Regulation and Function of the IL-1 Family Cytokine IL-1F9 in Human Bronchial Epithelial Cells

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1 Online Data Supplement Regulation and Function of the IL-1 Family Cytokine IL-1F9 in Human Bronchial Epithelial Cells Regina T. Chustz 1, Deepti R. Nagarkar 1, Julie A. Poposki 1, Silvio Favoreto, Jr 1, Pedro C. Avila 1, Robert P. Schleimer 1, Atsushi Kato 1 1. Division of Allergy and Immunology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA Materials and Methods Reagents Recombinant human TNF, IL-1β, IL-1F6, IL-4, IL-6, IL-13, IL-17A (IL-17), IFN-λ1 (IL-29), oncostatin M (OSM) and IFN-γ were purchased from R&D systems (Minneapolis, MN). Recombinant human IL-1F9 was purchased from Axxora (San Diego, CA). Recombinant human IFN-β, synthetic bacterial lipoprotein Pam3CSK4, poly(i:c) (dsrna), LPS from Escherichia coli, serotype 0111:B4, recombinant flagellin from Salmonella typhimurium, synthetic diacylated lipoprotein FSL-1, R-837, CpG oligodeoxynucleotide M362 (CpG-C) and polymyxin B were purchased from InvivoGen (San Diego, CA). The small interfering RNAs (sirnas) against RELA (Hs_RELA_5), IRF-3 (Hs_IRF3_1), IL-1RL2 (Hs_IL1RL2_3 and Hs_IL1RL2_6), IL-1RAP (Hs_IL1RAP_5) and the no effect control (AllStars negative control sirna) were obtained from Qiagen (Valencia, CA). Cell culture, treatments and transfection Primary normal human bronchial epithelial cells (NHBE) were obtained from Lonza (Walkersville, MD). NHBE were maintained in serum-free bronchial epithelial cell growth medium (BEGM, Lonza). NHBE were plated in 24-well culture plates coated with collagen (Vitrogen; Collagen Biomaterials, Palo Alto, CA). Before stimulation, NHBE were cultured in BEGM without hydrocortisone for at least 24 h. Submerged NHBE were stimulated with one or more of the following at the indicated concentration: 100 ng/ml TNF, 100 ng/ml IL-1β, 100 ng/ml IL-4, 100 ng/ml IL-6, 100 ng/ml IL-13, 100 ng/ml IL-17, 100 ng/ml OSM, 1000 U/ml IFN-β, 10 ng/ml IFN-γ, 100 ng/ml IL-29, 1 μg/ml Pam3CSK4 (TLR2 ligand), 5 μg/ml dsrna (TLR3 ligand), 1 μg/ml LPS (TLR4 ligand) with 1% human AB serum (MP Biomedicals (Solon, OH)), 10 ng/ml Flagellin (TLR5 ligand), 1 μg/ml FSL-1 (TLR2/6 ligand), 10 μg/ml R-837 (TLR7 ligand) and 2 μg/ml CpG-C (TLR9 ligand) for 6 h. NHBE were preincubated with 0.01% DMSO (vehicle control) or 1 10 μg/ml cycloheximide (CHX) for 1 hour, and then stimulated with 50 ng/ml IL-17 or 5 μg/ml dsrna for 24 h. For sirna experiments, NHBE were seeded at 3*10 4 cells/well in 24-well culture plates and were cultured for 2 days. At 40-60% confluence, cells were transfected with sirna against RELA, IRF-3 or control RNA (AllStars negative control sirna) at 5 nm using HiPerFect transfection reagent (Qiagen) following the manufacturer s instructions. Viability of the cells was usually 80% in sirna transfected cells. The transfected cells were further grown for 48 h, and then stimulated with 5 μg/ml dsrna for 6 h. Rhinovirus serotype 16 (RV16) was a gift from W. Busse and E. Dick (University of Wisconsin, Madison). Submerged NHBE were infected with RV16 at a multiplicity of infection (MOI) of 2 at 33 C. After 6 h post infection, NHBE were washed with PBS to remove excess RV16, and then cells were further cultured for 18 h. In some experiments, NHBE were differentiated at an air-liquid interface (ALI) on

2 0.4 μm pore membrane 12 well transwell plates (Costar, Corning, NY). NHBE were cultured with BEGM on the collagen-coated membrane. When confluent, they were shifted to ALI culture by removing all of the apical medium, and maintaining 500 μl of ALI medium containing BEBM/Dulbecco's modified Eagle's medium (DMEM) supplemented with BEGM SingleQuot Kit (Lonza) and additional bovine serum albumin (1.5 μg/ml, Sigma-Aldrich (St. Louis, MO)) and retinoic acid (5 x 10-8 M, Sigma-Aldrich) in the basal chamber. The ALI medium was changed every other day for 3 weeks. Before stimulation, differentiated NHBE were cultured in ALI medium without hydrocortisone for 2 days. Differentiated NHBE were stimulated with 50 ng/ml TNF, 50 ng/ml IL-17, 2.5 μg/ml dsrna or their combination for 24 and 48 h on the apical side (100 μl) and basolateral side (500 μl). After 6 hours stimulation, medium was removed from the apical side. After 48 h stimulation, cells were washed with 500 μl of PBS from the apical side to collect all secreted protein in the apical area. Primary normal human lung fibroblasts (NHLF, Lonza) were maintained in fibroblast growth medium 2 (FGM2, Lonza). NHLF were seeded at 3*10 4 cells/well in 12-well culture plates and were cultured for 2 days. At 40-60% confluence, cells were transfected with sirna against IL-1RL2, IL-1RAP or control sirna at 20 nm using HiPerFect transfection reagent following the manufacturer's instructions. Viability of the cells was usually greater than 90% in sirna transfected cells. The transfected cells were further grown for 48 h and then stimulated with 250 ng/ml IL-1F9 or 1 ng/ml IL-1β for 3 h. Real-time PCR Total RNA was extracted using RNeasy (Qiagen) or NucleoSpin RNA II (Clontech, Mountain Vies, CA) and was treated with DNase I according to the manufacturer's instructions. Single-strand cdna was synthesized with SuperScript II reverse transcriptase (Invitrogen) and random primers. Real-time RT-PCR was performed with a TaqMan method using an Applied Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) in 15 μl reactions (7.5 μl of 2x TaqMan Master mix (Applied Biosystems), 400 nm each primer, and 200 nm TaqMan probe plus cdna). Primer and probe sets for eight genes, IL-1β (sense, 5'-TTCTTCGACACATGGGATAACG-3'; 5'- TCCCGGAGCGTGCAGTT-3'; FAM/black hole quencher 1 (BHQ1) labeled probe, 5'-TGTGCACGATGCACCTGTACGATCA-3' ), IL-1F6 (sense, 5'-CAGCTGAAGGAAAAGGATATAATGGA T-3'; 5'-GCCACTCTGGCTGTGGTAGAA-3'; FAM/BHQ1 labeled probe, 5'-CAACCAACCCGAGCCTGTGAAGTCC-3' ), IL-1F9 (sense, 5'-GCCCACATTGCAGCTAAAAGAG-3'; 5'-CAACCCGAGCCCGTGAAACCCTT-3'; FAM/BHQ1 labeled probe, 5'-CAACCAACCCGAGCCTGTGAAGTCC-3' ), IL-33 (sense, 5'-AGCTCTCTGAAACTTAGTTGATGGAA A-3'; 5'-TTCCTTCTCCAGTGGTAGCATTTG-3'; FAM/MGB labeled probe, 5'-CTGTGAGTCTTGGGTTGAGTA-3'), IL-8 (sense, 5'-CCACACTGCGCCAACACAGAAATTAT TG-3'; 5'-GCCCTCTTCAAAAACTTCTCCACAACC C-3'; FAM/BHQ1 labeled probe, 5'-AAGCTTTCTGATGGAAGAGAGCTCTG TC-3'), CXCL3 (sense, 5'-CTGCGCTGCCAGTGCTT-3'; 5'-ACCTTACATTCACACTTTGGATGTTC-3 '; FAM/BHQ1 labeled probe, 5'-CAGACACTGCAGGGAATTCACCTCA-3' ), CCL20 (sense, 5'-GCTCCTGGCTGCTTTGATG-3'; 5'-CAAAGTTGCTTGCTGCTTCTGA-3'; FAM/MGB labeled probe, 5'-CTGCTACTCCACCTCTGCGGCGA-3')

3 and β-actin (ACTB; sense, 5'-CTGGCCGGGACCTGACT-3'; 5'-GCAGCCGTGGCCATCTC-3'; FAM/MGB labeled probe, 5'-CACCACCACGGCCGA-3') were synthesized by Integrated DNA Technologies (Coralville, Iowa) or Applied Biosystems. Primer and probe sets for IL-1F5 (Hs _m1), IL-1F7 (Hs _m1), IL-1F8 (Hs _m1), IL-1F10 (Hs _m1), IL-1RL2 (Hs _m1), IL-1RAP (Hs _m1), RELA (Hs _m1), IRF-3 (Hs _m1) and G-CSF (Hs _m1) were purchased from Applied Biosystems. To determine the exact copy number of the target genes, quantified aliquots of purified PCR fragments of the target genes were serially diluted and used as standards in each experiment. Aliquots of cdna equivalent to 10 ng of total RNA were used for real-time PCR. The mrna expression levels were normalized to the median expression of a housekeeping gene (ACTB). Western blot analysis NHBE were stimulated with 50 ng/ml TNF, 50 ng/ml IL-17, 5 μg/ml dsrna or their combinations for 48 h. The supernatant was concentrated 20 times using Amicon Ultra-4 10,000 MWCO filters (Millipore). Cells were harvested in cell lysis buffer (Cell Signaling Technology, Beverly, MA) supplemented with halt protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL). Whole cell lysates and concentrated supernatants were dissolved in lane marker sample buffer (Thermo Fisher Scientific). To detect IL-1F6 and IL-1F9, 30 μg of whole cell lysates or 20 μl of supernatants were resolved on a 4-12% NuPAGE Bis-Tris Gel (Invitrogen) in an MES buffer system and then proteins were transferred onto a PVDF membrane (Bio-Rad, Hercules, CA). The membranes were blocked in blocking buffer for fluorescent western blotting (Rockland, Gilbertsville, PA) for 2 h and subsequently incubated overnight at 4 C with 0.5 μg/ml rat anti-human IL-1F9 mab (clone: , R&D systems) or 0.1 μg/ml biotinylated goat anti-human IL-1F6 antibody (R&D systems). Following primary antibody incubation, the membranes were washed repeatedly in PBS/0.1% Tween 20 and then labeled by 45 min incubation at room temperature with infrared secondary antibody IRDye 700DX-conjugated goat anti-rat IgG (1:5,000, Rockland) or IRDye 700DX-conjugated streptavidin (1:100,000, Rockland). Protein was detected on an Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE). NHLF were harvested at the indicated time points and then whole cell lysates were dissolved in cell lysis buffer supplemented with halt protease inhibitor cocktail and halt phosphatase inhibitor cocktail (Thermo Fisher Scientific). The cell lysates were then dissolved in NuPAGE LDS Sample Buffer (Invitrogen). To detect p38, JNK, ERK, NF-κB p65 and CREB, 30 μg of whole cell lysates were resolved on a 4-12% NuPAGE Bis-Tris Gel and then proteins were transferred onto a PVDF membrane. Blocked-membranes were incubated overnight at 4 C with primary antibodies (Cell Signaling Technology) and then were labeled with infrared secondary antibodies IRDye 700DX-conjugated donkey anti-rabbit IgG (1:10,000, Rockland), IRDye 800-conjugated donkey anti-mouse IgG (1:10,000, Rockland), AlexaFluor 680-conjugated goat anti-mouse IgG (1:10,000, Invitrogen) or IRDye 800-conjugated goat anti-rabbit IgG (1:10,000, Rockland). ELISA and cytometric bead array The concentrations of IL-1F6 and IL-1F9 protein in cell-free supernatants were measured using sandwich ELISAs. Plates were coated with 0.5 µg/ml mouse anti-human IL-1F6 mab (clone; , R&D systems) or 2 µg/ml rat anti-human IL-1F9 mab (clone; , R&D systems) and then 0.1 µg/ml biotinylated goat anti-human IL-1F6 (R&D systems) or 0.5 µg/ml biotinylated goat anti-human IL-1F9 (R&D systems) were used as a detection reagent. The minimal detection limits are 62.5 pg/ml and 750 pg/ml, respectively. The concentrations of IL-33 were measured using a commercial IL-33 ELISA kit (Invitrogen). The

4 minimal detection limit is 15.6 pg/ml. The concentrations of IL-8 and G-CSF were measured using a cytometric bead array assay (CBA, BD Biosciences, San Jose, CA). The minimal detection limit is 5 pg/ml. Statistical Analysis All data are reported as the mean ± SEM unless otherwise noted. Differences between groups were analyzed using the paired Student's t test and considered to be significant if p < Figure E1. Effect of sirna on the expression of target molecules in human bronchial epithelial cells. NHBE were transfected with sirna against control RNA, RELA, or IRF-3 at 5 nm for 48 hours and then stimulated with 5 μg/ml dsrna for 6 hours. The levels of mrnas were determined by real-time PCR. Efficiency of sirna against target molecules was expressed as a percentage of non-sirna transfected cells. The results are shown as the mean ± SEM of five independent experiments. * p < Figure E2. IL-1F9 induces the activation of MAPKs, NF-κB and CREB in human lung fibroblasts. NHLF were harvested at the indicated time points. To detect p38, JNK, ERK, NF-κB p65 and CREB, 30 μg of whole cell lysates were resolved on a 4-12% NuPAGE Bis-Tris Gel then proteins were transferred onto a PVDF membrane. Blocked-membranes were incubated over night at 4 C with primary antibodies and then were labeled with infrared secondary antibodies. Protein was detected on an Odyssey Infrared Imaging System. The results are representative of three separate experiments.

5 Supplemental Figure 1 Figure E1. Effect of sirna on the expression of target molecules in human bronchial epithelial cells.

6 Supplemental Figure 2 Figure E2. IL-1F9 induces the activation of MAPKs, NF-κB and CREB in human lung fibroblasts.