Differences in initial immunoprofiles between recurrent and nonrecurrent chronic rhinosinusitis with nasal polyps Thibaut Van Zele, M.D., Ph.D., Gabriele Holtappels, Philippe Gevaert, M.D., Ph.D., and Claus Bachert, M.D., Ph.D.
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ABSTRACT
Background: Surgery for chronic rhinosinusitis with nasal polyps (CRSwNPs) often fails because of recurrence of disease. So far, we do not know if specific cytokine profiles are linked to recurrence after functional endoscopic sinus surgery (FESS) or can predict recurrence. In this study we investigate the cytokine profile in CRSwNPs that underwent FESS for the first time and recurrent CRSwNPs. Methods: Tissue samples (n ⫽ 21) of CRSwNP patients with no recurrence after the first surgery were randomly selected out of 131 primary FESS surgeries and compared with tissue samples (n ⫽ 15) from patients who had a first and second surgery because of recurrence. Interleukin (IL)-1beta, IgE, specific IgE, IL-5, interferon (IFN) gamma, IL-6, IL-17, transforming growth factor (TGF) beta1, and myeloperoxidase were measured on tissue homogenates. Results: Levels of IgE, specific IgE to Staphylococcus aureus enterotoxin, eosinophilic cationic protein (ECP), and IL-5 were significantly increased in recurrent versus nonrecurrent CRSwNPs at the moment of the first surgery, whereas IL-17, IL-6, TGF-beta1,and IL-1beta did not show any significant difference. IFN-gamma protein levels were significantly higher in nonrecurrent CRSwNPs. The odds ratio for recurrence of CRSwNPs was reduced to 0.029, if IFN-gamma was present in tissue homogenates. Asthma and aspirin intolerance were significantly more frequent in the recurrent CRSwNPs compared with nonrecurrent CRSwNPs. Discussion: Nonrecurrent and recurrent CRSwNPs needing revision surgery have different types of inflammatory patterns. Nonrecurrent CRSwNPs exhibits a mixed pattern of T helper (Th) cytokines with significant higher levels of IFN-gamma and lower concentrations of IgE, ECP, and IL-5 as compared with recurrent CRSwNPs that had a predominant Th2 type of inflammation. (Am J Rhinol Allergy 28, 192–198, 2014; doi: 10.2500/ajra.2014.28.4033)
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hronic rhinosinusitis (CRS) is an inflammatory disease of the upper airways that affects 10–15% of U.S. and European populations.1 It causes considerable impairment of performance and loss of quality of life, as well as high socioeconomic costs. A clinical definition has been postulated based on symptoms, duration of symptoms, and nasal endoscopy.2 This clinical characterization is intended to cover and accurately describe the clinical manifestations of this disease. In this perspective, CRS is now differentiated into CRS with nasal polyps (CRSwNPs) or CRS without NPs (CRSsNPs) based on nasal endoscopy. However, this definition does not include the recent pathological or immunologic insights. Based on remodeling and inflammatory mediator profiles we could identify distinct disease entities within the CRSwNPs group. In white subjects CRSwNPs are considered to be orchestrated by T helper (Th) 2 cells, with interleukin (IL)-5 as major cytokine, resulting an in eosinophilic type of inflammation, associated with local IgE formation.3,4 In contrast to white subjects, it was reported that in Chinese patients CRSwNPs showed a Th1/Th17 polarization with low eosinophilic cationic protein (ECP) levels biased toward a neutrophilic inflammation.5 All of these features add to the complexity of CRSwNPs and raise the need for careful clinical and immunologic phenotyping of these patients. The treatment of CRSwNPs is a therapeutic challenge. Over time, endoscopic sinus surgery has evolved to be the treatment of choice when conservative treatment has failed. Studies have shown
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From the Upper Airway Research Laboratory, and Department of Otorhinolaryngology, Ghent University, Belgium Funded by a postdoctoral grant to T Van Zele from the Flemish Scientific Research Board, FWO, and grants to C Bachert, from the Flemish Scientific Research Board (FWO Nr. A12/5-HB-KH3 and G.0436.04), the Global Allergy and Asthma European Network (GA2LEN), and the Interuniversity Attraction Poles program (IUAP), Belgian State–Belgian Science Policy P6/35 The authors have no conflicts of interest to declare pertaining to this article Address correspondence to Thibaut Van Zele, M.D., Ph.D., Upper Airway Research Laboratory, and Department of Otorhinolaryngology, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium E-mail address: [email protected] Copyright © 2014, OceanSide Publications, Inc., U.S.A.
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that endoscopic sinus surgery can result in a prolonged reduction of nasal symptoms and improvement in quality of life. However, regardless of the surgical technique used a considerable number of patients will experience a recurrence. Recurrence rates range from 4 to 60%. Aspirin sensitivity, bronchial asthma, and eosinophil density9 are well-known factors associated with polyp recurrence. However, with an overall recurrence rate of 60% after endoscopic sinus surgery and a large heterogeneity in the recurrence rate within the group of CRSwNPs, it is clear that not all clinical and immunologic factors associated with recurrence are known. So far, little attention has been given to the immunologic phenotyping in respect to recurrence rate after endoscopic sinus surgery for CRSwNPs. In this study we aimed to investigate the inflammatory profile in patients with CRSwNPs who underwent surgery for the first time and if a specific inflammatory profile is associated with recurrence or if this inflammatory profile changes over time.
MATERIALS AND METHODS Patients Between 2001 and 2008, 234 white patients underwent functional endoscopic sinus surgery (FESS) for CRSwNPs. Ome hundred thirtyone patients were primary cases with no FESS in the medical history; of those 32 needed a second surgery because of massive recurrence of NPs after an average period of 4.75 years (Table 1). The indication for a second surgery was unsatisfactory symptom control after maximal medical treatment with visible polyposis bilaterally on endoscopic examination. In 15 of 32 patients sufficient nasal tissue was available for both the initial and the revision surgery for this study. A second group of NP patients with no recurrence after the first surgery was selected out of the 131 primary FESS surgeries (n ⫽ 21) based on the absence of recurrent polyps at subsequent outpatient visits with a median follow-up time of 6.33 years. In total 29 patients of the 131 patients were lost to follow-up. All nasal tissue was obtained during routine endonasal surgery in consecutive patients scheduled for surgical intervention unrelated to the study. The diagnosis of sinus disease was based on history, clinical examination, nasal endoscopy, and computed
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Table 1 Demographic characteristics of patients with CRSwNPs CRSwNPs No Recurrence (n ⴝ 21)
CRSwNPs Recurrence (n ⴝ 15)
Statistical Test and Result, p Values
47.55 (3.87) 6/21 9/21 5/21 0/21 0/21 6.33 (0.59) 16.5 (4.5) 5 (3) 2 (1) 1 (1) 2 (1) 0 (0)
50.23 (4.33) 13/15 4/15 11/15 8/15 1/15 4.75 (0.62) 23 (7) 5 (1.5) 3 (1) 0.5 (05) 3 (1.25) 0 (0)
0.94 0.32 0.67 0.001 ⬍0.0001 0.21 0.44 0.17 0.13 0.26 0.80 0.43 0.66
Age (median), yr Gender (M; n) Positive skin-prick test (n) Asthma (n) Aspirin intolerance (n) Chronic obstructive pulmonary disease (n) Years between surgery or last follow-up (SEM) CT Polyp score (Davos) Nasal congestion Rhinorrhea Loss of smell Headache
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Data are expressed as medians and interquartile rang. The level of significance was obtained by using the Fisher’s exact or 2⫺test for categorical outcomes and the Mann–Whitney test for quantitative outcomes and set at an ␣ ⫽ 0.05. CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps.
tomographic (CT) scanning of the sinuses all in accordance with the EP3OS guidelines.2 All patients in both the recurrent and the nonrecurrent CRSwNP group received the same postoperative treatment, which includes nasal saline douching, ointment, and intranasal corticosteroids as an ongoing treatment. The atopic status was evaluated by skin-prick tests to common inhalant allergens. A chest physician confirmed the diagnosis of asthma. None of the subjects used oral or nasal corticosteroids or antibiotic treatment 4 weeks before surgery. Approval by the Ethics Committees of Ghent University Hospital, Belgium, was obtained and written informed consent was obtained from each patient before inclusion in the study.
The NP tissue was processed according to methods already published.7 Briefly, snap-frozen tissues were added to 1 mL of 0.9% NaCl solution per every 0.1 g of tissue. The tissue was then homogenized with a mechanical homogenizer (B. Braun-Melsungen, Melsungen, Germany) at 1000 rpm for 5 minutes on ice. After homogenization, the suspensions were centrifuged at 3000 rpm for 10 minutes at 4°C, after which the supernatants were separated and stored at ⫺80°C until analysis for cytokines and mediators. Supernatants were assayed for IL-1, IL-5, interferon (IFN) ␥, IL-6, IL-17A, transforming growth factor (TGF) 1, and myeloperoxidase by using commercially available ELISA kits (Quantikine ELISA; R & D Systems, Minneapolis, MN; myeloperoxidase was from Oxis International, Portland, OR). Concentrations of ECP and total and specific IgE to staphylococcal superantigens (mixture of Staphylococcus enterotoxin A, Staphylococcus enterotoxin C, and toxic shock syndrome toxin 1) were measured by using the UniCAP system (Phadia, Uppsala, Sweden).
Statistical Analysis Statistical analysis was performed with SPSS 17 (SPSS, Inc., Chicago, IL). Statistical analysis was performed by the Mann-Whitney U two-tailed test for unpaired comparisons between groups. Paired comparisons within groups were performed with Wilcoxon signedrank test. Baseline variables were analyzed by one way ANOVA test or Fishers’ exact test. The significance level was set at ␣ ⫽ 0.05. After examining the distributions of the variables, logistic regression was used to estimate the risk of recurrence. Variables were also recalculated into tertiles using SPSS 17. Odds ratios (ORs) were calculated using logistic regression analysis.
Clinical Characteristics of Recurrent versus Nonrecurrent CRSwNPs
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RESULTS
Clinical characteristics and disease-specific symptom scores of all patients are summarized in Table 1. Both groups were comparable in terms of age, female/male ratio, atopy (positive skin-prick test), symptom scores, polyp score in the nasal cavity determined by means of endoscopy, and CT score showing that the typical disease characteristics of NPs were equivalent in both groups. However, asthma and aspirin intolerance were significantly more frequent in the recurrent CRSwNPs needing revision surgery compared with nonrecurrent CRSwNPs; 23.8% of nonrecurrent CRSwNPs had a history of asthma and none of those patients had a history of aspirin intolerance whereas 73.3% of recurrent CRSwNPs needing revision surgery had a history of asthma and 53.5% had a history of aspirin intolerance (p ⫽ 0.001 and p ⬍ 0.0001, respectively). In the nonrecurrent CRSwNP group the median follow-up time was 6.33 years and the time to second surgery in the recurrent CRSwNPs was 4.75 years (Table 1).
Inflammatory Mediators in Recurrent versus Nonrecurrent CRSwNPs Concentrations of mediators, cytokines, and IgE in tissue homogenates at the first surgery in recurrent and nonrecurrent CRSwNPs are shown in Fig. 1 and Table 2. Levels of IgE, specific IgE to Staphylococcus aureus enterotoxins (SEs), ECP, and IL-5 were significantly and impressively increased in recurrent CRSwNPs needing revision surgery versus nonrecurrent CRSwNP, whereas IL-17, IL-6, TGF-1, and IL-1 did not show any significant difference in levels of those proteins between both groups. In clear contrast, IFN-␥ protein levels were significantly up-regulated in nonrecurrent versus recurrent CRSwNPs. Cytokine data were also recalculated into tertiles and analyzed for recurrence rates (Table 3). The recurrence rates were significantly higher for the highest tertile levels of specific IgE to SEs (p ⬍ 0.0001), ECP (p ⫽ 0.002), and IL-5 (p ⫽ 0.017) and significantly lower for the highest tertile of IFN-␥ (p ⬍ 0.0001) when compared with the other tertiles. Interestingly, the middle tertile of IL-17 also showed a significant higher number of recurrences (p ⬍ 0.0001). If the inflammatory profiles are examined based on signature cytokines for Th1 (IFN-␥), Th2 (IL-5), Th17 (IL-17), and SE-exacerbated disease (SE-IgE), we observed different inflammatory phenotypes in nonrecurrent CRSwNPs versus recurrent CRSwNPs needing revision sur-
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Figure 1. Measurement of IgE, interferon (IFN) ␥, eosinophil cationic protein (ECP), interleukin (IL)-5, IL-17, and specific IgE to Staphylococcus aureus enterotoxins (SAEs) by ELISA and Unicap on tissue homogenates in chronic rhinosinusitis with nasal polyp (CRSwNP) patients that will have no recurrence and CRSwNP patients that will have a recurrence after the first surgery. Data are expressed in box-and-whisker plots that represent the median, the lower and upper quartile, and the minimum to the maximum value. Statistical analysis was performed using the Kruskal–Wallis test followed by a Mann– Whitney U test. The significance level was set at ␣ ⫽ 0.05.
Table 2 Concentrations of cytokines and inflammatory markers in recurrent and nonrecurrent CRSwNPs
IL-5 (pg/mL) IL-17 (pg/mL) IL-1 (pg/ml) IL-6 (pg/mL) IFN-␥ (pg/mL) MPO (ng/mL) TGF-1 (pg/mL) IgE (kU/L) SE IgE (kU/L) ECP (g/L) ECP/MPO ratio
CRSwNP No Recurrence (n ⴝ 21)
CRSwNP Recurrence (n ⴝ 15)
Nonparametric Group Comparisons, p Values
144.7 (9.9–417.4) 16.5 (12.5–83.0) 31.5 (12.2–162.6) 344.9 (192.6–835.6) 265.4 (64.4–458.1) 2604.2 (1841.9–10091.3) 6859.4 (2777.3–12135.9) 174.6 (105.7–373.7) 1.9 (1.9–4.0) 3077.2 (1973.0–7139.9) 1.8 (0.5–3.7)
482.8 (288.5–1198.7) 12.5 (12.5–12.5) 32.7 (10.0–494.0) 437.8 (260.0–2316.2) 42.9 (42.9–42.9) 2310.4 (1394.8–5362.4) 8281.0 (5454.1–14081.1) 466.9 (242.5–2491.5) 4.3 (1.9–6.9) 10612.0 (6898.6–14740.0) 4.1 (2.8–7.5)
0.005 0.202 0.705 0.438 0.001 0.796 0.461 0.017 0.001 0.001 0.047
Data are expressed as medians and interquartile ranges. Statistical analysis was performed using a Mann–Whitney U test. The significance level was set at ␣ ⫽ 0.05. CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps; IL ⫽ interleukin; IFN ⫽ interferon; MPO ⫽ myeloperoxidase; ECP ⫽ eosinophil cationic protein; SE ⫽ Staphylococcus enterotoxin; TGF-1 ⫽ transforming growth factor 1.
gery (Table 4). Nonrecurrent CRSwNPs rather showed a heterogeneous than a homogeneous Th pattern; 14 of 21 (66.6%) patients have a combination of Th1, Th2, and Th17 cytokine production. In the nonrecurrent CRSwNP group significantly more samples expressed
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IFN-␥ in the polyps compared with recurrent CRSwNPs needing revision surgery. On the other hand, recurrent CRSwNPs needing revision surgery showed a more homogeneous Th pattern. All of these samples were Th2⫹ and only 3 of 15 (20%) had a mixed-type
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The time to recurrence versus the IL-5/IFN-␥ ratio was calculated and is shown in Fig. 2. A Spearman rank correlation analysis gave a nonsignificant correlation between time to recurrence and IL-5/IFN␥ ratio (p ⫽ 0.552; R ⫽ ⫺0.128).
Table 3. Concentration levels of ECP, IL-5, specific IgE to SAEs, IFN-␥, and IL-17 were recalculated and expressed as tertiles
ECP
IL-5
Specific IgE SAE
IFN-␥
IL-17
Tertile
CRSwNPs No Recurrence
CRSwNPs Recurrence
p Value
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
11 5 4 10 6 4 14 3 4 3 6 11 6 4 10
0 7 8 1 6 8 0 7 8 11 2 0 0 13 2
0.002
Inflammatory Patterns in Recurrent CRSwNP Undergoing a Second Surgery
0.017
Levels of IgE; specific IgE to SE; ECP; and IL-5, IL-17, IL-6, TGF-1 and IL-1 did not show any significant change in levels between both recurrent CRSwNPs needing revision surgery at the first and second surgery (Fig. 3 and Table 5). If the inflammatory profiles are examined based on signature cytokines for Th1 (IFN-␥), Th2 (IL-5), Th17 (IL-17), and SE-exacerbated disease (SE-IgE), we observed that all samples remained Th2⫹ (100%); however, significantly more recurrent CRSwNPs became Th17⫹ at the second surgery (p ⫽ 0.02; Table 5). If Th17⫹ with Th17⫺ samples at second surgery were compared we observed that Th17⫹ recurrent CRSwNPs were associated with a significant decrease in ECP and IL-5 (p ⫽ 0.015 and p ⬍ 0.05, respectively) and a significant increase in IL-1 and IL-6 (p ⬍ 0.05; Table 6).
⬍0.0001 ⬍0.0001 ⬍0.0001
Table 4 Cytokine profile at first surgery in nonrecurrent vs recurrent CRSwNPs
IL-5 IFN-␥ IL-17 Mixed Th pattern
CRSwNPs Recurrence (n ⴝ 15)
Statistics 2
14/21 15/21 8/21 14/21
15/15 1/15 2/15 3/15
0.013 (6.21) ⬍0.0001 (14.9) 0.102 (2.67) 0.006 (7.65)
Values represent total number of samples with a cytokine concentration above the detection limit. The level of significance was obtained by using the Fisher’s exact or 2-test for categorical variables and set at a value of ␣ ⫽ 0.05. CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps; IL ⫽ interleukin; IFN ⫽ interferon; Th ⫽ T helper.
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inflammation (Th1–Th2 or Th2–Th17). In nonrecurrent CRSwNPs a significant higher number of patients showed a mixed pattern compared with recurrent CRSwNPs (p ⫽ 0.006; Table 4). ORs for recurrence were calculated for different cytokines and clinical characteristics. The OR for recurrence of NPs was found to be low in IFN-␥⫹ (OR, 0.047; CI, 0.0079–0.29) polyps. High ORs for recurrence were found if samples were IL-5⫹ (OR, 6.4; CI, 0.68–60.31) or were specific IgE to SE positive (OR, 2.667; CI, 0.661–10.751); however, the CI includes one that makes this OR just not significant. CRSwNPs that clinically had a history of asthma or aspirin intolerance also had high ORs for recurrence (OR, 8.8 and CI, 1.92–40.34, and OR, 22.8 and CI, 2.41–216.87, respectively). In recurrent CRSwNPs needing revision surgery patients with aspirin intolerance or asthma had significant higher levels of ECP compared with recurrent CRSwNPs without aspirin intolerance or asthma (online supplement Table E1).
Here, we show for the first time that nonrecurrent and recurrent CRSwNPs needing revision surgery have different types of inflammatory patterns. Nonrecurrent CRSwNPs exhibit a mixed pattern of Th cytokines with significantly higher levels of IFN-␥, whereas recurrent CRSwNPs needing revision surgery at the time of first surgery express a predominant Th2-type inflammation with increased concentrations of IL-5, ECP, IgE, and SE-IgE. The type of Th inflammation can predict whether a patient is likely to have a recurrence necessitating a second surgical intervention. In this study the expression of IL-5 protein within the polyp and presence of SE-IgE is linked to a recurrence after the first surgery whereas IFN-␥ is linked to no recurrent disease after the first surgery. The measurement of cytokines in mucosal samples has previously been shown to differentiate disease subtypes of CRS.2 It is estimated that ⬃83% of all white people with CRSwNPs are IL-5⫹, which was identified as the main positive determinant for eosinophilic inflammation in CRSwNPs.11 If we combine recurrent and nonrecurrent CRSwNPs in this study, 80.5% of polyps are IL-5⫹; however, in the subgroup of recurrent CRSwNPs needing revision surgery the frequency of a IL-5⫹ polyps is 100%, significantly higher compared with nonrecurrent CRSwNPs. Subsequently, there is also a significantly higher concentration of IL-5 in the recurrent group. These findings are in line with our previously published results that high IL-5–containing polyp tissues with either a lack of IL-17 or a lack of IFN-␥ are prone to develop the most severe eosinophilic inflammation with asthma comorbidity within the CRSwNPs.5 From this study it is clear that Th2-type inflammation and the associated eosinophilic inflammation are a major risk factor for recurrence and more severe disease spectrum. However, in other studies results about the relation between high eosinophilic inflammation and recurrence seems contradicting. Tosun et al.9 showed that eosinophil-rich NPs have a higher postoperative recurrence rate and the eosinophil density of NPs can be used to get an estimate of the postoperative recurrence risk. However, in a study performed by Eweiss the degree of eosinophilia in NPs did not differ significantly between patients showing recurrence and those not showing recurrence. However, they indicated that the degree of vascular cell adhesion molecule 1 expression in NPs was significantly higher in patients showing recurrence compared with those not showing recurrence. High levels of vascular cell adhesion molecule 1 expression in the polyp tissue, particularly those above 43.37% of the blood vessels, can thus be considered as an indicator of high tendency for recurrence.12 Recent results from our group show that the three major T-effector cell cytokines (IFN-␥, IL-5, and IL-17) also can coexist in the upper airway mucosa of a patient.11 In this study we could distinguish Th2,
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CRSwNPs No Recurrence (n ⴝ 21)
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DISCUSSION
Values represent total number of samples with levels in the first, second, or third tertile. The level of significance was obtained by using the Fisher exact or 2-test for categorical variables and set at a value of ␣ ⫽ 0.05. CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps; IL ⫽ interleukin; IFN ⫽ interferon; ECP ⫽ eosinophil cationic protein; SAE ⫽ Staphylococcus aureus enterotoxin.
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Th2/Th1, Th2/Th17, and Th2/Th1/Th17 NP subgroups. Here, we show that their presence and pattern differentiates mucosal inflammation and also affects the clinical outcome in terms of recurrence. Nonrecurrent CRSwNPs show a mixed pattern of T-cell effector cytokines. These patients with a mixed pattern of T-effector cell cytokines and especially if associated with the expression of IFN-␥ were
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Figure 2. Time to recurrence expressed in months versus ratio of interleukin (IL)-5 and interferon (IFN) ␥ in the recurrent chronic rhinosinusitis with nasal polyp (CRSwNP) needing revision surgery group.
Figure 3. Measurement of interleukin (IL)-5, eosinophil cationic protein (ECP), IL-6, and IL-1 by ELISA and Unicap on tissue homogenates in chronic rhinosinusitis with polyp (CRSwNP) patients with a recurrence needing revision surgery at their first and second surgery. At the second surgery, CRSwNP are subdivided into IL-17⫹ and IL-17⫺ CRSwNPs. Data are expressed in box-and-whisker plots that represent the median, the lower and upper quartile, and the minimum to the maximum value. Statistical analysis was performed using paired Wilcoxon signed-rank test. The significance level was set at ␣ ⫽ 0.05.
less likely to have a recurrence after the first surgery; this is reflected by an OR of 0.047 (CI, 0.0079–0.29) if IFN-␥ is present. Mucosal inflammation in NPs orchestrated by Th2 cytokines is characterized by an increased eosinophilic inflammation and formation of IgE antibodies and this phenotype is associated with comorbid asthma in patients with NPs.11 If IL-17 concentrations are calculated
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Table 5 Concentrations of cytokines and inflammatory markers in patients who underwent two FESS surgeries between 2001 and 2008 CRSwNP with Recurrence
First Surgery (n ⴝ 15)
Second Surgery (n ⴝ 15)
p Value
IL-5 (pg/mL) IL-17 (pg/mL) IL-1 (pg/mL) IL-6 (pg/mL) IFN-␥ (pg/mL) MPO (ng/mL) TGF-1 (pg/mL) IgE (kU/L) SE IgE (kU/L) ECP (g/L)
482.8 (288.5–1198.7) 12.5 (12.5–12.5) 32.7 (10.0–494.0) 437.8 (260.0–2316.2) 42.9 (42.9–42.9) 2310.4 (1394.8–5362.4) 8281.0 (5454.1–14081.1) 466.9 (242.5–2491.5) 4.3 (1.9–6.9) 10612.0 (6898.6–14740.0)
369.0 (48.5–665.3) 14.1 (12.5–81.4) 117.6 (53.3–1043.6) 1200.6 (679.0–3215.2) 42.9 (42.9–42.9) 7442.6 (2053.4–11942.4) 7008.1 (5079.2–13890.0) 863.5 (415.2–2581.7) 4.4 (1.9–10.1) 9617.4 (3602.5–12192.1)
0.140 0.214 0.211 0.156 0.593 0.05 0.925 0.334 0.508 0.532
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Comparison of the tissue homogenates of the first surgery vs second surgery. Data are expressed as medians and interquartile ranges. Statistical analysis was performed using a Mann–Whitney U test. The significance level was set at ␣ ⫽ 0.05. FESS ⫽ functional endoscopic sinus surgery; CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps; IL ⫽ interleukin; IFN ⫽ interferon; MPO ⫽ myeloperoxidase; ECP ⫽ eosinophil cationic protein; SE ⫽ Staphylococcus enterotoxin; TGF-1 ⫽ transforming growth factor 1. Table 6 Cytokine profile at second surgery compared with first surgery CRSwNP with recurrence
First surgery (n ⴝ 15)
Second surgery (n ⴝ 15)
p Value
IL-5 IFN-␥ IL-17 Mixed Th pattern
15/15 1/15 2/15 3/15
15/15 1/15 8/15 8/15
1.00 (0) 1.00 (0) 0.02 (5.40) 0.058 (3.59)
Values represent total number of samples with a cytokine concentration above the detection limit. The level of significance was obtained by using the Fisher exact or 2⫺test for categorical variables and set at a value of ␣ ⫽ 0.05. CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps; IL ⫽ interleukin; IFN ⫽ interferon; Th ⫽ T helper.
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into tertiles we show that a significantly higher number of CRSwNP patients with an average concentration of IL-17 have a recurrence when compared with low and high concentrations of IL-17. Apart from the expression of T-cell effector cytokines, CRSwNPs have a specific remodeling pattern. This remodeling is regulated by diverse mediators among which TGF-1 takes a central role through the attraction and induction of proliferation of fibroblasts and the up-regulation of extracellular matrix synthesis. Here, we could not show any difference in TGF-1 concentrations between recurrent and nonrecurrent CRSwNPs, suggesting that remodeling factors probably play a role, especially in the initiation of disease, as recently confirmed for CRSsNPs.13 However, TGF- does not seem to affect the later outcome or recurrences. We also show for the first time that the inflammatory pattern of CRSwNPs can change over time. In recurrent CRSwNPs needing revision surgery we observed that significantly more CRSwNPs became IL-17⫹ and this was associated with a significant increase in IL-6 and IL-1 and a significant decrease in IL-5 and ECP at the time of the second surgery. Th17 cells, through IL-17, induce the production of proinflammatory mediators, such as IL-1, and induce IL-6 and IL-8 secretion in fibroblasts and endothelial and epithelial cells. Of interest, the induction of IL-17 is associated with a decrease in IL-5 and ECP, suggesting a reduction of the eosinophilic inflammation; this change could partially account for the success of surgery, although this can only be assumed until proven in the patients without revision surgery. In our study clinical symptoms (nasal obstruction and loss of smell) and results of nasal endoscopy (typical appearance of polyps) and sinus CT scanning were perfectly comparable in the two polyp groups. There was no role for allergy as a predictive factor for
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recurrence. The only exceptions from this were asthma comorbidity and aspirin intolerance, which were significantly more frequent in recurrent CRSwNPs needing revision surgery versus nonrecurrent CRSwNPs. The high ORs for recurrence in case of comorbid asthma and aspirin intolerance in this study confirms prior findings that suggested that recurrence rates are higher in patients with asthma alone or with Samter’s triad.14–16 These studies also show that allergy was not a determinant of treatment failure and recurrence.14 Interestingly, CRSwNPs with asthma and especially CRSwNPs with aspirin intolerance have been shown to have highly intense tissue eosinophilia and up-regulated IL-5 and ECP concentrations. These inflammatory factors were also associated with recurrences as shown in the immunologic analyses of our CRSwNP patients. The recurrence rates that have been reported vary substantially for several reasons, including the inherent disease heterogeneity of CRSwNPs and CRSsNPs. Mendelsohn found that polyp recurrence rates at 5 years were 45 and 90% for patients with asthma and patients with Samter’s triad, respectively. Revision surgery rates at 5 years were 25 and 37% for patients with asthma and patients with Samter’s triad, respectively.17 Other studies have reported recurrence rates in short mean follow-up periods (⬍5 years) of between 21 and 66% for CRSwNPs. In these studies Samter’s triad, fungal mucin, and initial frontal sinus disease increased the rate of revision surgery. Although CRSwNPs has been considered and treated as one single clinical disease entity, this study underlines that CRSwNPs should be considered as different immunologic and clinical pheno- and endotypes. Categorizing patients into dichotomous groups of CRSwNPs and CRSsNPs has increased our understanding of the underlying mechanisms but it is obvious that this approach does not reflect the true complexity of the disease. The evaluation of inflammatory cell profiles (eosinophils versus neutrophils), the presence of differentiated T-effector cells (Th1, Th2, and Th17), provide us additional relevant information besides the clinical characteristics that are relevant for predicting recurrence after surgery.18 Although surgery may not cure the underlying mucosal disease, it may significantly alleviate symptoms and improve quality of life. Postoperatively, aggressive medical management is necessary to control the mucosal disease in an attempt to prevent or delay polyp recurrence in all patient subgroups. Our findings confirm that nasal polyposis is a recalcitrant condition and especially in patients with Samter’s triad can be difficult to manage, both medically and surgically. For the first time, we provide biomarkers that may allow predicting recurrent disease at the first surgery independent from clinical expression of disease, may indicate patients need for long-term follow-up, and may identify those who require innovative and targeted treatment.
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Hastan, D, Fokkens WJ, Bachert C, et al. Chronic rhinosinusitis in Europe—An underestimated disease. A GA2LEN study. Allergy 66: 1216–1223, 2011. Fokkens WJ, Lund VJ, Mullol J, et al. EPOS 2012: European position paper on rhinosinusitis and nasal polyps 2012. A summary for otorhinolaryngologists. Rhinology 50:1–12, 2012. Van Zele T, Claeys S, Gevaert P, et al. Differentiation of chronic sinus diseases by measurement of inflammatory mediators. Allergy 61: 1280–1289, 2006. Van Zele T, Gevaert P, Holtappels G, et al. Local immunoglobulin production in nasal polyposis is modulated by superantigens. Clin Exp Allergy 37:1840–1847, 2007. Zhang N, Van Zele T, Perez-Novo C, et al. Different types of T-effector cells orchestrate mucosal inflammation in chronic sinus disease. J Allergy Clin Immunol 122:961–968, 2008. Alobid I, Benítez P, Bernal-Sprekelsen M, et al. Nasal polyposis and its impact on quality of life: Comparison between the effects of medical and surgical treatments. Allergy 60:452–458, 2005. Dalziel K, Stein K, Round A, et al. Systematic review of endoscopic sinus surgery for nasal polyps. Health Technol Assess 7:1–159, 2003. Review. Ragab SM, Lund VJ, and Scadding G. Evaluation of the medical and surgical treatment of chronic rhinosinusitis: A prospective, randomised, controlled trial. Laryngoscope 114:923–930, 2004. Tosun F, Arslan HH, Karslioglu Y, et al. Relationship between postoperative recurrence rate and eosinophil density of nasal polyps. Ann Otol Rhinol Laryngol 119:455–459, 2010.
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Bachert C, Gevaert P, Holtappels G, et al. Total and specific IgE in nasal polyps is related to local eosinophilic inflammation. J Allergy Clin Immunol 107:607–614, 2001. Bachert C, Zhang N, Holtappels G, et al. Presence of IL-5 protein and IgE antibodies to staphylococcal enterotoxins in nasal polyps is associated with comorbid asthma. J Allergy Clin Immunol 126:962–968, 2010. Eweiss A, Dogheim Y, Hassab M, et al. VCAM-1 and eosinophilia in diffuse sino-nasal polyps. Eur Arch Otorhinolaryngol 266:377– 383, 2009. Van Bruaene N, C PN, Van Crombruggen K, et al. Inflammation and remodelling patterns in early stage chronic rhinosinusitis. Clin Exp Allergy 42:883–890, 2011. Young J, Frenkiel S, Tewfik MA, and Mouadeb DA. Long-term outcome analysis of endoscopic sinus surgery for chronic sinusitis. Am J Rhinol 21:743–747, 2007. Kim JE, and Kountakis SE. The prevalence of Samter’s triad in patients undergoing functional endoscopic sinus surgery. Ear Nose Throat J 86:396–399, 2007. Vento SI, Ertama LO, Hyto¨nen ML, et al. Nasal polyposis: Clinical course during 20 years. Ann Allergy Asthma Immunol 85:209–214, 2000. Mendelsohn D, Jeremic G, Wright ED, and Rotenberg BW. Revision rates after endoscopic sinus surgery: A recurrence analysis. Ann Otol Rhinol Laryngol 120:162–166, 2011. Van Bruaene N, Denycke L, Perez-Novo CA, et al. TGF-beta; signaling and collagen deposition in chronic rhinosinusitis. J Allergy Clin Immunol 124:253–259 2009. e
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ABSTRACT
Background: Surgery for chronic rhinosinusitis with nasal polyps (CRSwNPs) often fails because of recurrence of disease. So far, we do not know if specific cytokine profiles are linked to recurrence after functional endoscopic sinus surgery (FESS) or can predict recurrence. In this study we investigate the cytokine profile in CRSwNPs that underwent FESS for the first time and recurrent CRSwNPs. Methods: Tissue samples (n ⫽ 21) of CRSwNP patients with no recurrence after the first surgery were randomly selected out of 131 primary FESS surgeries and compared with tissue samples (n ⫽ 15) from patients who had a first and second surgery because of recurrence. Interleukin (IL)-1beta, IgE, specific IgE, IL-5, interferon (IFN) gamma, IL-6, IL-17, transforming growth factor (TGF) beta1, and myeloperoxidase were measured on tissue homogenates. Results: Levels of IgE, specific IgE to Staphylococcus aureus enterotoxin, eosinophilic cationic protein (ECP), and IL-5 were significantly increased in recurrent versus nonrecurrent CRSwNPs at the moment of the first surgery, whereas IL-17, IL-6, TGF-beta1,and IL-1beta did not show any significant difference. IFN-gamma protein levels were significantly higher in nonrecurrent CRSwNPs. The odds ratio for recurrence of CRSwNPs was reduced to 0.029, if IFN-gamma was present in tissue homogenates. Asthma and aspirin intolerance were significantly more frequent in the recurrent CRSwNPs compared with nonrecurrent CRSwNPs. Discussion: Nonrecurrent and recurrent CRSwNPs needing revision surgery have different types of inflammatory patterns. Nonrecurrent CRSwNPs exhibits a mixed pattern of T helper (Th) cytokines with significant higher levels of IFN-gamma and lower concentrations of IgE, ECP, and IL-5 as compared with recurrent CRSwNPs that had a predominant Th2 type of inflammation. (Am J Rhinol Allergy 28, 192–198, 2014; doi: 10.2500/ajra.2014.28.4033)
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hronic rhinosinusitis (CRS) is an inflammatory disease of the upper airways that affects 10–15% of U.S. and European populations.1 It causes considerable impairment of performance and loss of quality of life, as well as high socioeconomic costs. A clinical definition has been postulated based on symptoms, duration of symptoms, and nasal endoscopy.2 This clinical characterization is intended to cover and accurately describe the clinical manifestations of this disease. In this perspective, CRS is now differentiated into CRS with nasal polyps (CRSwNPs) or CRS without NPs (CRSsNPs) based on nasal endoscopy. However, this definition does not include the recent pathological or immunologic insights. Based on remodeling and inflammatory mediator profiles we could identify distinct disease entities within the CRSwNPs group. In white subjects CRSwNPs are considered to be orchestrated by T helper (Th) 2 cells, with interleukin (IL)-5 as major cytokine, resulting an in eosinophilic type of inflammation, associated with local IgE formation.3,4 In contrast to white subjects, it was reported that in Chinese patients CRSwNPs showed a Th1/Th17 polarization with low eosinophilic cationic protein (ECP) levels biased toward a neutrophilic inflammation.5 All of these features add to the complexity of CRSwNPs and raise the need for careful clinical and immunologic phenotyping of these patients. The treatment of CRSwNPs is a therapeutic challenge. Over time, endoscopic sinus surgery has evolved to be the treatment of choice when conservative treatment has failed. Studies have shown
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From the Upper Airway Research Laboratory, and Department of Otorhinolaryngology, Ghent University, Belgium Funded by a postdoctoral grant to T Van Zele from the Flemish Scientific Research Board, FWO, and grants to C Bachert, from the Flemish Scientific Research Board (FWO Nr. A12/5-HB-KH3 and G.0436.04), the Global Allergy and Asthma European Network (GA2LEN), and the Interuniversity Attraction Poles program (IUAP), Belgian State–Belgian Science Policy P6/35 The authors have no conflicts of interest to declare pertaining to this article Address correspondence to Thibaut Van Zele, M.D., Ph.D., Upper Airway Research Laboratory, and Department of Otorhinolaryngology, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium E-mail address: [email protected] Copyright © 2014, OceanSide Publications, Inc., U.S.A.
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that endoscopic sinus surgery can result in a prolonged reduction of nasal symptoms and improvement in quality of life. However, regardless of the surgical technique used a considerable number of patients will experience a recurrence. Recurrence rates range from 4 to 60%. Aspirin sensitivity, bronchial asthma, and eosinophil density9 are well-known factors associated with polyp recurrence. However, with an overall recurrence rate of 60% after endoscopic sinus surgery and a large heterogeneity in the recurrence rate within the group of CRSwNPs, it is clear that not all clinical and immunologic factors associated with recurrence are known. So far, little attention has been given to the immunologic phenotyping in respect to recurrence rate after endoscopic sinus surgery for CRSwNPs. In this study we aimed to investigate the inflammatory profile in patients with CRSwNPs who underwent surgery for the first time and if a specific inflammatory profile is associated with recurrence or if this inflammatory profile changes over time.
MATERIALS AND METHODS Patients Between 2001 and 2008, 234 white patients underwent functional endoscopic sinus surgery (FESS) for CRSwNPs. Ome hundred thirtyone patients were primary cases with no FESS in the medical history; of those 32 needed a second surgery because of massive recurrence of NPs after an average period of 4.75 years (Table 1). The indication for a second surgery was unsatisfactory symptom control after maximal medical treatment with visible polyposis bilaterally on endoscopic examination. In 15 of 32 patients sufficient nasal tissue was available for both the initial and the revision surgery for this study. A second group of NP patients with no recurrence after the first surgery was selected out of the 131 primary FESS surgeries (n ⫽ 21) based on the absence of recurrent polyps at subsequent outpatient visits with a median follow-up time of 6.33 years. In total 29 patients of the 131 patients were lost to follow-up. All nasal tissue was obtained during routine endonasal surgery in consecutive patients scheduled for surgical intervention unrelated to the study. The diagnosis of sinus disease was based on history, clinical examination, nasal endoscopy, and computed
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Table 1 Demographic characteristics of patients with CRSwNPs CRSwNPs No Recurrence (n ⴝ 21)
CRSwNPs Recurrence (n ⴝ 15)
Statistical Test and Result, p Values
47.55 (3.87) 6/21 9/21 5/21 0/21 0/21 6.33 (0.59) 16.5 (4.5) 5 (3) 2 (1) 1 (1) 2 (1) 0 (0)
50.23 (4.33) 13/15 4/15 11/15 8/15 1/15 4.75 (0.62) 23 (7) 5 (1.5) 3 (1) 0.5 (05) 3 (1.25) 0 (0)
0.94 0.32 0.67 0.001 ⬍0.0001 0.21 0.44 0.17 0.13 0.26 0.80 0.43 0.66
Age (median), yr Gender (M; n) Positive skin-prick test (n) Asthma (n) Aspirin intolerance (n) Chronic obstructive pulmonary disease (n) Years between surgery or last follow-up (SEM) CT Polyp score (Davos) Nasal congestion Rhinorrhea Loss of smell Headache
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Data are expressed as medians and interquartile rang. The level of significance was obtained by using the Fisher’s exact or 2⫺test for categorical outcomes and the Mann–Whitney test for quantitative outcomes and set at an ␣ ⫽ 0.05. CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps.
tomographic (CT) scanning of the sinuses all in accordance with the EP3OS guidelines.2 All patients in both the recurrent and the nonrecurrent CRSwNP group received the same postoperative treatment, which includes nasal saline douching, ointment, and intranasal corticosteroids as an ongoing treatment. The atopic status was evaluated by skin-prick tests to common inhalant allergens. A chest physician confirmed the diagnosis of asthma. None of the subjects used oral or nasal corticosteroids or antibiotic treatment 4 weeks before surgery. Approval by the Ethics Committees of Ghent University Hospital, Belgium, was obtained and written informed consent was obtained from each patient before inclusion in the study.
The NP tissue was processed according to methods already published.7 Briefly, snap-frozen tissues were added to 1 mL of 0.9% NaCl solution per every 0.1 g of tissue. The tissue was then homogenized with a mechanical homogenizer (B. Braun-Melsungen, Melsungen, Germany) at 1000 rpm for 5 minutes on ice. After homogenization, the suspensions were centrifuged at 3000 rpm for 10 minutes at 4°C, after which the supernatants were separated and stored at ⫺80°C until analysis for cytokines and mediators. Supernatants were assayed for IL-1, IL-5, interferon (IFN) ␥, IL-6, IL-17A, transforming growth factor (TGF) 1, and myeloperoxidase by using commercially available ELISA kits (Quantikine ELISA; R & D Systems, Minneapolis, MN; myeloperoxidase was from Oxis International, Portland, OR). Concentrations of ECP and total and specific IgE to staphylococcal superantigens (mixture of Staphylococcus enterotoxin A, Staphylococcus enterotoxin C, and toxic shock syndrome toxin 1) were measured by using the UniCAP system (Phadia, Uppsala, Sweden).
Statistical Analysis Statistical analysis was performed with SPSS 17 (SPSS, Inc., Chicago, IL). Statistical analysis was performed by the Mann-Whitney U two-tailed test for unpaired comparisons between groups. Paired comparisons within groups were performed with Wilcoxon signedrank test. Baseline variables were analyzed by one way ANOVA test or Fishers’ exact test. The significance level was set at ␣ ⫽ 0.05. After examining the distributions of the variables, logistic regression was used to estimate the risk of recurrence. Variables were also recalculated into tertiles using SPSS 17. Odds ratios (ORs) were calculated using logistic regression analysis.
Clinical Characteristics of Recurrent versus Nonrecurrent CRSwNPs
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RESULTS
Clinical characteristics and disease-specific symptom scores of all patients are summarized in Table 1. Both groups were comparable in terms of age, female/male ratio, atopy (positive skin-prick test), symptom scores, polyp score in the nasal cavity determined by means of endoscopy, and CT score showing that the typical disease characteristics of NPs were equivalent in both groups. However, asthma and aspirin intolerance were significantly more frequent in the recurrent CRSwNPs needing revision surgery compared with nonrecurrent CRSwNPs; 23.8% of nonrecurrent CRSwNPs had a history of asthma and none of those patients had a history of aspirin intolerance whereas 73.3% of recurrent CRSwNPs needing revision surgery had a history of asthma and 53.5% had a history of aspirin intolerance (p ⫽ 0.001 and p ⬍ 0.0001, respectively). In the nonrecurrent CRSwNP group the median follow-up time was 6.33 years and the time to second surgery in the recurrent CRSwNPs was 4.75 years (Table 1).
Inflammatory Mediators in Recurrent versus Nonrecurrent CRSwNPs Concentrations of mediators, cytokines, and IgE in tissue homogenates at the first surgery in recurrent and nonrecurrent CRSwNPs are shown in Fig. 1 and Table 2. Levels of IgE, specific IgE to Staphylococcus aureus enterotoxins (SEs), ECP, and IL-5 were significantly and impressively increased in recurrent CRSwNPs needing revision surgery versus nonrecurrent CRSwNP, whereas IL-17, IL-6, TGF-1, and IL-1 did not show any significant difference in levels of those proteins between both groups. In clear contrast, IFN-␥ protein levels were significantly up-regulated in nonrecurrent versus recurrent CRSwNPs. Cytokine data were also recalculated into tertiles and analyzed for recurrence rates (Table 3). The recurrence rates were significantly higher for the highest tertile levels of specific IgE to SEs (p ⬍ 0.0001), ECP (p ⫽ 0.002), and IL-5 (p ⫽ 0.017) and significantly lower for the highest tertile of IFN-␥ (p ⬍ 0.0001) when compared with the other tertiles. Interestingly, the middle tertile of IL-17 also showed a significant higher number of recurrences (p ⬍ 0.0001). If the inflammatory profiles are examined based on signature cytokines for Th1 (IFN-␥), Th2 (IL-5), Th17 (IL-17), and SE-exacerbated disease (SE-IgE), we observed different inflammatory phenotypes in nonrecurrent CRSwNPs versus recurrent CRSwNPs needing revision sur-
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Figure 1. Measurement of IgE, interferon (IFN) ␥, eosinophil cationic protein (ECP), interleukin (IL)-5, IL-17, and specific IgE to Staphylococcus aureus enterotoxins (SAEs) by ELISA and Unicap on tissue homogenates in chronic rhinosinusitis with nasal polyp (CRSwNP) patients that will have no recurrence and CRSwNP patients that will have a recurrence after the first surgery. Data are expressed in box-and-whisker plots that represent the median, the lower and upper quartile, and the minimum to the maximum value. Statistical analysis was performed using the Kruskal–Wallis test followed by a Mann– Whitney U test. The significance level was set at ␣ ⫽ 0.05.
Table 2 Concentrations of cytokines and inflammatory markers in recurrent and nonrecurrent CRSwNPs
IL-5 (pg/mL) IL-17 (pg/mL) IL-1 (pg/ml) IL-6 (pg/mL) IFN-␥ (pg/mL) MPO (ng/mL) TGF-1 (pg/mL) IgE (kU/L) SE IgE (kU/L) ECP (g/L) ECP/MPO ratio
CRSwNP No Recurrence (n ⴝ 21)
CRSwNP Recurrence (n ⴝ 15)
Nonparametric Group Comparisons, p Values
144.7 (9.9–417.4) 16.5 (12.5–83.0) 31.5 (12.2–162.6) 344.9 (192.6–835.6) 265.4 (64.4–458.1) 2604.2 (1841.9–10091.3) 6859.4 (2777.3–12135.9) 174.6 (105.7–373.7) 1.9 (1.9–4.0) 3077.2 (1973.0–7139.9) 1.8 (0.5–3.7)
482.8 (288.5–1198.7) 12.5 (12.5–12.5) 32.7 (10.0–494.0) 437.8 (260.0–2316.2) 42.9 (42.9–42.9) 2310.4 (1394.8–5362.4) 8281.0 (5454.1–14081.1) 466.9 (242.5–2491.5) 4.3 (1.9–6.9) 10612.0 (6898.6–14740.0) 4.1 (2.8–7.5)
0.005 0.202 0.705 0.438 0.001 0.796 0.461 0.017 0.001 0.001 0.047
Data are expressed as medians and interquartile ranges. Statistical analysis was performed using a Mann–Whitney U test. The significance level was set at ␣ ⫽ 0.05. CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps; IL ⫽ interleukin; IFN ⫽ interferon; MPO ⫽ myeloperoxidase; ECP ⫽ eosinophil cationic protein; SE ⫽ Staphylococcus enterotoxin; TGF-1 ⫽ transforming growth factor 1.
gery (Table 4). Nonrecurrent CRSwNPs rather showed a heterogeneous than a homogeneous Th pattern; 14 of 21 (66.6%) patients have a combination of Th1, Th2, and Th17 cytokine production. In the nonrecurrent CRSwNP group significantly more samples expressed
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IFN-␥ in the polyps compared with recurrent CRSwNPs needing revision surgery. On the other hand, recurrent CRSwNPs needing revision surgery showed a more homogeneous Th pattern. All of these samples were Th2⫹ and only 3 of 15 (20%) had a mixed-type
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The time to recurrence versus the IL-5/IFN-␥ ratio was calculated and is shown in Fig. 2. A Spearman rank correlation analysis gave a nonsignificant correlation between time to recurrence and IL-5/IFN␥ ratio (p ⫽ 0.552; R ⫽ ⫺0.128).
Table 3. Concentration levels of ECP, IL-5, specific IgE to SAEs, IFN-␥, and IL-17 were recalculated and expressed as tertiles
ECP
IL-5
Specific IgE SAE
IFN-␥
IL-17
Tertile
CRSwNPs No Recurrence
CRSwNPs Recurrence
p Value
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
11 5 4 10 6 4 14 3 4 3 6 11 6 4 10
0 7 8 1 6 8 0 7 8 11 2 0 0 13 2
0.002
Inflammatory Patterns in Recurrent CRSwNP Undergoing a Second Surgery
0.017
Levels of IgE; specific IgE to SE; ECP; and IL-5, IL-17, IL-6, TGF-1 and IL-1 did not show any significant change in levels between both recurrent CRSwNPs needing revision surgery at the first and second surgery (Fig. 3 and Table 5). If the inflammatory profiles are examined based on signature cytokines for Th1 (IFN-␥), Th2 (IL-5), Th17 (IL-17), and SE-exacerbated disease (SE-IgE), we observed that all samples remained Th2⫹ (100%); however, significantly more recurrent CRSwNPs became Th17⫹ at the second surgery (p ⫽ 0.02; Table 5). If Th17⫹ with Th17⫺ samples at second surgery were compared we observed that Th17⫹ recurrent CRSwNPs were associated with a significant decrease in ECP and IL-5 (p ⫽ 0.015 and p ⬍ 0.05, respectively) and a significant increase in IL-1 and IL-6 (p ⬍ 0.05; Table 6).
⬍0.0001 ⬍0.0001 ⬍0.0001
Table 4 Cytokine profile at first surgery in nonrecurrent vs recurrent CRSwNPs
IL-5 IFN-␥ IL-17 Mixed Th pattern
CRSwNPs Recurrence (n ⴝ 15)
Statistics 2
14/21 15/21 8/21 14/21
15/15 1/15 2/15 3/15
0.013 (6.21) ⬍0.0001 (14.9) 0.102 (2.67) 0.006 (7.65)
Values represent total number of samples with a cytokine concentration above the detection limit. The level of significance was obtained by using the Fisher’s exact or 2-test for categorical variables and set at a value of ␣ ⫽ 0.05. CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps; IL ⫽ interleukin; IFN ⫽ interferon; Th ⫽ T helper.
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inflammation (Th1–Th2 or Th2–Th17). In nonrecurrent CRSwNPs a significant higher number of patients showed a mixed pattern compared with recurrent CRSwNPs (p ⫽ 0.006; Table 4). ORs for recurrence were calculated for different cytokines and clinical characteristics. The OR for recurrence of NPs was found to be low in IFN-␥⫹ (OR, 0.047; CI, 0.0079–0.29) polyps. High ORs for recurrence were found if samples were IL-5⫹ (OR, 6.4; CI, 0.68–60.31) or were specific IgE to SE positive (OR, 2.667; CI, 0.661–10.751); however, the CI includes one that makes this OR just not significant. CRSwNPs that clinically had a history of asthma or aspirin intolerance also had high ORs for recurrence (OR, 8.8 and CI, 1.92–40.34, and OR, 22.8 and CI, 2.41–216.87, respectively). In recurrent CRSwNPs needing revision surgery patients with aspirin intolerance or asthma had significant higher levels of ECP compared with recurrent CRSwNPs without aspirin intolerance or asthma (online supplement Table E1).
Here, we show for the first time that nonrecurrent and recurrent CRSwNPs needing revision surgery have different types of inflammatory patterns. Nonrecurrent CRSwNPs exhibit a mixed pattern of Th cytokines with significantly higher levels of IFN-␥, whereas recurrent CRSwNPs needing revision surgery at the time of first surgery express a predominant Th2-type inflammation with increased concentrations of IL-5, ECP, IgE, and SE-IgE. The type of Th inflammation can predict whether a patient is likely to have a recurrence necessitating a second surgical intervention. In this study the expression of IL-5 protein within the polyp and presence of SE-IgE is linked to a recurrence after the first surgery whereas IFN-␥ is linked to no recurrent disease after the first surgery. The measurement of cytokines in mucosal samples has previously been shown to differentiate disease subtypes of CRS.2 It is estimated that ⬃83% of all white people with CRSwNPs are IL-5⫹, which was identified as the main positive determinant for eosinophilic inflammation in CRSwNPs.11 If we combine recurrent and nonrecurrent CRSwNPs in this study, 80.5% of polyps are IL-5⫹; however, in the subgroup of recurrent CRSwNPs needing revision surgery the frequency of a IL-5⫹ polyps is 100%, significantly higher compared with nonrecurrent CRSwNPs. Subsequently, there is also a significantly higher concentration of IL-5 in the recurrent group. These findings are in line with our previously published results that high IL-5–containing polyp tissues with either a lack of IL-17 or a lack of IFN-␥ are prone to develop the most severe eosinophilic inflammation with asthma comorbidity within the CRSwNPs.5 From this study it is clear that Th2-type inflammation and the associated eosinophilic inflammation are a major risk factor for recurrence and more severe disease spectrum. However, in other studies results about the relation between high eosinophilic inflammation and recurrence seems contradicting. Tosun et al.9 showed that eosinophil-rich NPs have a higher postoperative recurrence rate and the eosinophil density of NPs can be used to get an estimate of the postoperative recurrence risk. However, in a study performed by Eweiss the degree of eosinophilia in NPs did not differ significantly between patients showing recurrence and those not showing recurrence. However, they indicated that the degree of vascular cell adhesion molecule 1 expression in NPs was significantly higher in patients showing recurrence compared with those not showing recurrence. High levels of vascular cell adhesion molecule 1 expression in the polyp tissue, particularly those above 43.37% of the blood vessels, can thus be considered as an indicator of high tendency for recurrence.12 Recent results from our group show that the three major T-effector cell cytokines (IFN-␥, IL-5, and IL-17) also can coexist in the upper airway mucosa of a patient.11 In this study we could distinguish Th2,
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Values represent total number of samples with levels in the first, second, or third tertile. The level of significance was obtained by using the Fisher exact or 2-test for categorical variables and set at a value of ␣ ⫽ 0.05. CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps; IL ⫽ interleukin; IFN ⫽ interferon; ECP ⫽ eosinophil cationic protein; SAE ⫽ Staphylococcus aureus enterotoxin.
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Th2/Th1, Th2/Th17, and Th2/Th1/Th17 NP subgroups. Here, we show that their presence and pattern differentiates mucosal inflammation and also affects the clinical outcome in terms of recurrence. Nonrecurrent CRSwNPs show a mixed pattern of T-cell effector cytokines. These patients with a mixed pattern of T-effector cell cytokines and especially if associated with the expression of IFN-␥ were
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Figure 2. Time to recurrence expressed in months versus ratio of interleukin (IL)-5 and interferon (IFN) ␥ in the recurrent chronic rhinosinusitis with nasal polyp (CRSwNP) needing revision surgery group.
Figure 3. Measurement of interleukin (IL)-5, eosinophil cationic protein (ECP), IL-6, and IL-1 by ELISA and Unicap on tissue homogenates in chronic rhinosinusitis with polyp (CRSwNP) patients with a recurrence needing revision surgery at their first and second surgery. At the second surgery, CRSwNP are subdivided into IL-17⫹ and IL-17⫺ CRSwNPs. Data are expressed in box-and-whisker plots that represent the median, the lower and upper quartile, and the minimum to the maximum value. Statistical analysis was performed using paired Wilcoxon signed-rank test. The significance level was set at ␣ ⫽ 0.05.
less likely to have a recurrence after the first surgery; this is reflected by an OR of 0.047 (CI, 0.0079–0.29) if IFN-␥ is present. Mucosal inflammation in NPs orchestrated by Th2 cytokines is characterized by an increased eosinophilic inflammation and formation of IgE antibodies and this phenotype is associated with comorbid asthma in patients with NPs.11 If IL-17 concentrations are calculated
May–June 2014, Vol. 28, No. 3
Delivered by Ingenta to: Economics Dept IP: 141.101.132.233 On: Tue, 28 Jun 2016 17:03:14 Copyright (c) Oceanside Publications, Inc. All rights reserved. For permission to copy go to https://www.oceansidepubl.com/permission.htm
Table 5 Concentrations of cytokines and inflammatory markers in patients who underwent two FESS surgeries between 2001 and 2008 CRSwNP with Recurrence
First Surgery (n ⴝ 15)
Second Surgery (n ⴝ 15)
p Value
IL-5 (pg/mL) IL-17 (pg/mL) IL-1 (pg/mL) IL-6 (pg/mL) IFN-␥ (pg/mL) MPO (ng/mL) TGF-1 (pg/mL) IgE (kU/L) SE IgE (kU/L) ECP (g/L)
482.8 (288.5–1198.7) 12.5 (12.5–12.5) 32.7 (10.0–494.0) 437.8 (260.0–2316.2) 42.9 (42.9–42.9) 2310.4 (1394.8–5362.4) 8281.0 (5454.1–14081.1) 466.9 (242.5–2491.5) 4.3 (1.9–6.9) 10612.0 (6898.6–14740.0)
369.0 (48.5–665.3) 14.1 (12.5–81.4) 117.6 (53.3–1043.6) 1200.6 (679.0–3215.2) 42.9 (42.9–42.9) 7442.6 (2053.4–11942.4) 7008.1 (5079.2–13890.0) 863.5 (415.2–2581.7) 4.4 (1.9–10.1) 9617.4 (3602.5–12192.1)
0.140 0.214 0.211 0.156 0.593 0.05 0.925 0.334 0.508 0.532
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Comparison of the tissue homogenates of the first surgery vs second surgery. Data are expressed as medians and interquartile ranges. Statistical analysis was performed using a Mann–Whitney U test. The significance level was set at ␣ ⫽ 0.05. FESS ⫽ functional endoscopic sinus surgery; CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps; IL ⫽ interleukin; IFN ⫽ interferon; MPO ⫽ myeloperoxidase; ECP ⫽ eosinophil cationic protein; SE ⫽ Staphylococcus enterotoxin; TGF-1 ⫽ transforming growth factor 1. Table 6 Cytokine profile at second surgery compared with first surgery CRSwNP with recurrence
First surgery (n ⴝ 15)
Second surgery (n ⴝ 15)
p Value
IL-5 IFN-␥ IL-17 Mixed Th pattern
15/15 1/15 2/15 3/15
15/15 1/15 8/15 8/15
1.00 (0) 1.00 (0) 0.02 (5.40) 0.058 (3.59)
Values represent total number of samples with a cytokine concentration above the detection limit. The level of significance was obtained by using the Fisher exact or 2⫺test for categorical variables and set at a value of ␣ ⫽ 0.05. CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps; IL ⫽ interleukin; IFN ⫽ interferon; Th ⫽ T helper.
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into tertiles we show that a significantly higher number of CRSwNP patients with an average concentration of IL-17 have a recurrence when compared with low and high concentrations of IL-17. Apart from the expression of T-cell effector cytokines, CRSwNPs have a specific remodeling pattern. This remodeling is regulated by diverse mediators among which TGF-1 takes a central role through the attraction and induction of proliferation of fibroblasts and the up-regulation of extracellular matrix synthesis. Here, we could not show any difference in TGF-1 concentrations between recurrent and nonrecurrent CRSwNPs, suggesting that remodeling factors probably play a role, especially in the initiation of disease, as recently confirmed for CRSsNPs.13 However, TGF- does not seem to affect the later outcome or recurrences. We also show for the first time that the inflammatory pattern of CRSwNPs can change over time. In recurrent CRSwNPs needing revision surgery we observed that significantly more CRSwNPs became IL-17⫹ and this was associated with a significant increase in IL-6 and IL-1 and a significant decrease in IL-5 and ECP at the time of the second surgery. Th17 cells, through IL-17, induce the production of proinflammatory mediators, such as IL-1, and induce IL-6 and IL-8 secretion in fibroblasts and endothelial and epithelial cells. Of interest, the induction of IL-17 is associated with a decrease in IL-5 and ECP, suggesting a reduction of the eosinophilic inflammation; this change could partially account for the success of surgery, although this can only be assumed until proven in the patients without revision surgery. In our study clinical symptoms (nasal obstruction and loss of smell) and results of nasal endoscopy (typical appearance of polyps) and sinus CT scanning were perfectly comparable in the two polyp groups. There was no role for allergy as a predictive factor for
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recurrence. The only exceptions from this were asthma comorbidity and aspirin intolerance, which were significantly more frequent in recurrent CRSwNPs needing revision surgery versus nonrecurrent CRSwNPs. The high ORs for recurrence in case of comorbid asthma and aspirin intolerance in this study confirms prior findings that suggested that recurrence rates are higher in patients with asthma alone or with Samter’s triad.14–16 These studies also show that allergy was not a determinant of treatment failure and recurrence.14 Interestingly, CRSwNPs with asthma and especially CRSwNPs with aspirin intolerance have been shown to have highly intense tissue eosinophilia and up-regulated IL-5 and ECP concentrations. These inflammatory factors were also associated with recurrences as shown in the immunologic analyses of our CRSwNP patients. The recurrence rates that have been reported vary substantially for several reasons, including the inherent disease heterogeneity of CRSwNPs and CRSsNPs. Mendelsohn found that polyp recurrence rates at 5 years were 45 and 90% for patients with asthma and patients with Samter’s triad, respectively. Revision surgery rates at 5 years were 25 and 37% for patients with asthma and patients with Samter’s triad, respectively.17 Other studies have reported recurrence rates in short mean follow-up periods (⬍5 years) of between 21 and 66% for CRSwNPs. In these studies Samter’s triad, fungal mucin, and initial frontal sinus disease increased the rate of revision surgery. Although CRSwNPs has been considered and treated as one single clinical disease entity, this study underlines that CRSwNPs should be considered as different immunologic and clinical pheno- and endotypes. Categorizing patients into dichotomous groups of CRSwNPs and CRSsNPs has increased our understanding of the underlying mechanisms but it is obvious that this approach does not reflect the true complexity of the disease. The evaluation of inflammatory cell profiles (eosinophils versus neutrophils), the presence of differentiated T-effector cells (Th1, Th2, and Th17), provide us additional relevant information besides the clinical characteristics that are relevant for predicting recurrence after surgery.18 Although surgery may not cure the underlying mucosal disease, it may significantly alleviate symptoms and improve quality of life. Postoperatively, aggressive medical management is necessary to control the mucosal disease in an attempt to prevent or delay polyp recurrence in all patient subgroups. Our findings confirm that nasal polyposis is a recalcitrant condition and especially in patients with Samter’s triad can be difficult to manage, both medically and surgically. For the first time, we provide biomarkers that may allow predicting recurrent disease at the first surgery independent from clinical expression of disease, may indicate patients need for long-term follow-up, and may identify those who require innovative and targeted treatment.
American Journal of Rhinology & Allergy
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May–June 2014, Vol. 28, No. 3
Delivered by Ingenta to: Economics Dept IP: 141.101.132.233 On: Tue, 28 Jun 2016 17:03:14 Copyright (c) Oceanside Publications, Inc. All rights reserved. For permission to copy go to https://www.oceansidepubl.com/permission.htm
