Activated pyrin domain containing 3 (NLRP3) inflammasome in neutrophilic chronic rhinosinusitis with nasal polyps (CRSwNP)

Chronic rhinosinusitis with nasal polyps (CRSwNPs) is a common chronic nasal inflammatory disease with high heterogeneity worldwide. The endotypes of CRSwNP are divided into eosinophilic (EOS) infiltration, TH2 response, neutrophilic (NEU) infiltration, and mixed TH2/TH17 responses.1Zhang Y. Gevaert E. Lou H. Wang X. Bachert C. Zhang N. et al.Chronic rhinosinusitis in Asia.J Allergy Clin Immunol. 2017; 140: 1230-1239Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Moreover, in the eosinophilic subjects, 35% showed a mixed eosinophil and neutrophil infiltration.2Wen W. Liu W. Zhang L. Bai J. Li H. Xu G. et al.Increased neutrophilia in nasal polyps reduces the response to oral corticosteroid therapy.J Allergy Clin Immunol. 2012; 129: 1522-1528Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar Detailed mechanisms regarding regulation of different types of inflammatory cells await further investigation. NLR family, pyrin domain containing 3 (NLRP3) inflammasomes characterized as multiprotein signaling complexes play important roles in both the innate and adaptive immune response. The NLRP3 and its downstream IL-1β were identified to be potent inducers of neutrophilic inflammation in various inflammatory diseases.3Haneklaus M. O’Neill L.A. NLRP3 at the interface of metabolism and inflammation.Immunology Rev. 2015; 265: 53-62Crossref PubMed Scopus (205) Google Scholar At the same time, a positive regulation between the NLRP3 and TH2 program was indicated in several independent research studies.4Ting J.P. Harton J.A. NLRP3 moonlights in TH2 polarization.Nat Immunol. 2015; 16: 794-796Crossref PubMed Scopus (14) Google Scholar,5Bruchard M. Rebe C. Derangere V. Togbe D. Vegran F. Ghiringhelli F. et al.The receptor NLRP3 is a transcriptional regulator of TH2 differentiation.Nat Immunol. 2015; 16: 859-870Crossref PubMed Scopus (37) Google Scholar Our study, together with Bruchard et al’s report, showed that NLRP3 was highly expressed in nasal polyps (NPs) from subjects with CRSwNP.6Lin H. Li Z. Lin D. Zheng C. Zhang W. Role of NLRP3 inflammasome in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps.Inflammation. 2016; 39: 2045-2052Crossref PubMed Scopus (9) Google Scholar However, the role of the NLRP3 inflammasome in CRSwNP remains unclear. We therefore evaluated the expression profile of the NLRP3 inflammasome in NPs and examined its relationship with NP endotypes. We enrolled 82 patients with CRSwNP, 32 patients with chronic rhinosinusitis without nasal polyps (CRSsNP), and 35 healthy control subjects to address this issue. The clinical characteristics are listed in Table E1 in this article’s Online Repository at www.jacionline.org. The study protocols were approved by ethics committee boards, and all subjects gave written informed consent. Expression of NLRP3 inflammasomes and the proinflammatory cytokines IL-1β and IL-4 was examined in nasal tissues. In addition, expression of NLRP3 and IL-1β in response to specific inflammatory stimulation was measured in cultured single-cell suspension of NPs. Details of the materials and methods used in this study are presented in the Methods section in this article’s Online Repository at www.jacionline.org. Our results showed that mRNA levels of NLRP3, NALP1, NLRC4, and IL-1β were significantly higher in NPs than in uncinate tissues from control subjects, patients with CRSsNP, or patients with CRSwNP (P < .05; see Fig E1, A-D, in this article’s Online Repository at www.jacionline.org). No significant differences in apoptosis speck protein (ASC) or caspase-1 mRNA expression were observed in NPs and uncinate tissues between patients with CRSsNP and CRSwNP (Fig E1, E and F). To evaluate whether the NLRP3 inflammasome and IL-1β are expressed in different polyp tissue subsets, we determined the presence of eosinophils, neutrophils, NLRP3, and IL-1β in nasal tissues by hematoxylin and eosin for eosinophils and immunohistochemical staining for neutrophils, NLRP3, and IL-1β (Fig 1, A). On the basis of defined cutoff values (see Fig E2 and Table E2 in this article’s Online Repository at www.jacionline.org), we divided the polyp tissues into 4 subtypes: eosinophilic and neutrophilic (EOS+NEU+), noneosinophilic and neutrophilic (EOS−NEU+), eosinophilic and nonneutrophilic (EOS+NEU−), and noneosinophilic and nonneutrophilic (EOS−NEU−). We observed that the staining intensity and the mean number of the NLRP3 inflammasome and IL-1β were significantly increased in polyp tissues from patients with CRSwNP with EOS+NEU+ and EOS−NEU+ subsets than in polyp tissues from patients with CRSwNP with EOS+NEU− and EOS−NEU− subsets (P < .01; Fig 1, A-C). Confirming the histologic findings, we found that polyp tissues from the EOS+NEU+ and EOS+NEU− CRSwNP subsets exhibited significantly higher NLRP3 mRNA and protein levels than those from the EOS+NEU− and EOS−NEU− CRSwNP subsets (P < .001, Fig 1, D and E). Although the mRNA level and precursor protein (pro–IL-1β, p37) of IL-1β were not different between the 4 CRSwNP subsets (Fig 1, F), the activation protein (p17) and the secretion of IL-1β were significantly increased in the EOS+NEU+ and EOS−NEU+ CRSwNP subsets than in the EOS+NEU− and EOS−NEU− CRSwNP subsets (P < .001, Fig 1, E and G). These results indicated that the higher expression of NLRP3 in the EOS+NEU+ and EOS−NEU+ subsets lead to an increase in IL-1β protein expression, whereas this situation was not apparent in the EOS+NEU− and EOS+NEU− CRSwNP subsets. Antigen stimulation and inflammation, which are important pathologic environments for different CRSwNP subsets, can activate the NLRP3 inflammasome. To establish the direct relationship between Toll-like receptor (TLR) agonists, proinflammatory cytokines, and neutrophil infiltration through the NLRP3–IL-1β axis in CRSwNP, we examined the effects of TLR activation and proinflammatory cytokine stimulation on inflammasome complex expression in a cultured single-cell suspension of NPs and performed neutrophil chemotaxis in vitro. As shown in Fig 2, A-J, NLRP3, IL-1β mRNA, and protein levels were significantly increased after stimulating the cultured NP single-cell suspension with TLR agonists and proinflammatory cytokines, especially TLR2, TLR3, TLR4 agonists LPS and polyinosinic:polycytidylic acid (poly I:C) and IL-4 (P < .01). Of note, IL-4, a typical TH2 cytokine, which is beyond our expectations, induced high NLRP3 and promoted subsequent IL-1β secretion in single-cell suspension of NPs as well (P < .01) To clarify which type of cell expresses NLRP3, we determined the cellular localization of NLRP3 by immunofluorescence. Then, we found that NLRP3 was colocalized with CD68 and MUC5AC in the human NP tissue (see Fig E3 in this article’s Online Repository at www.jacionline.org). This finding indicated that the macrophages and epithelial cells were the main source of NLRP3 in the poly tissues. Moreover, we examined the inflammasome complex mRNA or protein levels in the macrophage or epithelial cells from patients with CRSwNP and found that these responded to TLR agonists and proinflammatory cytokines. Then, we found that the TLR agonists (LPS and poly I:C) and proinflammatory cytokines (IL-4) increased the mRNA and protein levels of NLRP3/IL-1β (see Fig E4 in this article’s Online Repository at www.jacionline.org), consistent with the results of the mixed-cell experiments. Next, we found that the mRNA levels of TLR2, TLR3, and TLR4 had the highest expression in polyp tissues from EOS−NEU+, followed by EOS+NEU+, and the lowest expression was in EOS+NEU− and EOS−NEU− CRSwNP, as in our previous studies7Wei Y. Xia W. Ye X. Fan Y. Yang P. Li H. et al.The antimicrobial protein short palate, lung, and nasal epithelium clone 1 (SPLUNC1) is differentially modulated in eosinophilic and noneosinophilic chronic rhinosinusitis with nasal polyps.J Allergy Clin Immunol. 2014; 133: 420-428Abstract Full Text Full Text PDF PubMed Google Scholar (P < .001; see Fig E5, A-C, in this article’s Online Repository at www.jacionline.org). Accordingly, mRNA levels of TH2 cytokines IL-4, IL-5, and IL-13 were significantly higher in EOS+NEU+ and EOS+NEU− polyp tissues than in EOS−NEU+ and EOS−NEU− polyp tissues (P < .001; Fig E5, D-F). However, mRNA expressions of IFN-γ and IL-17a were not significantly different between the 4 CRSwNP subsets (Fig E5, G and H). The results suggested that antigen stimulation was the major factor leading to elevated NLRP3 and IL-1β expressions in EOS+NEU+ and EOS−NEU+ CRSwNP subsets; moreover, IL-4 enhanced this effect in the EOS+NEU+ subset. Furthermore, the supernatant of the NP single-cell treatment with LPS, poly I:C, IL-4, and ATP significantly chemotaxis more neutrophil compared with control, and these trends could be suppressed by small-molecule small-interfering RNA inhibiting NLRP3 or neutralizing antibody against IL-1β (Fig 2, K; see Fig E6, A-C, in this article’s Online Repository at www.jacionline.org; P < .001). Moreover, we did the neutrophils’ chemotaxis by culture supernatant from different treatments with macrophage or epithelial cells from patients with CRSwNP. Then, we found that LPS, poly I:C, and IL-4 also could chemotaxis neutrophils in the macrophage or epithelial cells (see Fig E7 in this article’s Online Repository at www.jacionline.org), consistent with the results of mixed-cell experiments. Inhibition of NLRP3 expression in single-cell suspension of NPs abrogated the effect of LPS, poly I:C, or IL-4 exposure on IL-1β secretion and neutrophil recruitment, indicating that NLRP3 is a central component in both tissue eosinophilia and neutrophilia. Meanwhile, the inhibitor of NLRP3 or abolished downstream IL-1β maturation played a potential role in the control of chronic nasal mucosal inflammation. Because glucocorticoid treatment is recommended as the primary treatment for patients with CRSwNP, we found that the treatment with glucocorticoids significantly downregulated NLRP3 mRNA and IL-1β protein levels and alleviated the chemotactic neutrophils by IL-4 but not for LPS, poly I:C, and ATP (Fig 2, K; see Fig E6, D, and Fig E8 in this article’s Online Repository at www.jacionline.org; P < .001). This finding indicates that glucocorticoid treatment might partially exert its anti-inflammatory effect by inhibiting the NLRP3 inflammasome and IL-1β expressions and reducing local mucosal neutrophilic infiltration. In summary, these studies suggest that the NLRP3 inflammasome and its associated downstream IL-1β were significantly correlated and upregulated in high-neutrophil–infiltrated NPs, including both mixed eosinophil and neutrophil infiltration subjects and singular neutrophil infiltration subjects. The antigen-stimulated high expression of NLRP3 and secretory IL-1β may be the key factor in neutrophilic inflammation of EOS−NEU+ and EOS+NEU+ CRSwNP subsets. Meanwhile, IL-4–induced NLRP3 expression promotes mixed eosinophilic and neutrophilic infiltration in the EOS+NEU+ CRSwNP. Gurung et al8Gurung P. Karki R. Vogel P. Watanabe M. Bix M. Lamkanfi M. et al.An NLRP3 inflammasome-triggered Th2-biased adaptive immune response promotes leishmaniasis.J Clin Invest. 2015; 125: 1329-1338Crossref PubMed Scopus (89) Google Scholar reported that NLRP3 was the trigger of an adaptive TH2-biased immune response upon parasite infection. In contrast, Niebuhr et al’s published data9Niebuhr M. Baumert K. Heratizadeh A. Satzger I. Werfel T. Impaired NLRP3 inflammasome expression and function in atopic dermatitis due to Th2 milieu.Allergy. 2014; 69: 1058-1067Crossref PubMed Scopus (45) Google Scholar from subjects with atopic dermatitis indicated that the function of the NLPR3 inflammasome was impaired in keratinocytes under a dominant TH2 milieu. It is possible that NLRP3 inflammasome signaling exerts different roles in different types of cells, under certain disease settings. IL-4–NLRP3 signaling interactions may amplify IL-1β production in single-cell suspension of NPs and modulate neutrophil chemotaxis, thus contributing to NP pathogenesis in vivo. These studies indicate that the NLRP3 pathway may be used as a therapeutic approach to target a certain CRSwNP subset. This study was approved by the local ethics committee boards, and written informed consent was obtained from each subject. Adult patients with CRS (CRSwNP, n = 82; CRSsNP, n = 32) and 35 control subjects were randomly recruited from the First Affiliated Hospital of Sun Yat-sen University. CRSwNP and CRSsNP diagnoses were made according to the current European position paper on rhinosinusitis and NPs.E1Wen W. Liu W. Zhang L. Bai J. Li H. Xu G. et al.Increased neutrophilia in nasal polyps reduces the response to oral corticosteroid therapy.J Allergy Clin Immunol. 2012; 129: 1522-1528Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, E2Wei Y. Xia W. Ye X. Fan Y. Yang P. Li H. et al.The antimicrobial protein short palate, lung, and nasal epithelium clone 1 (SPLUNC1) is differentially modulated in eosinophilic and noneosinophilic chronic rhinosinusitis with nasal polyps.J Allergy Clin Immunol. 2014; 133: 420-428Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Skin prick tests were performed to evaluate the atopic status of the patients. Asthma was diagnosed by a pneumologist on the basis of disease history and evaluation of airway responsiveness. The polyp tissues were sampled from patients with CRSwNP, and the uncinated tissues were sampled from patients with CRS and control subjects. The demographic data of all subjects enrolled in the study are listed in Table E1. Tissue samples were divided into 3 pieces: the first was frozen in liquid nitrogen and stored at –80°C for subsequent RNA extraction, the second was used for protein extraction, and the third was fixed overnight in a freshly prepared fixative containing 4% paraformaldehyde in PBS (pH 7.4) and embedded in paraffin wax for histologic assessment. In addition, single-cell suspension of human NPs was isolated and cultured from fresh polyp tissues. Different CRSwNP subsets were identified by eosinophilic and/or neutrophilic cells counted in hematoxylin and eosin and neutrophil elastase (NEU) immunohistochemically staining. After counting the number of positive cells, the cutoff value for the eosinophilic phenotype was set at 16 eosinophilic cells/hpf (200×), which is equivalent to the mean value in the noneosinophilic subjects (5.86) plus 2 times the SD (4.60 × 2 = 9.2; cutoff value, 5.86 + 9.2 = 15.06). The cutoff value for the neutrophilic phenotype was set at 17 neutrophilic cells/hpf (200×), which is equivalent to the mean value in the nonneutrophilic subjects (6.37) plus 2 times the SD (4.87 × 2 = 9.74; cutoff value, 6.37 + 9.74 = 16.44). On the basis of defined cutoff values, we divided the polyp tissues into 4 subtypes: EOS+NEU+, EOS−NEU+, EOS+NEU−, and EOS−NEU−. As shown in Table E2 and Figure E2, the median number of eosinophils per hpf in the polyp tissues was 34.4 (interquartile range [IQR], 21.2-49.8) for EOS+NEU+, 4.3 (IQR, 0.6-15.2) for EOS−NEU+, 28.6 (IQR, 18.2-43.2) for EOS+NEU−, and 3.4 (IQR, 0.6-15.8) for EOS−NEU, respectively. The median number of neutrophils per hpf in the polyp was 34 (IQR, 21.8-53.6) for EOS+NEU+, 40.5 (IQR, 25.4-57.8) for EOS−NEU+, 4.4 (IQR, 0.5-16.9) for EOS+NEU−, and 4.7 (IQR, 0.6-15.8) for EOS−NEU−, respectively. The demographic data of these subjects are listed in Table E2. The mRNA expression levels were evaluated by using quantitative RT-PCR analysis. Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, Calif) according to the manufacturer’s instructions. The total RNA was then used to synthesize the first-strand cDNA using the 5X Prime Script Kit (Takara, Syuzou, Shiga, Japan). Quantitative PCR was performed on the ABI7500 system (Invitrogen). For the relative quantification of mRNA expression, the expression of β-actin served as an internal control. Perfecta SYBR Green (Invitrogen) was used as a DNA intercalator dye to monitor amplified DNA quantification. The amplification conditions were 40 cycles for 2-stage PCR (95°C for 12 seconds and 60°C for 1 minute). The following primers are listed in Table E3. Hematoxylin and eosin and immunohistochemical staining for NEU, NLRP3, and IL-1β was performed according to the manufacturer’s instructions. Rabbit anti-NEU antibody (Abcam, Cambridge, Mass; ab68672, 1:200), mouse anti-NLRP3 antibody (Enzo, New York, NY; ALX-804-818, 1:100), rabbit anti–IL-1β antibody (Abcam, ab2105, 1:100), mouse IgG1 isotype (Abcam, ab81216, 1:100), and rabbit IgG isotype (Abcam, ab37415, 1:100) were used as the primary antibodies according to the manufacturer’s instructions (Fig E9). Each section was incubated with secondary antibody and then with horseradish peroxidase–labeled streptavidin complex (Dako, Glostrup, Denmark). Distribution of peroxidase was revealed by incubating the sections in a solution containing 3% 3, 3-diaminobenzidine tetrahydrochloride before being counterstained with hematoxylin and cover slipped. Negative control studies were performed using isotype control IgG and IgG1 to replace the primary antibody in appropriate concentration. The histological analyses were performed in a double-blind manner. The immunoreactivity of 5 randomly selected epithelial or subepithelial areas (200×) on each section of the subjects was quantitatively scored using Image-Pro Plus 6.0 software (Media Cybernetics), and the average OD value was calculated.E1Wen W. Liu W. Zhang L. Bai J. Li H. Xu G. et al.Increased neutrophilia in nasal polyps reduces the response to oral corticosteroid therapy.J Allergy Clin Immunol. 2012; 129: 1522-1528Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, E2Wei Y. Xia W. Ye X. Fan Y. Yang P. Li H. et al.The antimicrobial protein short palate, lung, and nasal epithelium clone 1 (SPLUNC1) is differentially modulated in eosinophilic and noneosinophilic chronic rhinosinusitis with nasal polyps.J Allergy Clin Immunol. 2014; 133: 420-428Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, E3Lin H. Li Z. Lin D. Zheng C. Zhang W. Role of NLRP3 inflammasome in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps.Inflammation. 2016; 39: 2045-2052Crossref PubMed Scopus (19) Google Scholar, E4Barac A. Pekmezovic M. Spiric V.T. Trivic A. Marinkovic J. Arsenijevic V.A. et al.Chronic rhinosinusitis: association of recalcitrant nasal polyposis and fungal finding in polyp’s single-cell suspension.Eur Arch Otorhinolaryngol. 2015; 272: 3727-3734Crossref PubMed Scopus (8) Google Scholar Nasal polyp tissues embedded in paraffin were cut into 4-μm sections and placed onto glass slides. Immunofluorescence staining was performed as previously described.E5Hong H.Y. Chen F.H. Sun Y.Q. Xu R. Li H.B. Shi J.B. et al.Local IL-25 contributes to Th2-biased inflammatory profiles in nasal polyps.Allergy. 2018; 73: 459-469Crossref PubMed Scopus (46) Google Scholar The tissues were incubated with a primary antibody overnight at 4°C, including mouse anti-CD68 (1:100, Abcam, ab955), mouse anti-MUC5AC (1:50, Santa Cruz Biotechnology, Dallas, Tex; sc-20118), goat anti-NLRP3 (1:100, Abacm, ab4207), rabbit anti–IL-1β (1:100, Abcam, ab2105), mouse IgG1 isotype control (Abcam, ab81216), goat IgG polyclonal isotype control (Abcam, ab37373), and rabbit IgG polyclonal isotype control (Abcam, ab37415). This was followed by the incubation with fluorescent-conjugated secondary antibodies for 50 minutes at room temperature, including donkey antimouse IgG H&L (Alexa Fluor 555, Abcam, ab150106) (1:500), donkey antigoat IgG H&L (Alexa Fluor 647, Abcam, ab150131) (1:500), and donkey antirabbit IgG H&L (Alexa Fluor 488, Abcam, ab150073) (1:500). All the secondary antibodies were purchased from Life Technologies (Carlsbad, Calif). Nuclear DNA was detected by incubation of 4'-6-diamidino-2-phenylindole, dihydrochloride (1:500, Roche, Basel, Switzerland) for 10 minutes at room temperature. Coverslips were mounted in ProLong Antifade Reagent. Images were captured with a confocal microscopy (Leica, Wetzlar, Germany; 780). The protein levels were evaluated by means of Western blot analysis as previously described.E1Wen W. Liu W. Zhang L. Bai J. Li H. Xu G. et al.Increased neutrophilia in nasal polyps reduces the response to oral corticosteroid therapy.J Allergy Clin Immunol. 2012; 129: 1522-1528Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, E2Wei Y. Xia W. Ye X. Fan Y. Yang P. Li H. et al.The antimicrobial protein short palate, lung, and nasal epithelium clone 1 (SPLUNC1) is differentially modulated in eosinophilic and noneosinophilic chronic rhinosinusitis with nasal polyps.J Allergy Clin Immunol. 2014; 133: 420-428Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar The protein extracts from nasal tissues underwent SDS–polyacrylamide gel electrophoresis and were transferred to a polyvinylidene fluoride membrane. The membranes were blocked and incubated with primary antibody (mouse anti-NLRP3 antibody, Enzo, ALX-804-818, 1:1000; rabbit anti–IL-1β antibody, Abcam, ab2105, 1:1000) and then incubated with the anti-mouse/rabbit IgG horseradish peroxidase secondary antibody and detected using enhanced chemiluminescence detection reagent (Millipore, Billerica, Calif). Western blot images were scanned and analyzed using GeneTools from stngene software (SYNGENE, Cambridge, United Kingdom). Polyp tissues were homogenized with 2 times their weight of PBS (mL/mg), and the final concentration was calculated from the weight of the tissue. The levels of IL-β in the homogenized supernatants of polyp tissues were measured by ELISA using a series of commercially available ELISA kits (IL-β, R&D Systems, Minneapolis, Minn; SLB50) according to the manufacturer’s instructions. Polyp tissues were collected from randomly selected patients with CRSwNP, and single-cell suspension of polyp tissues was isolated by collagenase Ⅴ digesting for 1 hour at 37°C. Then, we added RPMI 1640 medium containing 20% FBS to terminate digestion, filtered cell suspensions, and centrifuged at 400g for 5 minutes. We washed the cells with PBS (37°C, pH 7.4) and cultured them into RPMI 1640 medium (Gibco, Grand Island, NY) containing 10% FBS at 37°C with 5% CO2 as previously described.E1Wen W. Liu W. Zhang L. Bai J. Li H. Xu G. et al.Increased neutrophilia in nasal polyps reduces the response to oral corticosteroid therapy.J Allergy Clin Immunol. 2012; 129: 1522-1528Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, E2Wei Y. Xia W. Ye X. Fan Y. Yang P. Li H. et al.The antimicrobial protein short palate, lung, and nasal epithelium clone 1 (SPLUNC1) is differentially modulated in eosinophilic and noneosinophilic chronic rhinosinusitis with nasal polyps.J Allergy Clin Immunol. 2014; 133: 420-428Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, E3Lin H. Li Z. Lin D. Zheng C. Zhang W. Role of NLRP3 inflammasome in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps.Inflammation. 2016; 39: 2045-2052Crossref PubMed Scopus (19) Google Scholar, E4Barac A. Pekmezovic M. Spiric V.T. Trivic A. Marinkovic J. Arsenijevic V.A. et al.Chronic rhinosinusitis: association of recalcitrant nasal polyposis and fungal finding in polyp’s single-cell suspension.Eur Arch Otorhinolaryngol. 2015; 272: 3727-3734Crossref PubMed Scopus (8) Google Scholar After preparing a single-cell suspension, we added either 20 μL PBS (blank control), 20 μL TLR agonists (catalog no. and working concentration given in Table E4), or 20 μL recombinant proinflammatory cytokines (catalog no. and working concentration given in Table E5) to the medium immediately. The cells were harvested for assaying mRNA by PCR after 24 hours, assaying protein by western blotting after 48 hours, and the supernatants were collected for assaying IL-1β protein levels by ELISA after 72 hours. In some experiments, the single-cell suspension of polyp tissues was incubated with 20 μL PBS (blank control), 20 μL LPS, 10 μL poly I:C LMW (light molecular weight) + 10 μL poly I:C HMW (high molecular weight), 20 μL IL-4, or 5 mM ATP (Invitrogen, 18330-019; positive control) for 24 hours. Then, we added 60 μL PBS (blank control), 60 μL small-molecule small-interfering RNA inhibiting NLRP3 (20 nM, GenePharma, Shanghai, China), 60 μ neutralizing antibodies against IL-β (1 μg/mL, R&D Systems, MAB201), or 60 μ glucocorticoids (including 100 μM dexamethasone, 100 μM budesonide, and 100 μM fluticasone) and continued to culture for 48 hours. After 72 hours, the supernatant of treated single cells from polyp tissues was collected for the neutrophil chemotaxis experiment. PBMCs were isolated from buffy coats of blood from patients with CRSwNP by Ficoll density gradient centrifugation, as previously described.E6Zhao Q. Xiao X. Wu Y. Wei Y. Zhu L.Y. Kuang D.M. et al.Interleukin-17-educated monocytes suppress cytotoxic T-cell function through B7-H1 in hepatocellular carcinoma patients.Eur J Immunol. 2011; 41: 2314-2322Crossref PubMed Scopus (76) Google Scholar Monocytes were selected from PBMCs by anti-CD14 magnetic beads in a MACS column purification system (Miltenyi Biotech, Gladbach, Germany). To generate normal or activated macrophages, the purified monocytes were cultured for 6 to 7 days in complete RPMI medium supplemented with 40 ng/mL GM-CSF, and half of the culture medium was replaced on day 3 and day 5. Polyp tissues were collected from 4 randomly selected patients with CRSwNP, and human nasal epithelial cells (HNECs) were isolated, as we previously described.E7Lai Y. Chen B. Shi J. Palmer J.N. Kennedy D.W. Cohen N.A. Inflammation-mediated upregulation of centrosomal protein 110, a negative modulator of ciliogenesis, in patients with chronic rhinosinusitis.J Allergy Clin Immun. 2011; 128: 1207-1215Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar The NP tissue was cut into small pieces (3-5 mm) and incubated with dispase II (Sigma Aldrich, St Louis, Mo; D4693-1G) overnight and EDTA-trypsin (0.5%, Gibco, 25300-054) at 37°C with 5% CO2, 15 minutes, neutralized with Dulbecco modified Eagle medium (10% FBS). The tissue pieces thoroughly dispersed into a single-cell suspension, and were cultured in the 6-well plate (1 × 105 cells), with Dulbecco modified Eagle medium (10% FBS), at 37°C with 5% CO2, overnight. We drew the suspended cells and used them for the non-HNECs. The adherent cells were HNECs. The HNECs were cultured at air-liquid interface using Bronchial Epithelial Cell Growth Medium (Lonza, Zurich, Switzerland) in the polyester membrane transwell-clear inserts (Corning, Lowell, Mass), at 37°C with 5% CO2. After macrophages or HNECs achieved 70% to 80% confluence, 20 μL PBS (blank control), 20 μl LPS, 10 μl poly I:C LMW + 10 μL poly I:C HMW, and 20 μl IL-4 were added to the bottom chamber. The cells were harvested for assaying mRNA by PCR after 24 hours, assaying protein by western blotting after 48 hours, and the supernatants were collected for neutrophil chemotaxis after 72 hours. Neutrophils were isolated from buffy coats of blood from healthy donors by Ficoll density gradient centrifugation and red cell lysis. The polymorphonuclear neutrophils (PMNs) were selected by anti-CD16 magnetic beads in a MACS column purification system (Miltenyi Biotech) and checked by Trypan Blue exclusion staining (>97% viable). They were resuspended in RPMI 1640 medium (Gibco) containing 10% FBS (Sigma Aldrich). Neutrophil chemotaxis was assessed in a 48-well chemotaxis chamber (Neuro Probe), with a 5-μm polycarbonate filter between the upper and lower chambers and a 2-μm filter lining the bottom of the lower chamber. The lower chamber contained 50 μL of the supernatant of the polyp tissue single cells treated as described above or serum-free RPMI 1640 medium (control for random migration). The upper chamber contained 50 μL PMN suspension (3 × 106 cells/mL) with serum-free RPMI 1640 medium. The chambers were incubated for 1 hour at 37°C with 5% CO2. The upper chamber contents were discarded, and nonmigrated PMNs were wiped off the upper side of the 5-μm filter per the manufacturer’s instructions. Cells were counted on the lower side of the 5-μm filters under a light microscope (Olympas). Neutrophil migration can be expressed as PMNs/hpf. Continuous variables are expressed as means and the standard errors of the medians and IQRs. Data were analyzed using the nonparametric Mann-Whitney U test. The Spearman rank correlation test was used to analyze the correlations among different parameters. For in vitro assays, the data were expressed as the SEM of 3 independent experiments. One-way ANOVA and paired or unpaired Student t test were used for the statistical analysis. A P value of less than .05 was considered statistically significant.Fig E2Number of tissue eosinophils and neutrophils in EOS+NEU+, EOS+NEU−, EOS−NEU+, and EOS−NEU− polyp tissues. A, Eosinophils’ staining with H&E. B, Neutrophils’ staining with anti-NUE antibody. Positive cells per HPF, ×200 magnification. H&E, Hematoxylin and eosin. Results are expressed as the medians (IQRs). *P < .05 and ***P < .001, as determined by nonparametric Mann-Whitney U test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3The cellular localization of NLRP3 in NPs. To determine the cellular localization of NLRP3 by immunofluorescence, NLRP3 and IL-1β were colocalized with CD68 (A) and MUC5AC (B) in the human NP tissue.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4LPS, poly I:C, and IL-4–stimulated macrophage or epithelial cells from patients with CRSwNP. A and B, mRNA levels of NLRP3 and IL-β in macrophage. C and D, mRNA levels of NLRP3 and IL-β in epithelial cells. E, Protein levels of NLRP3/IL-1β in macrophage or epithelial cells. Con, Control. Results represent mean values (SEMs) from 3 independent experiments. ***P < .001, as determined by unpaired Student t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5mRNA expression of the TLR2, TLR3, TLR4, IL-4, IL-5, IL-13, IFN-γ, and IL-17a in 4 CRSwNP subsets. mRNA expression of the (A) TLR2, (B) TLR3, (C) TLR4, (D) IL-4, (E) IL-5, (F) IL-13, (G) IFN-γ, and (H) IL-17a, in 4 CRSwNP subsets. ns, No statistical significance. Results are expressed as the medians (IQRs). **P < .01 and ***P < .001, as determined by nonparametric Mann-Whitney U test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E6Neutrophils’ chemotaxis by cultured supernatant from different treatments’ single cells of polyp tissues. Representative neutrophil images by NP single-cell supernatant treatment with (A) LPS, poly I:C, IL-4, and ATP, (B) presence of small-molecule siRNA inhibiting NLRP3 (si-NLRP3), (C) neutralizing antibody against IL-1β (anti–IL-1β), and (D) glucocorticoid DXM. DXM, dexamethasone; siRNA, small-interfering RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E7Neutrophils’ chemotaxis by culture supernatant from LPS, poly I:C, and IL-4 with macrophage or epithelial cells from patients with CRSwNP. A, Representative neutrophil images by NP single-cell supernatant treatment with LPS, poly I:C, and IL-4. B, Summary graphs. Results represent mean values (SEMs) from 3 independent experiments. ***P < .001, as determined by unpaired Student t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E8mRNA expression of NLRP3 and IL-1β in the single-cell suspension of NPs with glucocorticoid treatment. NLRP3 and IL-1β mRNA levels after stimulation with glucocorticoid alone (A and E) or glucocorticoid in combination with IL-4 (B and F), LPS (C and G), and poly I:C (D and H). Bud, Budesonide; Dex, dexamethasone; Flu, fluticasone; ns, no statistical significance. Results represent mean values (SEMs) from 3 independent experiments. *P < .05, **P < .01, and ***P < .001, as determined by unpaired Student t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E9Immunohistochemically isotype staining for NEU, IL-1β, and NLRP3. A, Neutrophils staining with isotype control. B, Neutrophils staining with anti-NUE antibody. C, IL-1β staining with isotype control. D, IL-1β staining with anti–IL-1β. E, NLRP3 staining with isotype control. F, NLRP3 staining with anti-NLRP3. Red arrow indicates positive cells (×200 magnification).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Demographic characteristics of subjectsCharacteristicControl subjectsPatients with CRSsNPPatients with CRSwNPNo.353282Tissue sampledUncinateUncinateUncinate (34)/NPs (82)Age (y), mean (IQR)27.8 (22-55)33.8 (21-57)30.6 (21-58)Atopy, n (%)4 (11.43)5 (15.63)19 (23.17)∗P < .05, compared with control subjects.Asthma, n (%)02 (6.25)8 (10.98)Aspirin sensitivity, no.002 (2.44)Sex: male/female17/ 1815/1739/43CT Scores, mean ± SDNA5.56 ± 3.5315.79 ± 10.03Total IgE (IU/mL), mean ± SD11.95 ± 3.9688.46 ± 44.81101.74 ± 51.54∗P < .05, compared with control subjects.Blood eosinophlis (109/L), mean ± SD0.02 ± 0.050.18 ± 0.190.35 ± 0.38∗P < .05, compared with control subjects.Methodologies used Tissue IHC353234/82 Tissue mRNA353234/82 Tissue WB121413/15CT, Computed tomography; IHC, immunohistochemically; NA, not applicable/available; WB, Western blot.∗ P < .05, compared with control subjects. Open table in a new tab Table E2Characteristics of patients with EOS+NEU+, EOS+NEU−, EOS−NEU+ and EOS−NEU− CRSwNP subsetsCharacteristicEOS+NEU+ subsetsEOS+NEU− subsetsEOS−NEU+ subsetsEOS−NEU− subsetsNo.20202121Age (y), mean (IQR)31.8 (22-57)29.4 (21-56)30.2 (21-58)30.8 (23-55)Atopy, n (%)9 (45)∗P < .05, compared with control subjects.7 (35)∗P < .05, compared with control subjects.2 (9.5)1 (4.67)Asthma, n (%)4 (20)∗P < .05, compared with control subjects.4 (20)∗P < .05, compared with control subjects.NANAAspirin sensitivity, n1 (5)1 (5)NANASex: male/female11/910/1012/910/11CT Scores, mean ± SD18.67 ± 11.8614.12 ± 8.978.82 ± 5.605.54 ± 3.52Total IgE (IU/mL), mean ± SD110.52 ± 55.99104.48 ± 52.9388.54 ± 44.8574.58 ± 37.78Blood eosinophlis (109/L), mean ± SD0.45 ± 0.49∗P < .05, compared with control subjects.0.41 ± 0.45∗P < .05, compared with control subjects.0.22 ± 0.240.19 ± 0.21Methodologies used Tissue IHC20202121 Tissue mRNA20202121 Tissue WB7767CT, Computed tomography; IHC, immunohistochemically; NA, not applicable/available; WB, Western blot.∗ P < .05, compared with control subjects. Open table in a new tab Table E3Primer sequences for quantitative PCR analysisPrimerForwardReverseNLRP35′- AAGGAAGTGGACTGCGAGAA-3′5′-ACGTTCGTCCTTCCTTCCTT -3′IL-1β5′-GGGCCTCAAGGAAAAGAATC-3′5′-TTCTGCTTGAGAGGTGCTGA-3′NALP15′-GCAGTGCTAATGCCCTGGAT-3′5′-GAGCTTGGTAGAGGAGTGAGG-3′NLRC45′-ATCTTTCGGGATAGGTTCCTGT-3′5′-ACACGATCTCTGTTATAGGAGCA-3′ASC5′-TGGATGCTCTGTACGGGAAG-3′5′-CCAGGCTGGTGTGAAACTGAA-3′Caspase-15′-TTTCCGCAAGGTTCGATTTTCA-3′5′-GGCATCTGCGCTCTACCATC-3′TLR25′-GGTCCTGTGCCACCGTTTC-3′5’-GATGTTCCTGCTGGGAGCTTT-3’TLR35′-TGATGCTCCGAAGGGTGG-3′5′-CAGGGTTTGCGTGTTTCC-3′TLR45′-GGATGAGGACTGGGTAAGGAATG-3′5′-CACACCGGGAATAAAGTCTCTGTA-3′IL-45′-TTGCTGCCTCCAAGAACACAACTG-3′5′-TTCCTGTCGAGCCGTTTCAGGAAT-3′IL-55′-TCTACTCATCGAACTCTGCTGA-3′5′-CCCTTGCACAGTTTGACTCTC-3′IL-135′-TGGTCAACATCACCCAGAACCAGA-3′5′-AGCCTGACACGTTGATCAGGGATT-3′IFN-γ5′-TGCAGGTCATTCAGATGTAGCGGA-3′5′-TGTCTTCCTTGATGGTCTCCACACTC-3′IL-17a5′-CAACCGATCCACCTCACC-3′5′-AGCCCACGGACACCAGTA-3′β-Actin5′-AAGTGTGACGTTGACATCCG-3′5′-GATCCACATCTGCTGGAAGG-3′ASC, Apoptosis speck protein. Open table in a new tab Table E4TLR agonistsAgonistCompanyCatalog no.IngredientReceptorsWorking concentrationPam3CSK4Invivogentlrl-pmsSynthetic triacylated lipopeptideTLR1/2100 ng/mLPGNInvivogentlrl-pgns2PeptidoglycanTLR210 μg/mLPoly I:C LMWInvivogentlrl-picwPolyinosinic-polycytidylic acid, from 0.2 kb to 1 kbTLR3100 ng/mLPoly I:C HMWInvivogentlrl-picPolyinosinic-polycytidylic acid, from 1.5 kb to 8 kbTLR3100 ng/mLLPSInvivogentlrl-pb5lpsLPSTLR4100 ng/mLFLA-STInvivogentlrl-stflaRecombinant Flagellin from S typhimuriumTLR5100 ng/mLFSL-1Invivogentlrl-fslSynthetic lipopeptide contained a diacylated cysteine residueTLR2/6100 ng/mLImquimod-R837Invivogentlrl-imqsAn imidazoquinoline amine analog to guanosineTLR7/81 μg/mLssRNAInvivogentlrl-lrna40Single-stranded RNATLR7/81 μg/mLODN2359Invivogentlrl-2395Synthetic oligonucleotidesTLR91 μMHMW, High molecular weight; LMW, light molecular weight; ssRNA, single strand RNA. Open table in a new tab Table E5Recombinant proinflammatory cytokinesCytokinesCompanyCatalog no.Working concentrationIL-1βInvivogenrcyec-hil1b100 ng/mLIL-4Invivogenrcyec-hil4100 ng/mLIL-5Sino Biological Inc15673-HNCE100 ng/mLIL-13Peprotech200-13100 ng/mLIL-17aInvivogenrcyc-hil17a100 ng/mLIFN-γInvivogenrcyec-hifng100 ng/mLTGF-βInvivogenrcyc-htgfb1100 ng/mL Open table in a new tab CT, Computed tomography; IHC, immunohistochemically; NA, not applicable/available; WB, Western blot. CT, Computed tomography; IHC, immunohistochemically; NA, not applicable/available; WB, Western blot. ASC, Apoptosis speck protein. HMW, High molecular weight; LMW, light molecular weight; ssRNA, single strand RNA. CorrigendaJournal of Allergy and Clinical ImmunologyVol. 148Issue 3PreviewWith regard to the article in the March 2020 issue entitled “Activated pyrin domain containing 3 (NLRP3) inflammasome in neutrophilic chronic rhinosinusitis with nasal polyps (CRSwNP)” (J Allergy Clin Immunol 2020;145:1002-5.e16), the authors report some errors in Figs E6 and E7. The authors report that, after publication, it was discovered that the image of si-NLRP3 control in Fig E6, B, was duplicated with the image of DXM control in Fig E6, D, while omitting the actual data of the DXM control. Full-Text PDF