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Modulation of interleukin-1β-induced inflammatory responses by a synthetic cationic innate defence regulator… Turner-Brannen, Emily; Choi, Ka-Yee; Lippert, Dustin N; Cortens, John P; Hancock, Robert E; El-Gabalawy, Hani; Mookherjee, Neeloffer Aug 11, 2011

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RESEARCH ARTICLE Open AccessModulation of interleukin-1b-inducedinflammatory responses by a synthetic cationicinnate defence regulator peptide, IDR-1002, insynovial fibroblastsEmily Turner-Brannen1, Ka-Yee Choi1,2, Dustin ND Lippert1, John P Cortens1, Robert EW Hancock3,Hani El-Gabalawy1 and Neeloffer Mookherjee1,2*AbstractIntroduction: Innate defence regulator (IDR) peptides are synthetic cationic peptides, variants of naturallyoccurring innate immune effector molecules known as host defence peptides. IDR peptides were recentlydemonstrated to limit infection-associated inflammation selectively without compromising host innate immunefunctions. This study examined the impact of a 12-amino acid IDR peptide, IDR-1002, in pro-inflammatory cytokineinterleukin (IL)-1b-induced responses in synovial fibroblasts, a critical cell type in the pathogenesis of inflammatoryarthritis.Methods: Human fibroblast-like synoviocytes (FLS) were stimulated with IL-1b in the presence and absence of IDR-1002. Production of enzyme matrix metalloproteinase-3 (MMP-3) and IL-1-receptor antagonist (IL-1RA) wasmonitored by enzyme-linked immunosorbent assay (ELISA), and various chemokines were evaluated by usingmultiplex cytometric bead array. Transcriptional responses were analyzed by quantitative real-time PCR. The impacton IL-1b-induced proteome was investigated by quantitative proteomics by using isobaric tags. IL-1b-inducedpathways altered by IDR-1002 implicated by the proteomics analyses were further investigated by using variousimmunochemical assays. Cellular uptake of the peptide was monitored by using a biotinylated IDR-1002 peptidefollowed by microscopy probing with streptavidin-Alexa Fluor.Results: This study demonstrated that IDR-1002 suppressed the production of IL-1b-induced MMP-3 and monocytechemotactic protein-1 (MCP-1); in contrast, IDR-1002 enhanced the production of IL-1RA, without neutralizing allchemokine responses. IDR-1002 altered the IL-1b-induced proteome primarily by altering the expression ofmembers of nuclear factor kappa-B (NF-B) and c-Jun N-terminal kinase (JNK) pathways. The proteomics data alsosuggested that IDR-1002 was altering the transcription factor HNF-4a-mediated responses, known to be critical inmetabolic regulation. With various immunochemical assays, it was further demonstrated that IL-1b-induced NF-B,JNK, and p38 mitogen-activated protein kinase (MAPK) activations were significantly suppressed by IDR-1002.Conclusions: This study demonstrates the ability of an innate immune-modulatory IDR-peptide to influence the IL-1b-induced regulatory pathways and selectively to suppress inflammatory responses in synovial fibroblasts. Theresults of this study provide a rationale for examining the use of IDR-peptides as potential therapeutic candidatesfor chronic inflammatory diseases such as inflammatory arthritis.* Correspondence: mookherj@cc.umanitoba.ca1Manitoba Centre for Proteomics and Systems Biology, Department ofInternal Medicine, University of Manitoba, 799 John Buhler Research Centre,715 McDermot Avenue, Winnipeg, MB, R3E3P4, CanadaFull list of author information is available at the end of the articleTurner-Brannen et al. Arthritis Research & Therapy 2011, 13:R129http://arthritis-research.com/content/13/4/R129© 2011 Turner-Brannen et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.IntroductionCationic host defense peptides (HDPs) are naturallyoccurring effector molecules of innate immunity. Thesepeptides are 12 to 50 amino acids in length, with a netpositive charge ranging from +2 to +7 with up to 50%hydrophobic amino acids [1]. HDPs exhibit a wide vari-ety of immunomodulatory functions and delicately mod-ulate inflammatory responses without compromising theelements of immunity required for resolution of infec-tions [2-8]. HDPs exhibit anti-inflammatory effects bysuppressing certain pro-inflammatory pathways, upregu-lating anti-inflammatory mechanisms (for example, IL-10), and intervening in the activation of nuclear factor(NF)-B via multiple mechanisms [3]. A broad spectrumof cationic HDPs are expressed in human synovium tis-sues with differential expression patterns under inflam-matory conditions [9]. However, the role of HDPs insynovium biology is not well characterized. It has beensuggested that induction of HDPs by vitamin D mayplay a role in the protection against autoimmune dis-eases such as rheumatoid arthritis (RA) [10]. Therefore,HDPs and their derivatives are attractive candidates formodulating the inflammatory responses in chronicinflammatory disorders, including in inflammatoryarthritis.HDPs are widely diverse in sequence and structure,and this wide repertoire provides an extensive templatefor designing short synthetic peptides with optimizedactivities and reduced cytotoxicities [11-13]. The syn-thetic variants of HDP are known as innate defence reg-ulator (IDR) peptides [14]. Two IDR peptides, IDR-1and IDR-1002, have been shown to protect againstinfections largely by modulating innate immuneresponses of the host and upregulating anti-inflamma-tory mechanisms [15,16]. To our knowledge, no studiesto date have investigated the potential of IDR peptidesin limiting inflammation in immune-mediated chronicinflammatory disorders such as inflammatory arthritis.The complex pathophysiology of arthritis involvessynergistic interplay primarily between mesenchymalcells such as fibroblast-like synoviocytes (FLS) andimmune cells (for example, macrophages and T-lympho-cytes). Activation of FLS by pro-inflammatory cytokinesresults in the production of inflammatory cytokines,chemokines, and matrix-degrading matrix metallopepti-dases (MMPs), which lead to the destruction of articularcartilage and bone [17]. TNF-a and IL-1b are twoinflammatory cytokines that are well defined as criticalinflammatory mediators in arthritis [18]. TNF-a is pro-posed to be the dominant pro-inflammatory cytokine inthe inflammatory manifestations of synovitis, whereasIL-1b is thought to be important in the destructivepotential of chronic joint inflammation [19,20]. IL-1binduces the production of MMP-3 in cell types such asFLS, chondrocytes, and macrophages in arthritic joints,and the subsequent elevated level of MMP-3 mediatescartilage and bone destruction, directly contributing tothe pathogenesis of the disease [21]. Efficacious thera-peutic strategies have been developed to target each ofthese cytokines. Despite this, a major considerationregarding therapeutic agents targeting inflammatorycytokines such as TNF-a is the increased associated riskof infections and neoplasm [22,23]. This highlights theneed for the development of alternate strategies for themanagement of chronic inflammatory arthropathies. Wehypothesized that one such strategy would be to exam-ine the use of selectively immune-modulatory agentssuch as IDR peptides [24].In this study, we demonstrated that a 12-amino-acidIDR peptide, IDR-1002 [16], suppressed IL-1b-mediatedcellular responses in human FLS, especially MMP-3 andMCP-1 production. However, IDR-1002 did not neutra-lize all IL-1b-induced chemokine responses that arerequired for resolution of infections. In contrast, thispeptide enhanced the production of negative regulatorsof IL-1b (for example, IL-1-receptor antagonist (IL-1RA). These observations were consistent with the para-digm of the selective anti-inflammatory mechanism ofhost defense and IDR peptides [3,15,16]. We exploredthe molecular mechanism of regulation of IL-1b-inducedresponses by IDR-1002 in FLSs, by using quantitativeproteomics and other immunochemical assays. Wedemonstrated that IDR-1002 suppressed IL-1b-inducedNF-B, c-Jun kinase (JNK), and p38 mitogen-activatedprotein kinase (MAPK) activation in synovial fibroblasts.This study provides a rationale for further examiningthe use of IDR peptides as potential therapeutics for themanagement of inflammatory arthritis and possiblyother diseases characterized by chronic inflammation.Materials and methodsCell isolation and cultureSynovial tissues were obtained from patients withosteoarthritis (OA) or rheumatoid arthritis (RA) withinformed consent in accordance with a protocolapproved by the Institutional Review Board at the Uni-versity of Manitoba. Human FLS were isolated from thesynovial tissues, as previously described [25]. In brief,the tissues were digested with 1 mg/ml collagenase and0.05 mg/ml hyaluronidase (Sigma Aldrich) in Hanks’balanced salt solution (Gibco; Invitrogen Inc., Burling-ton, ON, Canada) for 1 to 2 hours at 37°C. Cells werewashed and cultured in DMEM media (Gibco), supple-mented with sodium pyruvate and nonessential aminoacids (referred to as complete DMEM media henceforth)containing 10% (vol/vol) fetal bovine serum (FBS) in aTurner-Brannen et al. Arthritis Research & Therapy 2011, 13:R129http://arthritis-research.com/content/13/4/R129Page 2 of 14humidified incubator at 37°C and 10% CO2. Isolatedhuman FLS (ex vivo) were seeded at 2 × 104 cells/ml,either 0.5 ml per well in 48-well tissue-culture plate, or3 ml per well in six-well tissue-culture plate, as required,and cultured in complete DMEM media containing 10%(vol/vol) FBS overnight. The following day, the culturemedia was changed to complete DMEM containing 1%(vol/vol) FBS before the addition of the various stimu-lants. A rabbit synoviocyte cell line HIG-82 (ATCCCRL-1832) was cultured in Ham’s F-12 growth mediumcontaining glutamine (Gibco) supplemented withsodium pyruvate (referred to as complete F-12 mediahenceforth), containing 10% (vol/vol) FBS in a humidi-fied incubator at 37°C and 5% CO2. Confluent humanFLS or HIG-82 cells were trypsinized with 1:3 dilutionof 0.5% trypsin-EDTA (Invitrogen) in Hanks’ balancedsalt solution. Cellular cytotoxicity was evaluated bymonitoring the release of lactate dehydrogenase (LDH)by using a colorimetric detection kit (Roche Diagnostics,Laval, QC, Canada).Peptides and recombinant cytokinesRecombinant human cytokines TNF-a and IL-1b wereobtained from eBioscience, Inc (San Diego, CA, USA).IDR-1002 peptide (VQRWLIVWRIRK-NH2) [16] wassynthesized by using F-moc chemistry at the NucleicAcid/Protein Synthesis Unit of University of BritishColumbia, Vancouver, BC, Canada, and IDR-1 peptide(KSRIVPAIPVSLL-NH2) [15] was obtained from Gen-Script USA Inc. (Piscataway, NJ, USA). The peptideswere resuspended in endotoxin-free water, aliquoted,and stored at -20°C. Based on previous studies demon-strating the anti-inflammatory and anti-infective proper-ties of the IDR peptides, in in vitro cell studies and invivo models of various infections models [15,16], and onpreliminary dose-titration studies, standard doses wereused for IDR-1002 (100 μg/ml) and IDR-1 (200 μg/ml)for all experiments.ELISA and multiplex flow cytometryTissue culture (TC) supernatants were centrifuged at1,500 g for 7 minutes to obtain cell-free samples, ali-quoted, and stored at -20°C until further use. Produc-tion of MMP-3 was monitored by using Quantikinehuman MMP-3 (total) ELISA kit (R&D Systems, Inc.Minneapolis, MN, USA), as per the manufacturer’sinstructions. Production of IL-1RA was monitored inthe TC supernatants by using specific antibody pairsfrom eBioscience, Inc. The production of chemokinesIL-8, RANTES, MIG, MCP-1, IP-10 was determined byusing a preconfigured multiplex BDCytometric BeadArray (CBA) human chemokine kit by using the FACSCalibur flow cytometer (BD Biosciences, Mississauga,ON, Canada) as per the manufacturer’s instructions.The concentration of the cytokines or chemokines inthe TC supernatants was evaluated by establishing astandard curve with serial dilutions of the recombinanthuman cytokines or chemokines, as required.Quantitative real-time (qRT-PCR)Human FLS were stimulated with either IL-1b (10 ng/ml), IDR-1002, or the combination of IL-1b and IDR-1002, for 2 hours. RNA was isolated by using the Qia-gen RNeasy kit as per the manufacturer’s instructions.Gene expression was subsequently analyzed with qRT-PCR by using SuperScript III Platinum Two-Step qRT-PCR Kit with SYBR Green (Invitrogen), according to themanufacturer’s instructions, in the ABI PRISM 7300sequence-detection system (Applied Biosystems). Foldchanges were calculated by the comparative Ct method[26], after normalization with 18sRNA. The list of pri-mers used is shown in Table 1.Quantitative proteomics using isobaric tag for relativeand absolute quantitation (iTRAQ)Amine-modifying iTRAQ reagents multiplex kit(Applied Biosystems) was used for relative quantitationof proteins in human FLS stimulated with IL-1b in thepresence and absence of IDR-1002 compared with unsti-mulated (control) cells. Human FLS (2 × 104/ml) wereseeded in a total volume of 3 ml per well in a six-welltissue-culture plate in complete DMEM media contain-ing 10% FCS. The cells were allowed to adhere over-night. The following day, the media was changed to 3ml complete DMEM containing 1% FCS per well. Thecells were either unstimulated or treated with IL-1b (10ng/ml) in the presence or absence of IDR-1002. Thepeptide was added 45 min before stimulation with IL-1b. After 24 hours of stimulation, the cells were washedwith cold PBS and lysed in 250 μl of buffer containing10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA,1% NP-40, and protease inhibitor cocktail (Sigma-Aldrich), on ice for 30 minutes with intermittent vortex-ing. Cells were centrifuged at 10,000 g for 10 minutes at4°C. Total protein content was estimated in each celllysate by using micro BCA assay (Pierce; Thermo Scien-tific, Rockford, IL, USA) with a bovine serum albumin(Sigma-Aldrich) standard curve. The samples were acet-one precipitated at -20°C overnight. Proteins were dis-solved in 20 μl of iTRAQ dissolution buffer (AppliedTable 1 Summary of primers used for quantitative real-time PCRGene Forward primer Reverse PrimerIL-1RA ttggaaggctctgaacctca ctgaaggcttgcatcttgctSIGIRR ctcagagccatgccaggt cctcagcacctggtcttca18sRNA gtaacccgttgaaccccatt ccatccaatcggtagtagcgTurner-Brannen et al. Arthritis Research & Therapy 2011, 13:R129http://arthritis-research.com/content/13/4/R129Page 3 of 14Biosystems) and further processed as per the manufac-turer’s instructions. In brief, proteins were reduced andthe cysteines blocked by using the reagents in the kit,followed by digestion of the protein samples with pro-vided trypsin solution overnight at 37°C. The trypsin-digested protein samples were labelled with the iTRAQisobaric tags as follows: unstimulated (control) samplewas labeled with iTRAQ isobaric tag 115; IL-1b-stimu-lated sample, with tag 116; and the isobaric tag 117 wasused for labeling the sample obtained from cells treatedwith IL-1b in the presence of IDR-1002. The contentsfrom each of the iTRAQ reagent-labeled sample wascombined together in 1:1 ratio and processed for nano-flow liquid chromatography coupled to tandem massspectrometry (LC-MS/MS) by using a QStar Elite massspectrometer (ABSciex, Toronto, ON, Canada).Monitoring activation of NF-BRabbit synoviocyte HIG-82 cells were transiently trans-fected with pNFB-MetLuc2-Reporter Vector (ClontechLaboratories Inc., Mountain View, CA, USA) or the pro-vided control vector as per the manufacturer’s instruc-tions. Various stimulants were added to the transfectedcells in culture media containing 1% (vol/vol) FBS. Thecells were stimulated with recombinant human IL-1b inthe presence and absence of IDR-peptides, either IDR-1002 or IDR-1, for 6 hours. The peptides were added atthe time of cytokine stimulation. The activation of NF-B was monitored by using the Ready-To-GlowSecreted NF-B Luciferase Reporter Assay (Clontech) asper the manufacturer’s instructions.Human OA FLS were stimulated with IL-1b in thepresence and absence of IDR-peptides, IDR-1002 andIDR-1. The peptides were added either 45 minutesbefore, or at the time of cytokine stimulation. Nuclearextracts were prepared by using NE-PER extractionreagents (Thermo Fisher Scientific) as per the manufac-turer’s instructions. Nuclear extracts (5 μg) wereresolved on 4% to 12% NuPAGE Bis-Tris gels (Invitro-gen) and probed with antibodies specific for either NF-B subunit p50 (Cell Signaling Technology) or antibodyto b-actin (Thermo Fisher Scientific) by usingimmunoblots.Monitoring functional JNK activityHuman FLS (5 × 104/ml) were seeded in a total volumeof 20 ml per 75-cm2 tissue-culture flask in completeDMEM media containing 10% (vol/vol) FBS for eachcondition. The cells were allowed to adhere overnight.The next day, the media was changed to 10 ml completeDMEM containing 1% (vol/vol) FBS. The cells wereeither unstimulated or treated with IL-1b (10 ng/ml) inthe presence or absence of IDR-1002 for 15 minutes.IL-1b is known to induce JNK activation after 15minutes in human FLSs [27]. Total protein concentra-tion was evaluated for each cell lysate by using microBCA (Thermo Scientific). Kinase activity specific to JNKwas monitored by using the JNK activity assay kit(Abcam Inc.) as per the manufacturer’s instructions. Inbrief, 20 μg of total protein per cell lysate was used forimmunoprecipitation (IP) by using a JNK-specific anti-body. The eluate was treated with c-Jun substrate andATP mixture. Subsequent phosphorylation of the c-Junsubstrate was evaluated by probing immunoblots withanti-phospho-c-Jun (Ser73)-specific antibody.Monitoring p38 MAPK activityHuman FLS (5 × 104/ml) were seeded in a total volumeof 20 ml per 75-cm2 tissue culture flask in completeDMEM media containing 10% (vol/vol) FBSs for eachcondition, allowed to adhere overnight, followed bychanging the media to 1% (vol/vol) FBS. The cells wereeither unstimulated or treated with IL-1b (10 ng/ml) inthe presence or absence of either IDR-1002 or IDR-1for 15 minutes. The peptides were added either (a) 45minutes before cytokine stimulation, or (b) simultaneouswith cytokine stimulation. The p38 MAPK activationhas been demonstrated in human FLSs on stimulationwith IL-1b for 15 minutes [28]. Total protein concentra-tion was evaluated for each cell lysate by using microBCA (Thermo Scientific). Kinase activity specific to p38MAPK was monitored by using the p38 MAPK activityassay kit (Cell Signaling Technology) as per the manu-facturer’s instructions. In brief, 10 μg of total proteinper cell extract was used for IP by using a p38 MAPK-specific monoclonal antibody. Kinase activity was evalu-ated by treating the IP eluates in the presence of ATPand kinase substrate ATF-2 fusion protein. Phosphoryla-tion of the substrate ATF-2 was monitored with Wes-tern blot by using a phospho-ATF-2 (Thr76) antibody.ImmunoblotsThe IP eluates or nuclear extracts were electrophoreti-cally resolved on a 4% to 12% NuPAGE Bis-Tris gels(Invitrogen Corporation), followed by transfer to nitro-cellulose membranes (Millipore). The nitrocellulosemembranes were blocked with TBST (20 mM Tris pH7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% (vol/vol) skimmed milk powder. Affinity-purified HRP-linkedanti-rabbit secondary antibody was used for detection.The membranes were developed with Amersham ECLdetection system (GE Healthcare, Baie d’Urfe, QC,Canada) according to the manufacturer’s instructions.MicroscopyA modified IDR-1002 was synthesized by incorporatinga C-terminal cysteine (IDR-1002C) to allow the presenceof a thiol group for biotinylation of the peptide. IDR-Turner-Brannen et al. Arthritis Research & Therapy 2011, 13:R129http://arthritis-research.com/content/13/4/R129Page 4 of 141002C was biotinylated, as previously described [29]. Inbrief, IDR-1002C was biotinylated by using desthiobiotinpolyethyleneoxide iodoacetamide (Sigma) as per themanufacturer’s instructions. Biotinylated IDR-1002 pep-tide (IDR-1002B) was purified by HPLC and confirmedby using MALDI mass spectrometry. To facilitate moni-toring cellular uptake of the peptide, 150 μl of humanFLS (2 × 104/ml) was seeded in 96-well glass-bottomNunc plates in DMEM containing 10% (vol/vol) FBS,overnight. The following day, the media was changed toDMEM containing 1% (vol/vol) FBS. The cells were sti-mulated with IDR-1002B (100 μg/ml) for 0, 15, or 30min. The cells were fixed by using 2% (vol/vol) para-for-maldehyde, the reaction quenched with 10 mM ethano-lamine and permeabilized with 0.1% Triton × 100. Thecells were washed in PBS and blocked with 3% (vol/vol)FBS in PBS. The cells were stained for actin by usingAlexa Fluor 546 phalloidin (Invitrogen) and stained withStreptavidin Alexa Fluor 488 conjugate (Invitrogen) todetect biotin. The cells were counterstained withHoescht 33258 (Invitrogen) for nuclear staining.ResultsIDR-1002 suppressed IL-1b-induced MMP-3, but inducednegative regulators of IL-1b in human FLSIL-1b induces the production of MMP-3 in FLS, whichcontributes to the destruction of cartilage and bone inarthritic joints [21]. We therefore evaluated the impactof IDR peptides on IL-1b-induced MMP-3 production.Human FLS isolated from OA and RA synovial tissueswere stimulated with IL-1b (10 ng/ml) in the presenceand absence of IDR peptides. The peptides were addedat the time of cytokine stimulation. The peptides werenot cytotoxic to the FLS under any experimental condi-tion, as determined by monitoring the TC supernatantsfor the release of LDH after 24 hours of stimulation(data not shown). TC supernatants were monitored after24 hours of stimulation for MMP-3 production byELISA. IL-1b-induced MMP-3 production was quantita-tively similar in OA and RA FLS (Figure 1). IL-1b-induced MMP-3 was significantly suppressed by 70% ±8% (P < 0.05) in the presence of IDR-1002 in OA FLS(Figure 1a). IL-1b-induced MMP-3 was also significantlysuppressed by 61% ± 14% (P < 0.05) in the presence ofIDR-1002 in RA FLS (Figure 1b). In contrast, IDR-1 [15]did not significantly suppress IL-1b-induced MMP-3production in either OA or RA FLS (Figure 1). Previousstudies have shown that human primary OA FLS afterstimulation with a pro-inflammatory cytokine such asIL-1b, upregulates the expression of MMP-3, MMP-13,MMP-1, various chemokines, activates transcription fac-tor NF-B, induces the phosphorylation of ERK, p38,and JNK MAPK, and phosphorylation of AKT, all criti-cal in the induction of inflammatory responses [30,31].Therefore, we proceeded to investigate further the cellu-lar responses and molecular mechanism of IDR-1002 byusing human OA FLS stimulated with pro-inflammatorycytokines. Our approach was consistent with other stu-dies that have used similar ex vivo methods with FLSsfrom OA tissues to investigate potential therapeutic tar-gets and molecular mechanisms of candidate therapeu-tics for anti-inflammatory interventions [31,32].Further to investigate cellular responses in the pre-sence of IDR-1002, human OA FLS were stimulatedwith pro-inflammatory cytokines, and the TC superna-tant was monitored for MMP-3 production after 24hours and IL-1RA production after 48 hours. MMP-3production was increased by threefold on stimulation ofFLS with cytomix, a combination of 10 ng/ml each ofIL-1b and TNF-a (Figure 2a), compared with IL-1balone (Figure 1a). This elevated level of cytomix-inducedMMP-3 was also suppressed by 56% ± 10% (P < 0.05)by the peptide IDR-1002 (Figure 2a). IDR-1002 by itselfdid not induce MMP-3 production above the back-ground amount observed in unstimulated control FLS(Figures 1 and 2a). In contrast, IDR-1002 synergisticallyenhanced the production of IL-1b-induced IL-1RA bythreefold, which was not seen with IDR-1 peptide (Fig-ure 2b). Consistent with this, on monitoring geneexpression of endogenous inhibitors of IL-1b [33], it wasdemonstrated that IDR-1002 by itself induced the geneexpression of IL-1RA by > 10-fold, and did not suppressthe IL-1b-induced gene expression of IL-1RA (Figure2c). Similarly, gene expression of another negative regu-lator of IL-1b, SIGIRR (single Ig IL-1R related molecule,also known as TIR8) was induced more than ninefold (P< 0.05) by the peptide IDR-1002 relative to thatobserved in cells stimulated with IL-1b (Figure 2d).Taken together, these results indicated that IDR-1002suppressed IL-1b-induced pro-inflammatory MMP-3production and in contrast enhanced the expression ofnegative regulators of IL-1b, in human FLS.IDR-1002 altered the IL-1b-induced proteome in humanFLSAs an approach to globally defining the impact of IDR-1002 on IL-1b-induced protein production, we under-took a quantitative proteomic analysis. Human OA FLSwere pretreated with IDR-1002 45 min before IL-1b (10ng/ml) stimulation for 24 hours. The TC supernatantswere monitored for MMP-3 and chemokine production.IDR-1002 significantly (P < 0.01) suppressed IL-1b-induced MMP-3 production by 80% (Figure 3a) andsuppressed chemokine MCP-1 production by > 60%(Figure 3b) after 24 hours. However, IL-1b-induced neu-trophil chemokine IL-8 production (Figure 3c) was onlymodestly suppressed (by 20%, P < 0.05) by the peptide.These observations were consistent with previousTurner-Brannen et al. Arthritis Research & Therapy 2011, 13:R129http://arthritis-research.com/content/13/4/R129Page 5 of 14studies demonstrating that HDP can selectively suppressinflammation without abrogating certain chemokineresponses that are required for cell movement andrecruitment essential to combat infectious assault [3].The FLS lysates were labeled with iTRAQ reagents;isobaric tag 115 for unstimulated (control) samples, tag116 for IL-1b-stimulated cells, and the tag 117 for cellstreated with the combination of IL-1b and IDR-1002.Samples from three independent donors were individu-ally examined by LC-MS/MS. The mass spectrometrydata were analyzed by using ProteinPilot (Applied Bio-systems). Only those proteins that were identified in atleast two of the three independent experiments with95% confidence were selected for further analysis. Intotal, 517 proteins were identified from at least two ofthe three independent samples stimulated with IL-1b.Proteins were defined to be induced if the relative ratioscompared with the unstimulated controls (fold change)were at least mean ± 1.3 standard deviations (whichmeant that 20% of the population was differentiallyexpressed). Forty-eight of the 517 proteins were definedto be induced on stimulation with IL-1b. Of these 48IL-1b-induced proteins, 11 proteins were found to besuppressed by IDR-1002 between 20% and 60% (Addi-tional file 1, Table s1). To define immunity-related path-ways that may be involved in the alteration of IL-1b-induced responses in the presence of IDR-1002, weundertook a network-based approach. The eleven IL-1b-induced protein candidates that were suppressed byIDR-1002 (Additional file 1, Table S1) were submittedto InnateDB biomolecular interaction database, whichfacilitates systems-level analysis of mammalian immunegenes and protein products [34].The computational network analysis demonstratedthat several members of NF-B and the mitogen-acti-vated protein kinase-8 (MAPK8) pathways were directinteractors of the selected protein candidates (Additionalfile 1, Table S2). Four of the 11 selected proteins partici-pated in interactions with candidates known to partici-pate in NF-B activation, including (i) IBE; whichactivates NF-B via TRAF-2 [35], (ii) TRAF-6; an NF-B regulator that plays a critical role in human autoim-mune diseases including arthritis [36], (iii) TNF-receptorsuperfamily member TNFRSF21; which activates NF-Band MAPK8 pathways [37,38], and (iv) mitogen-acti-vated protein kinase kinase kinase-14 (MAP3K14), alsocalled the NF-kappa-beta-inducing kinase (NIK); whichactivates NF-B via TRAF-2 [39]. Several members ofthe c-Jun N-terminal kinases of the JNK pathway [40],MAPK8, MAPK8IP1, and TNFRSF21, were also identi-fied in the interaction protein network of IL-1b-inducedcandidates that were suppressed by IDR-1002 in humanFLS cells (Additional file 1, Table S2). Another interest-ing observation from this computational analysis wasthat genes encoding for four of the 11 selected proteincandidates had binding sites for the transcription factorhepatocyte nuclear factor (HNF)-4a (Additional file 1,Table S2).IDR-1002 inhibited IL-1b-induced activation of JNK andp38 MAPKThe network-based interrogation of the proteomics dataindicated that members of the JNK pathways (for exam-ple, MAPK8, MAPK8IP1) were modulated by the pep-tide IDR-1002. Therefore, we evaluated the impact ofIDR-1002 on IL-1b-induced activation of JNK in humanMMP-3 (ng/ml) 0510152025NS051015202530NSCtrl                              IL-1ECtrl                                   IL-1EA.                                                              B.+ IDR-1002- IDR peptide+ IDR-1Figure 1 Evaluation of MMP-3 production in OA and RA FLS. Human fibroblast-like synoviocytes (FLS) isolated from either (a), osteoarthritis(OA), or (b), rheumatoid arthritis (RA) synovial tissues were stimulated with pro-inflammatory cytokine IL-1b (10 ng/ml) in the presence andabsence of IDR peptides. Tissue-culture supernatants were monitored for MMP-3 production by ELISA after 24-hour stimulation. Results shownare an average of at least three independent biologic experiments performed with cells isolated from synovial tissues obtained fromindependent donors ± standard error (*P < 0.05). IDR, innate defence regulator; IL, interleukin; MMP-3, matrix metalloproteinase-3.Turner-Brannen et al. Arthritis Research & Therapy 2011, 13:R129http://arthritis-research.com/content/13/4/R129Page 6 of 14MMP-3 (ng/ml)A.                                                                                      C.D.010203040506070 *Ctrl                IL1-B + TNF-ACtrl             IL-1 050100150200250300350400450IL-1RA (pg/ml)*02468101214160510152025**p < 0.07IL-1 IDR-1002          IL1-+ IDR-1002 IL-1RASIGIRRFold Change Relative to un-stimulated cells normalized to 1 Fold Change Relative to cells stimulated with IL-1BIL-1B IDR-1002         IL1-+ IDR-1002 **+ IDR-1002- IDR peptide+ IDR-1B.BB BBFigure 2 Evaluation of MMP-3 and IL-1RA production, and transcriptional response of IL-1RA and SIGIRR . Human fibroblast-likesynoviocytes (FLS) were stimulated with pro-inflammatory cytokines either IL-1b (10 ng/ml) or a combination of IL-1b and TNF-a (10 ng/mleach), in the presence and absence of IDR peptides. The peptides were added at the time of cytokine stimulation. Tissue-culture supernatantswere monitored for (a), MMP-3 production after 24-hour stimulation, or (b), IL-1RA after 48 hours, with ELISA. Transcriptional responses for (c), IL-1RA, and (d), SIGIRR, were evaluated with quantitative real-time PCR in human FLS cells stimulated with IL-1b (10 ng/ml) in the presence andabsence of IDR-1002 after 2 hours. Results shown are an average of at least three independent biologic experiments performed with cellsisolated from synovial tissues obtained from independent donors ± standard error (*P < 0.05). IDR, innate defence regulator; IL, interleukin; MMP-3, matrix metalloproteinase-3.Turner-Brannen et al. Arthritis Research & Therapy 2011, 13:R129http://arthritis-research.com/content/13/4/R129Page 7 of 14FLS. To monitor JNK activation, human OA FLS weretreated with IL-1b in the presence and absence of IDR-1002 for 15 minutes, followed by IP by using a JNK-spe-cific antibody. The IP eluates were treated with c-Junsubstrate in the presence of ATP. Phosphorylation ofthe c-Jun substrate was evaluated with an anti-phospho-c-Jun (Ser73)-specific antibody as a measure of JNKactivity. We reproducibly demonstrated that IL-1b-induced JNK activation was abrogated in the presenceof IDR-1002 in human FLS (Figure 4a).Another member of the MAPK family, p38 MAPK,plays a critical role in inflammatory diseases, includingin RA [41,42]. Also, p38 MAPK has been demonstratedto be involved in the immunomodulatory mechanism ofboth IDR peptides, IDR-1 and IDR-1002 [15,16]. There-fore, we evaluated the impact of both these peptides(that is, IDR-1002 and IDR-1) on IL-1b-induced p38MAPK activation in human FLS. IL-1b activates p38MAPK in FLS after 15 minutes of stimulation [28].Human OA FLS were stimulated with IL-1b in the pre-sence and absence of the IDR peptides for 15 minutes.p38 MAPK activity was evaluated by using IP with p38MAPK (Thr180/Tyr182) monoclonal antibody. The IPeluates were treated with p38 MAPK substrate ATF-2 inthe presence of ATP. Phosphorylation of the substratewas monitored in immunoblots, probing with a phos-pho-ATF-2 (Thr76)-specific antibody as a measure ofp38 MAPK activity. We conclusively demonstrated thatIDR-1002 abrogated IL-1b-induced p38 MAPK activityin human FLS, whereas peptide IDR-1 had a limitedeffect (Figure 4b).IDR-1002 inhibited IL-1b-induced activation of NF-BThe network-based interrogation of the proteomics datashowed that members of the NF-B pathway werealtered by the peptide IDR-1002. Therefore, to confirmthe proteomics data, we evaluated IL-1b-induced activa-tion of NF-B in the presence and absence of IDR-1002in synovial fibroblasts. To monitor NF-B direct activa-tion, a rabbit synovial fibroblast cell line (HIG82) wastransiently transfected with pNFB-MetLuc2-ReporterVector (Clontech). The cells were stimulated with IL-1b(10 ng/ml each), in the presence and absence of eitherIDR-1002 or IDR-1. The activation of NF-B was moni-tored after 6 hours of stimulation by using the Ready-To-Glow Secreted NF-B Luciferase Reporter Assay05101520MMP-3 (ng/ml)Ctrl                  IL-1 IL-1 +1002051015202530IL-8 (ng/ml)0123456MCP-1 (ng/ml)+ IL-1- IL-1Ctrl                       IDR 1002Ctrl                       IDR 1002A.                                                              B.                                               C.B BBBFigure 3 Monitoring IL-1b-induced MMP-3 and chemokine production on pretreatment with IDR-1002. Human fibroblast-likesynoviocytes (FLS) were pretreated with IDR-1002 for 45 minutes before stimulation with IL-1b (10 ng/ml) for 24 hours. Tissue-culturesupernatants were monitored for (a) MMP-3 production with ELISA, and chemokines, (b) MCP-1, and (c), IL-8 production by using theBDCytometric Bead Array (CBA) preconfigured human chemokine multiplex system with FACS Caliburflow cytometer (BD Biosciences). Resultsshown are an average of three independent biologic experiments performed with cells isolated from synovial tissues of three different donors ±standard error (*P < 0.05, **P < 0.01). IDR, innate defence regulator; IL, interleukin; MMP-3, matrix metalloproteinase-3.Turner-Brannen et al. Arthritis Research & Therapy 2011, 13:R129http://arthritis-research.com/content/13/4/R129Page 8 of 14(Clontech) as per the manufacturer’s instructions. Pep-tide IDR-1 by itself activated NF-B, which was notobserved by the peptide IDR-1002 (Figure 5a). IL-1b-induced NF-B activation was significantly (P < 0.05)neutralized by IDR-1002 (the levels were below thoseobserved in control unstimulated cells), but not by thepeptide IDR-1 (Figure 5a).We also monitored NF-B activation in primaryhuman OA FLS by evaluating the nuclear translocationof NF-B subunit p50 after stimulation with IL-1b inthe presence and absence of IDR-peptides. IDR-1002significantly suppressed IL-1b-induced nuclear translo-cation of NF-B p50, whereas IDR-1 did not (Figure 5b).IDR-1002 was internalized in human FLSPrevious studies demonstrated cellular uptake of catio-nic HDP in monocytic cells, dendritic cells, and epithe-lial cells, and implicated that inhibition of cellularuptake/endocytic mobilization interferes with cellularresponses mediated by these peptides [43-45]. In thisstudy, we wanted to monitor whether the peptide IDR-1002 was internalized in human FLS. Recent studieshave demonstrated cellular uptake of HDP and IDRpeptides by using biotinylated peptides and haveshown that C-terminal cysteine modification and bioti-nylation does not alter the immune-modulatory activityof these peptides [29,43]. Consistent with this, intracel-lular receptors have been recently demonstrated forHDP LL-37 and IDR-1 [29,43]. Therefore, we used abiotinylated IDR-1002 (IDR-1002B) to monitor cellularuptake in human OA FLS after 15 and 30 minutes ofstimulation. These time points were selected becausewe demonstrated that IDR-1002 altered IL-1b-inducedcell signaling after 15 minutes of stimulation (Figure4). In this study, we showed that IDR-1002B was effec-tively taken up in human FLS after 15 minutes of sti-mulation and that the cellular localization was largelycytosolic (Figure 6).DiscussionIn this study, we examined the use of innate immune-modulatory IDR peptides in limiting IL-1b-inducedinflammatory responses in synovial fibroblasts. Wedemonstrated that a 12-amino-acid IDR peptide, IDR-1002 [16], controlled IL-1b-induced inflammatoryresponses in synovial fibroblasts largely by interveningwith IL-1b-induced NF-B, JNK, and p38 MAPK activa-tion. A recent study demonstrated the ability of IDR-1002 to protect against bacterial infections largely bymodulating host immune responses [16]. A distinctadvantage of the class of IDR peptides is the likelihoodthat these agents can control inflammation while main-taining elements of innate immunity required for effi-cient anti-infective mechanisms [3,14,15,46], which isspeculated to be distinct from current anti-TNFtherapies.In this study, we showed that IDR-1002 significantlysuppressed IL-1b-induced MMP-3 and MCP-1 proteinproduction in FLS. MMP-3 (stromelysin 1) is known tobe elevated in both OA and RA, and it promotes thedestruction of matrix components of the joints [21].MCP-1 is a monocyte chemoattractant highly expressedin the synovial fluid and tissues of RA patients [47]. Pro-duction of MCP-1, either by macrophages or by FLS,results in an autocrine or paracrine stimulation of cellswithin the synovial microenvironment, resulting in over-all extracellular matrix degradation [48]. For example,MCP-1 increases collagenase activity and induces MMP-3 release from chondrocytes [48]. Therefore, significantsuppression of IL-1b-induced production of both MMP-3 and MCP-1 in human FLS demonstrates the therapeu-tic potential of IDR-1002.-phospho-c-Jun(Ser73)A.B.Ctrl IL-1B+IDR-1002IL-1BCtrlIL-1B+IDR-1002IL-1B(i)     (ii) (i)     (ii)IL-1B+IDR-1- phospho-ATF-2(Thr76)IPIPFigure 4 Evaluation of JNK and p38 MAPK activation. (a)Human fibroblast-like synoviocytes (FLS) were stimulated with IL-1b(10 ng/ml) in the presence and absence of IDR-1002 for 15 minutes.Immunoprecipitation (IP) was performed by using 20 μg of celllysates with a JNK-specific antibody. The IP eluates were incubatedwith c-Jun substrate and ATP mixture, and the kinase activityspecific to JNK was monitored by monitoring the phosphorylationof the substrate by using an anti-phospho-c-Jun (Ser73)-specificantibody. (b) Human FLS were stimulated with IL-1b (10 ng/ml) inthe presence and absence of either IDR-1002 or IDR-1 for 15minutes. The peptides were added either (i) 45 minutes before or(ii) at the time of cytokine stimulation. IP was done by using 10 μgof cell lysates with a p38-specific antibody, and the IP eluates wereincubated with substrate ATF-2 protein and ATP mixture. Kinaseactivity specific to p38 MAPK was evaluated in immunoblotsprobing with a phospho-ATF-2 (Thr76)-specific antibody. Theimmunoblots shown are representative of three independentexperiments performed with cells isolated from synovial tissues ofthree different donors. IDR, innate defence regulator; IL, interleukin;JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated proteinkinase; MMP-3, matrix metalloproteinase-3.Turner-Brannen et al. Arthritis Research & Therapy 2011, 13:R129http://arthritis-research.com/content/13/4/R129Page 9 of 14We showed that even though IDR-1002 significantlysuppressed IL-1b-induced MMP-3 production (Figures 1and 3a), and chemokine MCP-1 production (Figure 3b),the peptide did not significantly suppress the expressionof an anti-infective neutrophil chemokine (that is, IL-8production in human FLS; Figure 3c). In contrast, IDR-1002 induced gene expression of endogenous inhibitorof IL-1b (for example, IL-1RA (Figure 2c) and enhancedIL-1b-induced production of IL-1RA protein (Figure 2b)in human FLS). It should be noted that recombinant IL-1RA (anakinra) has been extensively explored as apotential therapy for RA [33]. This selective and differ-ential modulation of inflammatory responses by theIDR-peptide is consistent with previous studies. Forexample, both HDP and IDR peptides can modestlyinduce classic pro-inflammatory responses, such as cer-tain chemokine production in macrophages required foranti-infective immunity [3,15,16]. In contrast, these pep-tides significantly induce anti-inflammatory mediatorssuch as IL-10 [3,15,16]. These peptides result in a netbalancing of inflammation. In this study, we showedsuch “selective” modulation of cytokine-mediatedinflammatory response by IDR-1002 in synovial fibro-blasts. Thus, based on previous study [16] and thisstudy, we can speculate that IDR-1002 can exhibit a tar-geted immune-modulatory activity on both immunecells (for example, macrophages [16]), and mesenchymalcells, such as FLS.We demonstrated that IDR-1002 modulated IL-1b-induced responses in synovial fibroblasts primarily byintervening with the activation of JNK and p38 MAPK(Figure 4) and NF-B (Figure 5) signaling pathways. Inthis study, it was demonstrated that IDR-1002 suppressedIL-1b-induced proteins that are regulated by the JNK andNF-B pathways (Additional file 1, Table S2). Consistentwith this, by using immunochemical assays, we showedthat IDR-1002 abrogated both IL-1b-induced JNK andp38 MAPK activity in human FLS (Figure 4), and signifi-cantly suppressed the IL-1b-induced activation of NF-Bin synovial fibroblasts (Figure 5). These results are con-sistent with previous studies that have demonstrated thatboth HDP and IDR-peptides can influence MAPK path-ways and selectively modulate pathogen-induced NF-Bregulation [3,15,16,49]. IL-1b-induced JNK and p38MAPK are critical in the induction of MMPs and subse-quent tissue destruction in arthritis [42,50]. Conse-quently, both JNK and p38 MAPK are defined as avaluable therapeutic targets for arthritis [51-53]. It shouldbe noted that HDP (for example, the human cathelicidinLL-37) by itself can transiently and differentially activatethe NF-B subunits [49]. However, in this study, IDR-1002 did not induce NF-B activity by itself in synovialfibroblasts at the time point monitored (Figure 5a).Taken together, this is consistent with the paradigm of“selective” immunomodulation of inflammatoryresponses by HDP and IDR-peptides (that is, suppressionof excessive activation of NF-B in the presence of exo-genous infectious/inflammatory stimuli, while maintain-ing transient NF-B activity), overall resulting in a netbalanced inflammation.00.511.522.533.544.5MillionsNF-NB activationCtrl                          IL-1E*NS+ IDR-1- peptides+ IDR-1002(i)    (ii)Ctrl    IL-1EIL-1E+ IDR-1IL1E+ IDR-1002- NF-NB p50- E-actinA.                                                                                                B.Figure 5 Monitoring activation of NF-B. (a) Rabbit synovial fibroblast HIG-82 cells were transiently transfected with pNFB-MetLuc2-ReporterVector (Clontech). The transfected cells were stimulated with IL-1b (10 ng/ml), in the presence and absence of either IDR-1002 or IDR-1. Theactivation of NF-B was monitored after 6 hours of stimulation by using Ready-To-Glow Secreted NF-B Luciferase Reporter Assay (Clontech) asper the manufacturer’s instructions. Results shown are an average of at least three independent experiments ± standard error (*P < 0.05; NS,nonsignificant). (b) Nuclear extracts of human fibroblast-like synoviocytes (FLS) stimulated with IL-1b (10 ng/ml) in the presence and absence ofeither IDR-1002 or IDR-1 were probed with NF-B p50 antibody or b-actin antibody in immunoblots. IDR-1002 was added either (i) 45 minutesbefore, or (ii) at the time of cytokine stimulation. Result shown is a representative blot of three independent experiments performed with FLSobtained from three different donors. IDR, innate defence regulator; IL, interleukin; NF-B, nuclear factor-kappaB.Turner-Brannen et al. Arthritis Research & Therapy 2011, 13:R129http://arthritis-research.com/content/13/4/R129Page 10 of 14An interesting observation from the computationalanalysis of the proteomics data in this study was theimplication of the involvement of transcription factorHNF-4a-targets in IDR-1002-mediated anti-inflamma-tory activity (Additional file 1, Table S2). A publicationin 2009 by Chenomx Inc. describing the analysis of ser-ologic metabolite profiles in RA, demonstrated the inter-connectedness between the IL-1b-induced inflammatoryprotein networks and the transcription factor HNF-4a-mediated signaling. The modulation of HNF-4a-targetelements by IDR peptides warrants further investigation[54].In this study we also demonstrated cellular uptake andcytosolic localization of IDR-1002 in human FLS (Figure6). Cellular uptake and endocytic mobilization of catio-nic HDP has been shown in monocytic cells and epithe-lial cells, and it was previously suggested that cellularuptake may be essential for immune-modulatory activityfor cationic peptides [43,44]. Even though someintracellular interacting protein partners and putativecell-surface receptors have been identified for bothhuman HDP LL-37 and IDR-1 [29,43,55], the mechan-ism of receptor interaction for IDR peptides is yet to becompletely elucidated. A recent study suggested thatIDR-1002 activity may be mediated by a Gi-coupledreceptor [16]; however, no direct interacting proteinpartner or receptor has been defined for IDR-1002.Taken together, based on previous studies and thisstudy, we can speculate the action of IDR-1002 on FLSas follows. IDR-1002 may be internalized by FLS eitherinteracting with putative cell surface receptors or, morelikely, by inserting directly into the cell membrane, asproposed by previous studies for cationic peptides[45,56]. A vesicle-mediated uptake pathway, as pre-viously implicated for HDP [44,45,56], is likely to facili-tate the internalization of IDR-1002, and subsequentinteraction with putative intracellular interacting proteinpartners or receptors, as described for other HDP and50 ¾ mFigure 6 Evaluating cellular uptake of IDR-1002. Human fibroblast-like synoviocytes (FLS) were stained fluorescently to demonstrate theuptake of IDR-1002. The cells were stimulated with 100 μg/ml biotinylated IDR-1002 (IDR-1002B). Cells were either (a), not treated with peptide,or stimulated with IDR-1002B for (b), 0 minutes, (c), 15 minutes, or (d), 30 minutes. Cells were washed, fixed, and stained for actin by usingAlexa Fluor 546 phalloidin (red) and stained with Streptavidin Alexa Fluor 488 conjugate to detect IDR-1002B (green). Hoescht 33258 was usedto counterstain the nucleus (blue). The scale bar in (d) represents a length of 50 μm. IDR, innate defence regulator.Turner-Brannen et al. Arthritis Research & Therapy 2011, 13:R129http://arthritis-research.com/content/13/4/R129Page 11 of 14IDR peptides [29,43]. Identification of intracellularreceptors for IDR-1002 in mesenchymal cells such ashuman FLS warrants further investigation and is beyondthe scope of this article. Interaction of the peptide withputative intracellular protein partners may be facilitatingalteration of innate immune signaling pathways, overallresulting in the modulation of inflammatory responsesby IDR-1002 in synovial fibroblasts. Results from thisstudy provide evidence that supports further researchinto the development of IDR-peptides as potential thera-peutics in immune-mediated chronic inflammatory dis-eases such as RA.ConclusionsThis study demonstrates that an IDR peptide, based onnaturally occurring innate immune effector HDP, canselectively modulate IL-1b-induced responses in synovialfibroblasts. This study showed that IDR-1002 peptidesuppressed IL-1b-induced inflammatory responses byaltering the IL-1b-induced proteome, suppressed IL-1b-mediated activation of NF-B, MAPK p38, and JNK,suppressed IL-1b-induced MMP-3 and MCP-1 produc-tion, but did not neutralize the production of all chemo-kines that are required for resolution of infections. Incontrast, the peptide enhanced the expression of anti-inflammatory endogenous inhibitors of IL-1b (for exam-ple, IL-1RA). Overall, this study showed that an IDR-peptide can selectively modulate immune-mediated“sterile” inflammation in mesenchymal cells such asFLS. This study provides a new potential application ofinnate immune IDR-peptides as selective immune-mod-ulatory agents that are likely to control inflammationwithout hampering anti-infective immunity. We proposethat IDR-peptides represent valuable candidates thatshould be explored further as potential therapeutics forinflammatory arthritis and other diseases characterizedby chronic inflammation.Additional materialAdditional file 1: Supplementary Table 1. IL-1b-induced proteinssuppressed in the presence of IDR peptide, IDR-1002. Humanfibroblast-like synoviocytes (FLS) were stimulated with IL-1b (10 ng/ml) inthe presence and absence of IDR-1002 for 24 hours. The peptide wasadded 45 minutes before cytokine stimulation. The cell lysates wereprocessed for iTRAQ labelling by using three different isobaric tags. Threeindependent LC-MS/MS runs were performed on iTRAQ-labelled samplesfrom three independent donors. Protein candidates were selected only ifthey were detected in at least two of the three independent biologicexperiments. Eleven proteins induced by IL-1b were found to besuppressed by IDR-1002 between 20% and 60%. Supplementary Table2. Computational network-based analysis by using InnateDBbiomolecular network database. IL-1b-induced protein candidates thatwere found to be suppressed between 20% and 60% by IDR-1002 (11proteins) were submitted to InnateDB biomolecular interaction database[57]. This database was used to identify direct interactions between theselected 11 protein candidates and any known immunity-relatedproteins. The identified interactions are summarized, and the members ofNF-B and JNK pathways, and association of HNF-transcription factor, areindicated in bold in this table.AbbreviationsFLS: fibroblast-like synoviocytes; HDP: host defense peptide; IDR: innatedefence regulator; JNK: c-Jun N-terminal kinases; MAPK: mitogen-activatedprotein kinase; MCP-1: monocyte chemotactic protein-1; MMP-3: matrixmetalloproteinase-3; NF-κB: nuclear factor kappa-B; OA: osteoarthritis; RA:rheumatoid arthritis.AcknowledgementsThe authors gratefully acknowledge the funding agencies Health SciencesCentre Foundation (HSCF) Manitoba, and Manitoba Health ResearchCouncil (MHRC), Canada, for this research. REWH is funded by theCanadian Institutes of Health Research (CIHR) and the Grand Challenges inGlobal Health program through the Foundation of the National Institutesof Health and CIHR. The authors greatly appreciate the support of Dr. OlegKrohkin for Maldi mass spectrometry and critical discussions from Dr. JohnWilkins. The authors also appreciate the technical support of Keng Wongand Peyman Ezzatti. REWH and HEG are recipients of Canada ResearchChairs.Author details1Manitoba Centre for Proteomics and Systems Biology, Department ofInternal Medicine, University of Manitoba, 799 John Buhler Research Centre,715 McDermot Avenue, Winnipeg, MB, R3E3P4, Canada. 2Department ofImmunology, University of Manitoba, 471 Apotex Centre, 750 McDermotAvenue, Winnipeg, MB, R3E0T5, Canada. 3Centre for Microbial Diseases andImmunity Research, University of British Columbia, 2259 Lower Mall ResearchStation, Vancouver, BC, V6T1Z4, Canada.Authors’ contributionsETB performed all experiments, except proteomics and microscopy, andcontributed to writing the manuscript. KGC performed all experiments,except proteomics and microscopy. DNDL performed the microscopyexperiments. JPC performed the computational analysis for the proteomicsdata. REWH provided the IDR-1002 peptide and edited the manuscript. HEGprovided tissues for isolation of human FLS, provided intellectual input, andedited the manuscript. NM conceived the study, performed the proteomicsiTRAQ experiments, and wrote the manuscript. All authors contributed tothe conception and/or acquisition of data and analysis for this project, andto either drafting or revising the manuscript.Competing interestsThe authors declare that they have no competing interests.Received: 8 February 2011 Revised: 1 April 2011Accepted: 11 August 2011 Published: 11 August 2011References1. Hancock REW, Chapple DS: Peptide antibiotics. Antimicrob AgentsChemother 1999, 43:1317-1323.2. Allaker RP: Host defence peptides: a bridge between the innate andadaptive immune responses. Trans R Soc Trop Med Hyg 2008, 102:3-4.3. 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Hancock REW, Rozek A: Role of membranes in the activities ofantimicrobial cationic peptides. FEMS Microbiol Lett 2002, 206:143-149.57. Lynn DJ, Winsor GL, Chan C, Richard N, Laird MR, Barsky A, Gardy JL,Roche FM, Chan TH, Shah N, Lo R, Naseer M, Que J, Yau M, Acab M,Tulpan D, Whiteside MD, Chikatamarla A, Mah B, Munzner T, Hokamp K,Hancock RE, Brinkman FS: InnateDB: facilitating systems-level analyses ofthe mammalian immune response. Mol Syst Biol 2008, 4:218.doi:10.1186/ar3440Cite this article as: Turner-Brannen et al.: Modulation of interleukin-1b-induced inflammatory responses by a synthetic cationic innate defenceregulator peptide, IDR-1002, in synovial fibroblasts. Arthritis Research &Therapy 2011 13:R129.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitTurner-Brannen et al. Arthritis Research & Therapy 2011, 13:R129http://arthritis-research.com/content/13/4/R129Page 14 of 14

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