Postischemic Administration of IL-1α Neutralizing Antibody Reduces Brain Damage and Neurological Deficit in Experimental Stroke

HomeCirculationVol. 142, No. 2Postischemic Administration of IL-1α Neutralizing Antibody Reduces Brain Damage and Neurological Deficit in Experimental Stroke Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplementary MaterialsFree AccessLetterPDF/EPUBPostischemic Administration of IL-1α Neutralizing Antibody Reduces Brain Damage and Neurological Deficit in Experimental Stroke Luca Liberale, MD, Nicole R. Bonetti, MD, Yustina M. Puspitasari, BS, MD, Lena Schwarz, MS, Alexander Akhmedov, PhD, Fabrizio Montecucco, MD, PhD, Frank Ruschitzka, MD, Jürg H. Beer, MD, Thomas F. Lüscher, MD, John Simard, BS, Peter Libby, MD and Giovanni G. Camici, PhD Luca LiberaleLuca Liberale Center for Molecular Cardiology, University of Zürich, Schlieren, Switzerland (L.L, N.R.B., Y.M.P., L.S., A.A., J.H.B., T.F.L., G.G.C.). First Clinic of Internal Medicine, Department of Internal Medicine, University of Genoa, Italy (L.L.). Search for more papers by this author , Nicole R. BonettiNicole R. Bonetti Center for Molecular Cardiology, University of Zürich, Schlieren, Switzerland (L.L, N.R.B., Y.M.P., L.S., A.A., J.H.B., T.F.L., G.G.C.). Department of Internal Medicine, Cantonal Hospital of Baden, Switzerland (N.R.B., J.H.B.). Search for more papers by this author , Yustina M. PuspitasariYustina M. Puspitasari Center for Molecular Cardiology, University of Zürich, Schlieren, Switzerland (L.L, N.R.B., Y.M.P., L.S., A.A., J.H.B., T.F.L., G.G.C.). Search for more papers by this author , Lena SchwarzLena Schwarz Center for Molecular Cardiology, University of Zürich, Schlieren, Switzerland (L.L, N.R.B., Y.M.P., L.S., A.A., J.H.B., T.F.L., G.G.C.). Search for more papers by this author , Alexander AkhmedovAlexander Akhmedov Center for Molecular Cardiology, University of Zürich, Schlieren, Switzerland (L.L, N.R.B., Y.M.P., L.S., A.A., J.H.B., T.F.L., G.G.C.). Search for more papers by this author , Fabrizio MontecuccoFabrizio Montecucco IRCCS Ospedale Policlinico San Martino Genoa–Italian Cardiovascular Network, Italy (F.M.). First Clinic of Internal Medicine, Department of Internal Medicine and Centre of Excellence for Biomedical Research (CEBR), University of Genoa, Italy (F.M.). Search for more papers by this author , Frank RuschitzkaFrank Ruschitzka University Heart Center, Department of Cardiology, University Hospital Zurich, Switzerland (F.R., G.G.C.). Search for more papers by this author , Jürg H. BeerJürg H. Beer Center for Molecular Cardiology, University of Zürich, Schlieren, Switzerland (L.L, N.R.B., Y.M.P., L.S., A.A., J.H.B., T.F.L., G.G.C.). Department of Internal Medicine, Cantonal Hospital of Baden, Switzerland (N.R.B., J.H.B.). Search for more papers by this author , Thomas F. LüscherThomas F. Lüscher Royal Brompton and Harefield Hospitals and Imperial College, London, United Kingdom (T.F.L.). Search for more papers by this author , John SimardJohn Simard XBiotech, Austin, TX (J.S.). Search for more papers by this author , Peter LibbyPeter Libby Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA (P.L.). Search for more papers by this author and Giovanni G. CamiciGiovanni G. Camici Giovanni G. Camici, PhD, Center for Molecular Cardiology, University of Zurich, Wagistrasse 12, CH-8952 Schlieren, Switzerland. Email E-mail Address: [email protected] https://orcid.org/0000-0002-0523-0695 Center for Molecular Cardiology, University of Zürich, Schlieren, Switzerland (L.L, N.R.B., Y.M.P., L.S., A.A., J.H.B., T.F.L., G.G.C.). University Heart Center, Department of Cardiology, University Hospital Zurich, Switzerland (F.R., G.G.C.). Department of Research and Education, University Hospital Zurich, Switzerland (G.G.C.). Search for more papers by this author Originally published13 Jul 2020https://doi.org/10.1161/CIRCULATIONAHA.120.046301Circulation. 2020;142:187–189Treatment of patients who have experienced ischemic stroke remains a clinical challenge, mandating exploration of novel therapeutic strategies. Inflammation, and particularly IL-1 (interleukin-1) signaling, plays a pivotal role in the pathophysiology of ischemic stroke.1 Specifically, IL-1α mediates local inflammation at the site of dying cells, thus likely aggravating ischemic cerebral injury. This study tested the hypothesis that IL-1α inhibition can mitigate ischemic stroke. A human monoclonal antibody that specifically inhibits IL-1α has entered phase III trials for cancers.2 Because the human monoclonal anti–IL-1α antibody does not neutralize rodent IL-1α, this study used a specific anti-mouse IL-1α antibody. We evaluated the efficacy of antibody-mediated IL-1α neutralization in mice with ischemic stroke using a clinically relevant experimental approach. IL-1α inhibition started after transient cerebral ischemia, resembling the case in patients with acute stroke who undergo reperfusion therapy.1All procedures were approved by the local ethical committee for animal research and the Cantonal Veterinary Authority. This report complies with the initial STAIR (Stroke Therapy Academic Industry Roundtable) recommendations for standards regarding preclinical neuroprotective and restorative drug development, as permitted by local authorities.Twelve-week-old male C57BL/6 Wild-type mice underwent transient middle cerebral artery occlusion (tMCAO) for 45 minutes. Next, mice randomly received either anti-mouse IL-1α antibody (ie, Flo1-2a) or isotype control (XBiotech, Austin, TX) at different dosages (10 or 65 μg/g). Forty-eight hours after tMCAO, stroke volume was assessed by TTC (2,3,5-triphenyltetrazolium chloride) staining while neurological deficit was assessed by RotaRod test and Bederson score, as previously described.1 Immunohistological staining investigated the underlying mechanisms, as previously published.3In control antibody-treated mice exposed to tMCAO, tissue IL-1α levels rose in the ipsilateral hemisphere underscoring the pathophysiological relevance of this cytokine for stroke (Figure 1A). After 48 hours of reperfusion, mice treated with the lower dose of the anti–IL-1α antibody showed a minor reduction in infarct volume, as assessed by TTC staining (Figure 1B), although poststroke neurological deficit did not improve. Treatment with the higher dose of the anti–IL-1α antibody reduced stroke size by 36% compared with isotype control and improved neurological performance as determined by Bederson and RotaRod tests (Figure 1B through 1D).Download figureDownload PowerPointFigure 1. Postischemic treatment with anti–IL-1α antibody limits cerebral infarct size and lessens neurological deficit after transient middle cerebral artery occlusion through blunted endothelial activation and inflammation.A, Cerebral IL-1α (interleukin-1α) concentration rose in ipsilateral hemisphere of mice undergoing transient middle cerebral artery occlusion for 45 minutes followed by 48 hours reperfusion, as compared with the contralateral hemisphere (n=8; unpaired 2-tailed Student t test). B, Animals postischemically treated with monoclonal anti–IL-1α antibody showed a dose-dependent reduction of the stroke volumes as assessed on TTC (2,3,5-triphenyltetrazolium chloride)-stained coronal sections (n=7–11; 1-way ANOVA with Tukey post hoc test). Treatment with IL-1α neutralizing antibody at the dose of 65 μg/g improved poststroke neurological function as assessed by (C) Bederson-based neurological score or (D) RotaRod test (n=14; 2-way ANOVA with Sidak post hoc test), as compared to treatment with same dose of the isotype control. E, Immunostaining for P-selectin (yellow) and the endothelial marker VE (vascular endothelium)-cadherin (red) in ipsilateral hemispheres 48 hours after transient middle cerebral artery occlusion showed a significant reduction in the endothelial expression after postischemic treatment of mice with anti-IL-1α antibody as compared to controls. F, The expression of ICAM-1 (intracellular adhesion molecule; cyan) fell significantly in endothelial cells (cluster of differentiation 31 [CD31]; red) of the penumbra area of animals treated with anti IL-1α as compared to control antibody. G, The endothelial expression (CD31, red) of another adhesion molecule VCAM-1 (vascular cell adhesion molecule 1; blue) declined significantly with IL-1α blockade. H, Immunostaining for the activated microglia/macrophage marker Iba1 (ionized calcium binding adaptor molecule 1; red) in the penumbra area of brain 48 hours after tMCAO showed a significant reduction in counts of active mononuclear phagocyte in mice treated postischemically with anti–IL-1α antibody as compared with controls. I, Immunostaining quantification of MMP-9 (matrix metalloproteinase 9) levels (blue) normalized to total endothelial surface (CD31-positive area) also showed significantly lower levels of this mediator in the penumbra area of animals treated with anti–IL-1α as compared with control antibody. For E through I: representative staining above, quantification below; n=7–9; unpaired 2-tailed Student t test. *P<0.05, **P<0.01, ****P<0.0001.Postischemic blood–brain barrier (BBB) damage significantly influences stroke outcome. Immunohistochemical analysis of IgG extravasation demonstrated a slight nonsignificant trend toward increased blood–brain barrier permeability in anti–IL-1α-treated animals. Similarly, rates of hemorrhagic transformation as assessed macroscopically did not differ among the groups. In accord, the endothelial expression of occludin, claudin 5, and vascular endothelium-cadherin—regulators of paracellular blood-brain barrier permeability—did not differ between the treated and the control group.After stroke, the local rise of damage-associated molecular patterns and other inflammatory mediators, such as IL-1α, recruits circulating leukocytes to the damage site and facilitates their effector functions. Leukocyte migration depends on a complex pattern of adhesion molecules expressed by both brain microvascular endothelial cells and white blood cells including selectins, adhesion molecules of the immunoglobulin superfamily, and integrins. In accord with our recently published results showing that IL-1α directly mediates endothelial cell activation,4 confocal microscopy of the penumbra area demonstrated decreased endothelial expression of P-selectin, ICAM-1 (intracellular adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1) in animals treated with the IL-1α inhibitory antibody as compared with control littermates (Figure 1E through 1G). After ischemia, activation of resident immune cells of the brain (ie, microglia) as well as infiltration of monocytes from circulating pool contributes significantly to brain tissue damage.Postischemic IL-1α neutralization significantly decreased numbers of activated macrophages in the stroke area compared with control mice as assessed by Iba-1 (ionized calcium-binding adaptor protein-1) immunostaining (Figure 1H). On activation, macrophages secrete several proinflammatory mediators such as TNF-α (tumor necrosis factor α), ILs (interleukins), and MMPs (matrix metalloproteinases), thereby exacerbating cerebral parenchymal damage. Among these, MMP9 can exert direct neurotoxic effects. In line with the Iba-1 data, further immunohistochemical analysis of the penumbra area revealed reduced MMP9 tissue levels in animals receiving the IL-1α neutralizing antibody as compared with controls (Figure 1I).In summary, this study demonstrates (1) an important role for IL-1α in aggravation of stroke and (2) using a translationally relevant experimental protocol, the efficacy of selective postischemic IL-1α blockade in improving outcome after stroke in mice. A recent study reported neuroprotective effects in the ischemic mouse brain following systemic administration of subpathological IL-1α doses.5 IL-1α functions locally, primarily by autocrine and paracrine signaling. Indeed, IL-1α is mainly membrane-bound with hardly detectable circulating levels. As such, the properties of systemic IL-1α remain unknown, precluding the direct comparison between the present study and that of Salmeron et al.5 The availability of an antihuman IL-1α antibody already in clinical use offers further translational potential to the present observations. Randomized clinical trials should assess the potential of anti–IL-1α therapy as an adjuvant to interventional or thrombolytic treatment of acute ischemic strokes.Sources of FundingThe present work was supported by the Swiss National Science Foundation (to G.G.C. [310030_175546]), the Alfred and Annemarie von Sick Grants for Translational and Clinical Research Cardiology and Oncology to G.G.C., and the Foundation for Cardiovascular Research–Zurich Heart House. G.G.C. and F.P. are recipients of a Sheikh Khalifa’s Foundation Assistant Professorship at the Faculty of Medicine, University of Zurich. PL receives funding from the US National Heart, Lung, and Blood Institute (R01HL080472 and 1R01HL134892), the American Heart Association (18CSA34080399), and the RRM Charitable Fund.DisclosuresDr Libby is an unpaid consultant to, or involved in clinical trials for Amgen, AstraZeneca, Baim Institute, Beren Therapeutics, Esperion, Therapeutics, Genentech, Kancera, Kowa Pharmaceuticals, Medimmune, Merck, Norvo Nordisk, Merck, Novartis, Pfizer, Sanofi-Regeneron. Dr Libby is a member of scientific advisory board for Amgen, Corvidia Therapeutics, DalCor Pharmaceuticals, Kowa Pharmaceuticals, Olatec Therapeutics, Medimmune, Novartis, XBiotech, Inc. Dr Libby’s laboratory has received research funding in the last 2 years from Novartis. Dr Libby is on the Board of Directors of XBiotech, Inc. Dr Libby has a financial interest in Xbiotech, a company developing therapeutic human antibodies. Dr Libby’s interests were reviewed and are managed by Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict of interest policies. Dr Simard is an executive officer of XBiotech; he owns shares in the company and is the inventor of numerous patents relating to therapies targeting IL-1α. Drs Libby, Liberale, and Camici are coinventors on a provisional patent application that was filed in May 2019. The patent relates to the use of antibodies which specifically bind IL-1α to reduce various sequelae of ischemia-reperfusion injury to the central nervous system. The other authors report no conflicts.Footnoteshttps://www.ahajournals.org/journal/circThe data that support the findings of this study are available from the corresponding author upon reasonable request.The podcast and transcript are available as a Data Supplement at https://www.ahajournals.org/doi/suppl/10.1161/CIRCULATIONAHA.120.046301.Giovanni G. Camici, PhD, Center for Molecular Cardiology, University of Zurich, Wagistrasse 12, CH-8952 Schlieren, Switzerland. Email giovanni.[email protected]chReferences1. Liberale L, Diaz-Cañestro C, Bonetti NR, Paneni F, Akhmedov A, Beer JH, Montecucco F, Lüscher TF, Camici GG. Post-ischaemic administration of the murine Canakinumab-surrogate antibody improves outcome in experimental stroke.Eur Heart J. 2018; 39:3511–3517. doi: 10.1093/eurheartj/ehy286CrossrefMedlineGoogle Scholar2. Hickish T, Andre T, Wyrwicz L, Saunders M, Sarosiek T, Kocsis J, Nemecek R, Rogowski W, Lesniewski-Kmak K, Petruzelka L, et al. MABp1 as a novel antibody treatment for advanced colorectal cancer: a randomised, double-blind, placebo-controlled, phase 3 study.Lancet Oncol. 2017; 18:192–201. doi: 10.1016/S1470-2045(17)30006-2CrossrefMedlineGoogle Scholar3. Bonetti NR, Diaz-Cañestro C, Liberale L, Crucet M, Akhmedov A, Merlini M, Reiner MF, Gobbato S, Stivala S, Kollias G, et al. 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Kao M, Wu J, Cheung W, Chen J, Sun G, Ong W, Herr D and Lin T (2020) Clinacanthus nutans Mitigates Neuronal Death and Reduces Ischemic Brain Injury: Role of NF-κB-driven IL-1β Transcription, NeuroMolecular Medicine, 10.1007/s12017-020-08618-y, 23:1, (199-210), Online publication date: 1-Mar-2021. July 14, 2020Vol 142, Issue 2 Advertisement Article InformationMetrics © 2020 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.120.046301PMID: 32658615 Originally publishedJuly 13, 2020 Keywordsstrokecerebrovascular diseaseinterleukinsneuroprotective agentsinterleukin-1αblood brain barrierinflammationPDF download Advertisement SubjectsAnimal Models of Human DiseaseBasic Science ResearchInflammationPathophysiologyVascular Biology