comorbidities in general. However, from our data showing spontaneous involution pattern of disease extent and neutrophilic infiltration in the elderly patients with NE-NP, strategy for maximal medical treatment may be an option in these elderly NE-NP patients who are at high risk for surgical complication. Moreover, based on our data demonstrating a better postoperative course in elderly patients with NE-NP compared to the non-elderly, strategy for "minimally invasive surgery such as simple polypectomy" might be suggested in elderly NE-NP patients who have high risk for long-time surgery due to medical conditions such as heart diseases.
Lastly, this study failed to demonstrate age-related changes in E-NP in Korean population, while previous studies showed that elderly patients with Western NP, majority of which is E-NP, have higher prevalence of asthma and CT scores regardless of decreased ECP levels [3,4]. Possible explanation is that E-NP in Asian population showed somewhat different characteristics compared to Western E-NP. Previous studies demonstrated asthma prevalence of 4 to 26.9% and atopy of 21 to 44.3% in E-NP of East Asia, compared with over 50% asthma and atopy rates in the U.S. [7,8,25,26]. Majority of Asian NP is non-eosinophilic. Furthermore, E-NP in Asians seems less eosinophilic and less prevalent comorbidities of allergic diseases than those in Westerns, implying a different or less severe inflammatory mechanism compared with Western NP. Our study population showing asthma rate of 9.3% and atopy of 47% is consistent with previous Asian reports. Therefore, further investigation would be valuable regarding the endotype of E-NP according to geographic distribution.
In summary, this study demonstrates that elderly patients with NE-NP have lower number of neutrophilic infiltration and lower expression neutrophil-associated cytokines compared with the non-elderly. These findings correlated with better postoperative course.