The Epithelial Danger Signal IL-1α Is a Potent Activator of Fibroblasts and Reactivator of Intestinal Inflammation
2015; Elsevier BV; Volume: 185; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2015.02.018
ISSN1525-2191
AutoresMelania Scarpa, Sean P. Kessler, Tammy Sadler, Gail West, Craig R. Homer, Christine McDonald, Carol de la Motte, Claudio Fiocchi, Eleni Stylianou,
Tópico(s)Immune Response and Inflammation
ResumoIntestinal epithelial cell (IEC) death is typical of inflammatory bowel disease (IBD). We investigated: i) whether IEC–released necrotic cell products (proinflammatory mediators) amplify mucosal inflammation, ii) the capacity of necrotic cell lysates from HT29 cells or human IECs to induce human intestinal fibroblasts' (HIF) production of IL-6 and IL-8, and iii) whether IL-1α, released by injured colonocytes, exacerbated experimental IBD. Necrotic cell lysates potently induced HIF IL-6 and IL-8 production independent of Toll-like receptors 2 and 4, receptor for advanced glycation end-products, high-mobility group box 1, uric acid, IL-33, or inflammasome activation. IL-1α was the key IEC-derived necrotic cell product involved in HIF cytokine production. IL-1α–positive cells were identified in the epithelium in human IBD and dextran sulfate sodium (DSS)-induced colitis. IL-1α was detected in the stool of colitic mice before IL-1β. IL-1α enemas reactivated inflammation after DSS colitis recovery, induced IL-1 receptor expression in subepithelial fibroblasts, and activated de novo inflammation even in mice without overt colitis, after the administration of low-dose DSS. IL-1α amplifies gut inflammation by inducing cytokine production by mesenchymal cells. IL-1α–mediated IEC–fibroblast interaction may be involved in amplifying and perpetuating inflammation, even without obvious intestinal damage. IL-1α may be a target for treating early IBD or preventing the reactivation of IBD. Intestinal epithelial cell (IEC) death is typical of inflammatory bowel disease (IBD). We investigated: i) whether IEC–released necrotic cell products (proinflammatory mediators) amplify mucosal inflammation, ii) the capacity of necrotic cell lysates from HT29 cells or human IECs to induce human intestinal fibroblasts' (HIF) production of IL-6 and IL-8, and iii) whether IL-1α, released by injured colonocytes, exacerbated experimental IBD. Necrotic cell lysates potently induced HIF IL-6 and IL-8 production independent of Toll-like receptors 2 and 4, receptor for advanced glycation end-products, high-mobility group box 1, uric acid, IL-33, or inflammasome activation. IL-1α was the key IEC-derived necrotic cell product involved in HIF cytokine production. IL-1α–positive cells were identified in the epithelium in human IBD and dextran sulfate sodium (DSS)-induced colitis. IL-1α was detected in the stool of colitic mice before IL-1β. IL-1α enemas reactivated inflammation after DSS colitis recovery, induced IL-1 receptor expression in subepithelial fibroblasts, and activated de novo inflammation even in mice without overt colitis, after the administration of low-dose DSS. IL-1α amplifies gut inflammation by inducing cytokine production by mesenchymal cells. IL-1α–mediated IEC–fibroblast interaction may be involved in amplifying and perpetuating inflammation, even without obvious intestinal damage. 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Epigenetic silencing of tumor necrosis factor alpha during endotoxin tolerance.J Biol Chem. 2007; 282: 26857-26864Crossref PubMed Scopus (115) Google Scholar Necrotic cell lysates (NCLs; diluted 1:10 to 1:1000 in PBS), recombinant human HMGB-1 (10 pg/mL to 5 μg/mL; R&D Systems, Minneapolis, MN), IL-33 (0.01 pg/mL to 10 ng/mL; Peprotech), or recombinant IL-1α (0.1 to 1000 pg/mL) as controls were incubated with HIFs for 24 hours at 37°C, and supernatants were harvested for cytokine enzyme-linked immunosorbent assays (ELISAs). NCLs were prepared by standard methods.30Iyer S.S. Pulskens W.P. Sadler J.J. Butter L.M. Teske G.J. Ulland T.K. Eisenbarth S.C. Florquin S. Flavell R.A. Leemans J.C. Sutterwala F.S. Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome.Proc Natl Acad Sci U S A. 2009; 106: 20388-20393Crossref PubMed Scopus (538) Google Scholar, 31Sauter B. Albert M.L. Francisco L. Larsson M. Somersan S. Bhardwaj N. Consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells.J Exp Med. 2000; 191: 423-434Crossref PubMed Scopus (1216) Google Scholar The IEC line HT29, fresh IECs, lamina propria mononuclear cells, primary human polymorphonuclear neutrophils, and human monocytes were suspended in RPMI, 10% FBS medium (Lonza) containing 2.5% v/v penicillin, streptomycin, and amphotericin B, 1.5% v/v 1 mol/L HEPES buffer. THP1 cells were suspended in RPMI 10% v/v FBS medium containing 0.5 mmol/L 2-mercaptoethanol. Cells were washed, resuspended at 108 cells/mL in PBS, and subjected to five freeze–thaw cycles by placement sequentially in a liquid nitrogen/37°C water bath, followed by centrifugation for 30 minutes at 15,000 × g. The supernatants were collected and are hereafter referred to as NCLs. HIFs were incubated with NCL (at 1:20 dilution in the culture medium) for 24 hours, then supernatants were collected. Levels of human IL-6 and IL-8 (BD Biosciences, San Jose, CA) released into the HIF culture medium and of IL-1α (BioLegend, San Diego, CA) and IL-1β (BD Biosciences) in the NCLs were determined using ELISA kits following the manufacturer's protocols. For HIF Toll-like receptor (TLR) gene expression analysis, total RNA was extracted from nonstimulated HIFs and reverse-transcribed, and cDNA was amplified by RT-PCR. Five microliters of cDNA was amplified in the presence of 0.125 μmol/L each of the 5′ and 3′ primers (Biosynthesis, Lewisville, TX) and 1 U of Taq DNA polymerase (Roche Diagnostics Deutschland, Mannheim, Germany). PCR was performed in a DNA thermal cycler using preoptimized temperatures and times, with the following primers: TLR1: forward 5′-CTATACACCAAGTTGTCAGC-3′, reverse 5′-GTCTCCAACTCAGTAAGGTG-3′; TLR2: forward 5′-GCCAAAGTCTTGATTGATTGG-3′, reverse 5′-TTGAAGTTCTCCAGCTCCTG-3′; TLR3: forward 5′-AAATTGGGCAAGAACTCACAGG-3′, reverse 5′-GTGTTTCCAGCGCCGTGCTAA-3′; TLR4: forward 5′-TGGATACGTTTCCTTATAAG-3′, reverse 5′-GAAATGGAGGCACCCCTTC-3′; TLR5: forward 5′-CTAGCTCTTAATCCTGATG-3′, reverse 5′-CCATGTGAAGTCTTTGCTGC-3′; TLR6: forward 5′-GAGGAAGCCCACTAAAGGAC-3′, reverse 5′-GGGAGACAAAACAAAGATGGAC-3′; TLR7: forward 5′-TCACCCTCACCATTAACCAC-3′, reverse 5′-TTTCAGGGAGAGCACTTTTAAC-3′; TLR8: forward 5′-TGTGGAATGATGATGACAACAG-3′, reverse 5′-AGAAAGAAAGCCTTGTGCC-3′; and TLR9: forward 5′-GGAAAGAGGAAGGGGTGAAG-3′, reverse 5′-TGAGGGACAGGGATATGAGG-3′. As a positive control, RNA was obtained from peripheral blood mononuclear cells. Fifteen microliters of the PCR product were subjected to electrophoresis on 1.5% Agarose gel and stained with 0.5 μg/mL ethidium bromide, using 100-BP DNA ladder as a marker. Genomic DNA was extracted from HT29 NCLs by incubation in 700 μL of lysis buffer (20 mmol/L Tris, pH8, 20 mmol/L EDTA, 2% SDS, 0.5 mg/mL proteinase K) at 55°C overnight with gentle shaking. Samples were mixed with an equal volume of phenol: chloroform in phase-lock gel tubes. DNA was then precipitated with cold ethanol (100%, 700 μL) and incubated for 1 hour at room temperature or overnight at −20°C. DNA was pelleted at maximum speed in a microcentrifuge at 4°C for 5 minutes and decanted, and 500 μL of 70% ethanol was added to wash the pellet. Samples were spun in a microcentrifuge at maximum speed at room temperature for 5 minutes, and DNA pellets were air-dried and resuspended in distilled water. Histones were extracted from HT29 nuclei (0.5 to 1 × 108) by resuspension of the nuclear pellet in 0.4 normality sulfuric acid and gentle shaking for 2 to 16 hours at 4°C as previously described.32Shechter D. Dormann H.L. Allis C.D. Hake S.B. Extraction, purification and analysis of histones.Nat Protoc. 2007; 2: 1445-1457Crossref PubMed Scopus (716) Google Scholar Native chromatin was extracted from HT29 with the ChIP-It Express enzymatic kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions, with modifications as detailed previously.33Sadler T. Scarpa M. Rieder F. West G. Stylianou E. 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Briefly, cells were washed in ice-cold phosphate-buffered saline and scraped, on ice, in a hypotonic buffer containing additional proteinase and phosphatase inhibitors [buffer A: 10 mmol/L HEPES, pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol (DTT), 100 μmol/L phenanthroline, 1 μg/mL pepstatin, 100 μmol/L E64, 100 μmol/L DCI, 10 mmol/L NaF, 100 μmol/L Na3VO4, and 25 mmol/L β-glycerophosphate]. Cells were then lysed by incubating for 10 minutes on ice in 60 to 80 μL hypotonic buffer containing 0.2% NP40. Lysates were then centrifuged (10,000 × g at 4°C) for 10 minutes, and the supernatants were discarded. The pelleted nuclei were resuspended in 60 to 80 μL of a lysis buffer [buffer C: 20 mmol/L HEPES, pH 7.9, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L ethylenediaminetetraacetic acid (EDTA), 25% glycerol, and 100 μmol/L DCI] and were incubated at 4°C for 15 minutes. Lysed nuclei were vortexed then centrifuged (10,000 × g at 4°C) for 10 minutes and supernatants were snap frozen and stored at −80°C. RNA and/or proteins were depleted from NCLs by digestion with RNase (7 U/106 cells; 5 PRIME, Gaithersburg, MD) or proteinase K (20 μg/106 cells, 5 PRIME) for 1 hour at 37°C. RNA and protein content was evaluated by Agarose gel electrophoresis and SDS-PAGE, respectively. Complete digestion was confirmed in each case (data not shown). Spectrophotometry (NanoDrop; Thermo Scientific, Rochester, NY), was used to quantify RNA levels. One hour before stimulation with the NCLs, HIFs were incubated with the following antibodies: anti-TLR2 (5 μg/mL; InvivoGen), anti-TLR4 (5 μg/mL; maba2-htlr4; InvivoGen), anti–receptor for advanced glycation end-products (RAGE; 5 μg/mL; AF1145; R&D Systems), anti–IL-33 (0.01 pg/mL to 10 ng/mL; Peprotech), anti–IL-1 receptor (IL-1RI; 2.5 μg/mL; AF269; R&D Systems), anti–HMGB-1 (5 or 10 μg/mL), soluble RAGE (5 or 10 μg/mL), or an IgG control antibody (5 or 10 μg/mL). NCLs were also digested with uricase (10 U/mL) for 30 minutes at 25°C for removal of monosodium urate crystals before being diluted 1:20 and incubated with HIFs. Supernatants were harvested 24 hours later, and IL-6 and IL-8 ELISAs were performed. The functionality of TLR2 and TLR4 expressed by HIFs was tested by exposing the cells to their respective specific ligands, synthetic diacylated glycoprotein 1, a TLR2/TLR6 agonist (1 μg/mL), and LPS (1 μg/mL), in the presence or absence of neutralizing antibodies against TLR2 and TLR4. HIFs were preincubated for 1 hour with 5 μg/mL neutralizing anti-human TLR2 antibody (InvivoGen) followed by 1 μg/mL ligand fibroblast-stimulating ligand 1, or with 5 μg/mL neutralizing anti-human TLR4 antibody (InvivoGen) followed by 1 μg/mL ligand LPS for 24 hours. IL-6 and IL-8 levels in the supernatants were measured by ELISA. To neutralize IL-1α and IL-1β, NCLs were diluted 1:20 in cell medium and incubated for 1 hour with each specific anti–IL-1 antibody [1 μg/mL; anti–IL-1α (AF-200-NA) or anti–IL-1β (AF-201-NA); R&D Systems]. The IL-1–neutralized NCLs were then incubated with HIFs for 24 hours. The optimal antibody concentration was determined for each antibody by using concentrations ranging from 0.1 to 5 μg/mL. For neutralization of uric acid, NCLs were incubated with 10 U/mL uricase for 30 minutes at 25°C. At the end of each treatment, lysates were diluted 1:20 and incubated with HIFs for 24 hours. Transfection of HIFs and HT29 cells was performed by Nucleofection technology (Amaxa, Koln, Germany) according to the manufacturer's instructions, using a control siRNA (2 μmol/L; ON-TARGETplus nontargeting pool, D-001810-10-05; GE Dharmacon, Lafayette, CO) or an siRNA targeting human IL-1α (ON-TARGETplus SMARTpool siRNA, L-007952-00-0005; GE Dharmacon). Transfected HIFs were incubated with NCL (at 1:20 dilution in growth medium) 24 hours after transfection, and then supernatants were collected 24 hours later for cytokine measurements. Transfected HT29 cells were rendered necrotic 48 hours after transfection, as described in the Preparation of NCLs section. The efficiency of silencing was evaluated 48 hours after transfection by measurement of the levels of IL-1α and β-actin mRNA by real-time RT-PCR. The following primers were used: IL1A: forward 5′-AGTGCTGCTGAAGGAGATGCCTG-3′, reverse 5′-CCCTGCCAAGCACACCCAGTAG-3′; ACTB: forward 5′-CTGGACTTCGAGCAAGAGATG-3′, reverse 5′-AGTTGAAGGTAGTTTCGTGGATG-3′. Confluent HT29 cells were infected with log-phase cultures of either Shigella flexneri strain M90T or the noninvasive strain BS176 (gifts from Philippe Sansonetti, Institute Pasteur, Paris, France) at a multiplicity of infection of 50 for 8 hours. Cells were washed with PBS, and infection was continued in Dulbecco's modified Eagle's medium, 10% FBS supplemented with 100 μg/mL gentamicin, for an additional 16 hours. Medium was collected and centrifuged for 5 minutes at 250 × g, and supernatants were snap-frozen at −80°C before vacuum concentration and assessment of IL-1α release by ELISA. Infected cells were analyzed by fluorescence flow cytometry on an LSR II FACScan (BD Biosciences) to determine the number of necrotic cells using the Live/Dead Fixable Cell Stain Kit (L23105; Life Sciences, Grand Island, NY) according to manufacturer's instructions. Noninfected and dead cells were used as positive and negative controls, respectively, and 10,000 events were captured. The percentage of cells undergoing necrosis was calculated relative to infected control cells. HT29 cells (1 × 106) were seeded into 6-well plates for 24 hours. Cells were then washed and grown in medium containing 2% FCS for 24 hours before either being incubated in 2% growth medium or primed with 10 ng/mL to 1 μg/mL ultrapure LPS (InvivoGen) for 3 hours at 37°C. HT29 cultures were then incubated with either 100 ng/mL TNF-α, 5 mmol/L ATP, or 0.1 to 0.6 μg/mL monosodium urate crystals for 24 hours at 37°C. Supernatants were harvested, cytokine (IL-1α, IL-1β, and IL-8) levels were quantified by ELISA, and the remaining cells were lysed. Protein in cell lysates was quantified (BCA protein assay; Pierce, Rockford, IL), and all ELISA assays were normalized to lysate protein content. Mice (male; C57Bl/6; Jackson ImmunoResearch Laboratories, West Grove, PA) were conventionally housed, and all husbandry and treatments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Cleveland Clinic. For the induction of DSS colitis, mice were allowed to drink sterile, acidified water in the presence or absence of added 1.5% or 3.0% w/v DSS (MP Biomedicals, Solon, OH) ad libitum. Mice were euthanized on days 0, 3, 5, and 7 (three mice per group). In some experiments, mice were given water for 21 days after 7 days of DSS. On day 28, mice were administered a 45% ethanol enema 30 minutes before receiving an IL-1α enema (10 ng in 150 μL PBS). Mice were then euthanized 24 hours after the IL-1α enema. In each mouse, the colon from the cecum to the rectum was dissected and removed. The tissue was then fixed whole in Histochoice (Sigma, St. Louis, MO), paraffin-embedded, and sectioned for hematoxylin and eosin staining by the Lerner Research Institute, Histological Examination Core, Cleveland Clinic. The histopathological degree of inflammation in the colonic sections was scored as previously reported,35Kessler S. Rho H. West G. Fiocchi C. Drazba J. de la Motte C. Hyaluronan (HA) deposition precedes and promotes leukocyte recruitment in intestinal inflammation.Clin Transl Sci. 2008; 1: 57-61Crossref PubMed Scopus (65) Google Scholar and images were obtained using a microscope (Leica Microsystems, GmbH, Wetzlar, Germany), with Image-Pro Plus version 7 (Media Cybernetics, Rockville, MD) and Photoshop version 9 (Adobe Systems, San Jose, CA) software. Paraffin sections of mouse colon were rehydrated and incubated in a solution of Hanks' balanced salt solution (HBSS) containing 2% FBS for 30 minutes to block nonspecific antibody binding. Subsequently, the slides were incubated overnight at 4°C with 15 μg/mL specific goat polyclonal antibody against murine IL-1α (R&D Systems) diluted in HBSS + 2% FBS. The slides were washed three times in HBSS and then incubated with a solution containing Alexa 488–tagged donkey anti-goat IgG (1:1000; Molecular Probes, Eugene, OR) in HBSS + 2% FBS, for 1 hour at 25°C. The slides were washed three times in HBSS and coverslips mounted in Vectashield containing DAPI (Vector Laboratories, Inc., Burlingame, CA), sealed, and stored at −20°C until visualization. Images were obtained using a TCS-SP laser scanning confocal microscope (Leica). Deidentified human colonic tissue was obtained from individuals undergoing colonic resection, fixed in Histochoice (AMRESCO, Solon, OH), and embedded in paraffin. Antibody staining was performed on serial 4-μm sections on a Benchmark XT automated immunostainer (Ventana Medical Systems, Inc., Tucson, AZ) using an iVIEW DAB Detection Kit (Ventana) with no cell conditioning and minus the provided secondary. Primary antibody, polyclonal rabbit anti–IL-1α (catalog #PA5-25921; Thermo Scientific–Pierce) was diluted 1:50 and incubated with sections for 60 minutes followed by goat anti-rabbit biotinylated secondary antibody (Vector Laboratories) used at 1:200 for 8 minutes. Paraffin-embedded sections of HeLa cells were used as a positive control (not shown), and negative controls comprised HeLa and colon tissue incubated with secondary antibody only. Images were taken on a DMR upright microscope (Leica) with a Retiga EXi Cooled CCD camera with liquid crystal tunable RGB filter (QImaging, Surrey, BC, Canada) using Image-Pro
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