IL-36γ in chronic rhinosinusitis

Online Repository

The activation and function of IL-36γ in neutrophilic inflammation in chronic rhinosinusitis

Hai Wang, M.D.1*, Zhi-Yong Li, M.D.1*, Wen-Xiu Jiang, M.D.1, Bo Liao, M.D., Ph.D.1, Guan-Ting Zhai, M.D.1, Nan Wang, M.D., Ph.D.1, Zhen Zhen, M.D., Ph.D.2, Jian-Wen Ruan, M.D.1, Xiao-Bo Long, M.D., Ph.D.1, Heng Wang, M.D., Ph.D.1, Wei-Hong Liu, M.D. Ph.D.3, Geng-Tian Liang，M.D.4, Wei-Min Xu, M.D.5, Atsushi Kato, Ph.D.6, Zheng Liu, M.D., Ph.D.1

1Department of Otolaryngology-Head and Neck Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China

2Department of Otolaryngology-Head and Neck Surgery, Peking University First Hospital, Beijing, P.R. China

3Department of Otolaryngology-Head and Neck Surgery, Wuhan No.1 Hospital, Wuhan, P.R. China

4Department of Otolaryngology-Head and Neck Surgery, Wuhan No. 3 Hospital, Wuhan, P.R.China

5Department of Otolaryngology-Head and Neck Surgery, Wuhan Puai Hospital, Wuhan, P.R.China

6Division of Allergy and Immunology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, USA

* These authors contributed equally to the completion of this article.

Running Head: IL-36γ in chronic rhinosinusitis

For correspondence, please contact:

Zheng Liu, M.D., Ph.D.

Department of Otolaryngology-Head and Neck Surgery

Tongji Hospital, Tongji Medical College

Huazhong University of Science and Technology

No. 1095 Jiefang Avenue

Wuhan 430030, P.R. China

E-mail:

METHODS

Subjects

This study was approved by the Ethics Committee of Tongji Hospital of Huazhong University of Science and Technology and was conducted with written informed consent from patients. A total of 320 subjects including 27 patients with CRSsNP,93patients with eosinophilic CRSwNP, 98patients with non-eosinophilic CRSwNP, and 102control subjects were included in this study. The diagnosis of CRS was made according to the current American and European guidelines.E1, E2CRSwNP was defined as eosinophilic when the percentage of tissue eosinophils exceeded 10% of total infiltrating cells, as reported by our previous study.E3This cut-off was calculated as twice of the standard deviation of the mean of eosinophil percentage in controls.E3 Subjects undergoing septoplasty because of anatomic variations and without other sinonasal diseases were enrolled as control subjects. All subjects came from central China and patients with CRSwNP had bilateral nasal polyps. Atopic status was evaluated by using skin prick test with a panel of inhalant allergens common in our region and/or by using the ImmunoCAP to detect IgE antibodies against common inhalant allergens (Phadia, Uppsala, Sweden).E4, E5 The diagnosis of asthma was based on Global Initiative for Asthma 2006 guideline.E6 Oral glucocorticoid and intranasal steroid spray were discontinued at least 3 months and 1 month before surgery, respectively. Antileukotrienes were discontinued at least 1 month before inclusion. Patients with an acute upper respiratory tract infection and acute asthma episode within 4 weeks of entering the study, and patients under immunotherapy were excluded. In addition, patients who had a history of antrochoanal polyps, cystic fibrosis, primary ciliary dyskinesia, fungal sinusitis, immunodeficiency, or systemic vasculitis were excluded from this study, because these are discrete disorders with unique pathophysiology.None of subjects had a history of aspirin sensitivity. Diseased ethmoid sinus mucosal samples from CRSsNP patients,polyp tissues from CRSwNP patients, and inferior turbinate mucosal tissues from control subjects were collected during surgery, respectively.Human nasal epithelial cells (HNECs) were scraped from the mucosa of inferior turbinate of controls as previously described.E7More information is provided in the Online Repository including Table E1 and Table E2.

Histology and immunohistochemistry

Tissue samples were fixed in formaldehyde solution and embedded in paraffin. Paraffin sections (4 μm) were prepared from tissue blocks. After deparaffinization and rehydration, sections were stained with hematoxylin and eosin routinely. Immunohistochemical andimmunofluorescencestaining were performed as previously reported.E3,E8-E10Sections were subjected to heat-induced antigen retrieval using citric acid repair solution. The 3% hydrogen peroxidase was used for endogenous peroxidase inhibition and 5% bovine serum albumin was used to block nonspecific binding.E8-E10Sections werestained with primary antibodies shown in Table E3. Species and subtype-matched antibodies were used as negative controls. Fluorescence conjugated secondary antibodies used are listed in Table E4.For immunohistochemical staining, all antigens were detected by using the streptavidin-peroxidase complex method with a histostain-plus kit (Boster Biotechnology, Wuhan, China) according to the manufacturer’s instructions.E10Color development was achieved with 3’, 3’-diaminobenzidine, which rendered positive cells brown. Finally, sections were counterstained with hematoxylin and mounted. Consecutive sections were used to determine the relationship between myeloperoxidase (MPO) positive cells and the expression of elastase, chemokine (C-X-C motif) ligand (CXCL)1, CXCL2, CXCL8, IL-17A, and matrix metallopeptidase (MMP)-9. The relationships between IL-36 receptor (IL-36R) positive cells and MPO, major basic protein (MBP), CD4, CD8, CD11c, CD68, CD138 and tryptase positive cells were determined by double immunofluorescence staining. The number of positive infiltrating cells in lamina propria per high-power field (HPF) was counted by 2 independent physicians who were blind to the clinical data.E3,E10Ten HPFs were randomly selected and analyzed. Given the diffuse staining pattern, the intensity of positive staining in epithelial cells and blood vessels was analyzed by using image pro-plus 6.0 analysis software (Media Cybernetics, Inc. Silver Spring, MD, USA) and the results were presented as mean optical density value per unit area.E7

In situ hybridization

Nasal polyp tissue sections were analyzed for IL-36γ mRNA expression by in situ hybridization as previously reported.E11Briefly, after deparaffinization and rehydration, sections were subjected to antigen retrieval with 10 μg/mL proteinase K (Boster Biotechnology) at 37°C for 30 min. After washing with diethyl pyrocarbonate water twice, the sections were re-fixed in 1%polyformaldehyde for 20 min, followed by prehybridization at 37°C for 2 h. Hybridization reaction was then carried out with digoxin-labeled probe specific for IL-36γmRNA (1:100; Boster Biotechnology) at 37°C for 22 h. After washing with gradient salt-sodium citrate buffer (0.3 M sodium chloride and 0.03 M sodium citrate) thoroughly, sections were incubated with alkaline phosphatase conjugated mouse anti-digoxin antibody (1:400, Guge Biotechnology, Wuhan, China) at 37°C for 2 h.Color development was achieved with 4-nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate, which rendered positive cells dark blue. Sections were next processed for immunochemistry staining for MPO according to the methods described above.The sequence of oligonucleotide probe for IL-36γ was 5’-GGAAG GGTTT CACGG GCTCG GGTTG GC-3’.

Quantitative RT-PCR

Total RNA was extracted from tissues and cells by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as mentioned elsewhere.E3, E10, E121 μg of total RNA was reverse-transcribed to cDNA using a PrimeScript RT reagent kit (TaKaRa Biotechnology, Dalian, China). cDNA equivalent to 50 ng of total RNA was used for PCR assay. Quantitative PCR was performed by using the SYBR Premix Ex Taq kit (TaKaRa Biotechnology, Dalian, China) with specific primers (Table E5) on StepOnePlus (Applied Biosystems, Foster City, CA, USA). Amplification was as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 sec, specific annealing temperature for 10 sec, and 72 °C for 15 sec. After PCR, a melting curve was constructed by increasing the temperature from 65 to 95 °C with a temperature transition rate of 0.1°C/s. Relative gene expression was calculated by using the 2(-Delta Delta CT) method.E13A normal inferior turbinate mucosal sample and a control cell culture sample were used as the calibrator for tissue samples and cell culture samples, respectively. Beta-glucuronidase (GUSB)was used as a housekeeping gene for normalization of gene expression. No template sample was used as negative control.

Human nasal epithelial cells (HNECs) culture

HNECs were scraped from inferior turbinate of 6 control subjectsusing a sterile Rhino-Pro curette (Arlington Scientific Inc, Springville, UT)under endoscope and werecultured with an air-liquid interface method as stated elsewhere.E7, E12, E14, E15Briefly, the obtained HNECs were washed twice in Dulbecco modified Eagle medium (Thermo Fisher Scientific, Waltham, MA, USA) with penicillin/streptomycin and then grown submerged in bronchial epithelial cell basal medium (BEBM, Lonza, Walkersville, MD, USA) supplemented with SingleQuot Kit Suppl (Lonza) in a 6-well plates coated with rat tail collagen type I (Sigma-Aldrich, St. Louis, MO, USA) in a 5% CO2-humidified atmosphere at 37°C. Upon confluence, the cells were passaged to gel-coatedtranswell culture inserts (0.4 μm pore size; Becton Dickison, Franklin Lake, NJ, USA) and were grown in bronchial epithelial cell growth medium (Lonza). When they were confluent, medium from the apical side of the culture was removed and medium below the insert was replaced with an air-liquid interface medium consisting of BEBM/Dulbecco’s modified Eagle’s medium H (50: 50; Invitrogen). HNECs were maintained at air-liquid-interface culture for 21 days for the differentiation.E14 After the HNECs were differentiated, they were stimulated with IL-4, IL-6, IL-10, IL-12, IL-13, IL-17A, TNF-α, IL-1β, IFN-γ, IL-22,thymic stromal lymphopoietin (TSLP), and IL-33 at 10 ng/mL (R&D systems, Minneapolis, MN, USA), IL-25 (R&D system) at 50 ng/mL, poly (I:C) (dsRNA) at 25 μg/mL (Tocris,Bristol, UK), lipopolysaccharides (LPS) (InvivoGen, San Diego, CA, USA) at 1 μg/mL, staphylococcal enterotoxin B (SEB) (Millipore, Merck KGaA, Darmstadt, Germany) at 1 μg/mL, CpG (InvivoGen) at 2 μg/mL, and Dermatophagoides pteronyssinus group 1 (Der p1) at 10 μg/mL (Prospec, East Brunswick, NJ, USA) for 6 hours. After stimulation, cells were harvested for quantitative RT-PCR assay.

Peripheral blood neutrophil isolation

Neutrophils were prepared from heparinized peripheral blood from patients with CRSwNP (n = 8) and control group (n =31) using Ficoll and 3% dextran as described previously.E16Briefly, peripheral blood was mixed with equivoluminal3% dextran (Tianjin Haoyang Biological manufacture, Tianjin, China) and phosphate buffered saline (PBS), and allowed to subside for 30 min. The leukocyte containing supernatant was then carefully layered onto Ficoll-Hypaque gradient(TianjinHaoyang Biological manufacture). After centrifugation at 2000 rpm for 20 min, all the layers above the red cells were removed. The left cells were washed after incubating with hypotonic erythrocytes lysis to remove erythrocytes and then resuspended in RPMI 1640 medium (HyClone, Logan, Utah, USA) with 1% penicillin/streptomycin (Guge Biotechnology, Wuhan, China) and 10% FBS (HyClone) for cell culture or flow cytometry.The purity of isolated neutrophils was usually > 95% as determined by flow cytometric analysis of CD16+Siglec-8-positive cells (see Fig 4, A).

Peripheral blood neutrophil culture

Purified peripheral blood neutrophils were culturedin RPMI 1640 medium (Guge Biotechnology) supplemented with 10% fetal calf serum (FCS) (Gibico,Thermo Fisher Scientific, Waltham, MA, USA) in 24-well plates (1×105/mL).E17Cells were stimulated with IL-4, IL-6, IL-13,IL-17A, IL-22, TNF-α, IL-1β, IFN-γ and TSLP at 10 ng/mL (R&D systems), IL-25 at 50 ng/mL (R&D systems), LPS at 1 μg/mL (InvivoGen), SEB at 1 μg/mL (Millipore), CpG at 2 μg/mL(InvivoGen), and Der p1 at 10 μg/mL (Prospec). After 6 hours and 20 hoursstimulation, cells were harvested for PCR assay and flow cytometry, respectively.The concentration of Der p1 used for stimulation study was chosen based on our pilot dose-response experiment (Fig E9, B). To investigate the potential mechanism underlying Der p1 effect, purified blood neutrophils were stimulated by Der p1 (10 μg/mL; Prospec) with or without a specific protease-activated receptor (PAR)-2 antagonist, N1-3-methylbutyryl-N4-6-aminohexanoyl-piperazine (ENMD, 1.2 mM; Abcam, Cambridge, UK), for 6 hours and then harvested for RT-PCR analysis. In some experiments, neutrophils were cultured with IL-4, IL-17A, and IFN-γ at 10 ng/mL (R&D systems) for 20 hours in the presence of 25 nMphorbol 12-myristate 13-acetate (PMA; Sigma Aldrich), which was added 4 hours before culture supernatants were harvested for elastase activity assay.

Dispersed nasal polyp cells (DNPCs) preparation

DNPCs(n = 25) were prepared from nasal polyp tissues by means of mechanical dissociation with GentleMACSDissociator (MiltenyiBiotec, San Diego, CA, USA).E8 The resulting cell suspension was filtered 2 times through a 40 μm cell strainer (BD Biosciences, San Jose, CA, USA), then washed twice with a culture medium RPMI-1640medium (HyClone).E8Cell pellets were resuspended in erythrocyte lysis buffer and incubated for 5mins.After washing with PBS,cells were resuspended in the culture medium for cell culture. In order to exclude the influence of CD16+natural killer cells and dendritic cells on flow cytometric analysis, mononuclear cells were depleted from DNPCs by using Ficoll-Hypaque gradient (Tianjin Haoyang Biological manufacture) as previously described.E8, E18

Flow cytometry

For flow cytometry, cells were first stained with the Fixable Viability Stain 520 (BD Biosciences, San Jose, CA, USA) as a live/dead discriminator.E19Surface expression of receptors on isolated peripheral neutrophils, DNPCs depleted of mononuclear cells, and mononuclear cells from blood and nasal polyps was detected by incubating cells with goat polyclonal anti-human IL-36R antibody or rabbit monoclonal anti-human PAR-2 antibody on ice for 30 min. After wash, the cells were further stained with mouse anti-goat IgG-PerCP-Cy5.5, or donkey anti-rabbit IgG-FITC combined withAPC conjugated mouse monoclonal antibody against human CD16, PE-conjugated mouse monoclonal antibody against human Siglec-8, APC-conjugated mouse monoclonal antibody against humanCD3, PE-conjugated mouse monoclonal antibody against humanCD8, APC-Cy7-conjugated mouse monoclonal antibody against humanCD14,FITC-conjugated mouse monoclonal antibody against human Lineage, APC-conjugated mouse monoclonal antibody against humanHLA-DR,APC-Cy7-conjugated mouse monoclonal antibody against human CD11c, or/and PE-conjugated mouse monoclonal antibody against humanCD123for 30 min at 4°C in the dark.E17, E20Species and subtype-matched antibodies were used as controls. Neutrophils, monocytes, myeloid dendritic cells (DCs), and plasmacytoid DCs were identified as CD16+Siglec-8-, CD3-CD14+, Lineage-HLA-DR+CD11c+CD123-,and Lineage-HLA-DR+CD11c-CD123+ cells, respectively. In gated neutrophils, T cells, monocytes and DCs, the expression of IL-36R was analyzed. For intracellular staining of IL-17A, following CD16 and siglec-8 staining, cells were fixed and permeablized using the BD cytofix/cytoperm kit (BD Biosciences) for intracellular staining following manufacturer’s protocols, and cells were stained with FITC-conjugated mouse monoclonal antibody against human IL-17Afor 30min at 4°C as previously described.E17The detailed information of primary antibodies used are listed in Table E6. The stained cells were analyzed using LSRII flow cytometer (Becton Dickinson Biosciences, San Jose, CA, USA).Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

Western blot analysis

Total cellular protein was extracted from tissues and cells in modified radio-immunoprecipitation assay buffer (Guge Biotechnology) containing cocktail of protease inhibitors (Guge Biotechnology). Protein concentrations were quantified with the Bradford assay (Guge Biotechnology). Samples containing 30 μg of proteins and 2 μg recombinant full lengthIL-36γ treated with elastase or polyp elutes were denatured by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyl difluoride membranes (Guge Biotechnology). The membranes were incubated with 5% fat-free skim milk in Tris-buffered saline with Tween-20 (TBS-T; Guge Biotechnology) for 1 hour at room temperature and then incubated overnight with goat polyclonal antibody against human IL-36γ (1:4000, R&D Systems). After washing 3 times in TBS-T buffer, the membranes were incubated with horseradish peroxidase (HRP)-conjugated rabbitanti-goat IgG (1:4000, Guge Biotechnology) and finally processed by using an ECL chemiluminescence reaction kit (Cell Signaling, Danvers, Mass), followed by exposure on chemiluminescent film to visualize the proteins. β-actin quantification was used as an internal standard to correct for variations in total protein loading. Densitometric analysis of the blots was performed by using the software AlphaEase FC (Alpha Innotech, Silicon Valley, CA, USA).Full-length IL-36γ was identified as a single band around 19KDa. Cleaved IL-36γ was identified as discernable bands smaller than 17KDa.

Elastase activity assay

The detection of elastase activity in polyp elutes, DNPCs culturesupernatant, and neutrophil culture supernatant was carried out using a commercial kit (Biovision,Milpitas, CA, USA) under the instructions of manufacturer. Briefly, the diluted neutrophil elastase substrate-AFCwas added into50μLsamples. The neutrophil elastase substrate-AFC wasproteolytically cleaved by neutrophil elastase and released fluorophore AFC was measured immediately by using an automated fluorimeter (BIO-RAD, Hercules, CA, USA) at wavelengths of 380 nm (excitation) and 500 nm (emission) at 37˚C in kinetic mode in 10-20min. The corresponding values for the relative fluorescence unit (RFU) at 10 min and 20 min (RFU1 and RFU2, respectively) were obtained and the activity of neutrophil elastase is reflected by ΔRFU (ΔRFU = RFU2-RFU1).

DNPC culture

DNPCs depleted of mononuclear cells were resuspended in culture medium (RPMI 1640 supplemented with 10% FBS, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin)and were grown submerged in 24-well plates (2 × 106 /mL) in a 5% CO2-humidified atmosphere at 37 °C. DNPCs were stimulated with N-terminally truncated mature human IL-36γ (aa18-169) (R&D systems) or full-length human IL-36γ (aa1-169) (R&D systems) at 0, 1, 10 and 100 ng/mL for 6 and 20 hours. In some experiments, IL-36γ stimulation was conducted with 10 μM elastase inhibitor IV (Calbiochem, Shanghai, China) or 10−7 M dexamethasone (Sigma-Aldrich). Dexamethasone was added 2-hour before the stimulation. After 6-hour stimulation, cells were harvested for PCR assay. Culture supernatants were harvested for ELISA after 24-hour stimulation.In some experiments, cells were stimulated with full-length IL-36γ (100 ng/mL) or truncated IL-36γ (100 ng/mL) for 24 hours. Fifteen minutes after the stimulation,brefeldin A and monensin (2μL/mL, eBioscience,San Diego, CA, USA) were added. Cells were harvested for flow cytometric analysis of IL-17A expression in neutrophils.

Cytokine measurement

Snap-frozen sinonasal mucosal specimens were homogenized as previously described.E21, E22Briefly, 1 mL of 0.9% sodium chloride solution containing protease inhibitor cocktail (Guge Biotechnology) was added per every 0.1 g tissue. Tissue was homogenized on ice and centrifuged at 3000 rpmfor 10 minutes at 4 ºC.The supernatants were harvested and stored at -80 ºC.The concentrations of IL-5, IFN-γ and IL-17A in tissue homogenates were measured by means of Bio-Plex suspension chip technology (BIO-RAD, Hercules, CA, USA) according to the manufacturer’s instructions.E23Mucosal MPO (R&D Systems), IL-36α (CusabioLife Sciences, Wuhan, China), IL-36β (Cusabio Life Sciences), IL-36γ (CusabioLife Sciences), IL-36Ra (CusabioLife Sciences), and IL-38 (CusabioLife Sciences) were detected using commercially available ELISA kits. Mucosal eosinophilic cationic protein (ECP) levels weremeasured by using the UniCAP system (Pharmacia, Uppsala, Sweden).E21,E22Cytokine concentrations in supernatants of cultured DNPCs were measured with commercial ELISA kits (R&D Systems) specific for IL-17A, MMP-9, CXCL1, CXCL2, and CXCL8. Values of less than the detection limit were considered negative for categorical analysis and given a value equal to half of the detection limit for continuous analysis.E22Detection limits for ELISA assay and Bio-Plex assay were listed in Tables E7 and Table E8, respectively. The lower detection limit for ECP detected by UniCAP system was 2 μg/L.