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Extracellular vesicles as carriers of oxidation-specific epitopes

Extracellular vesicles as carriers of oxidation-specific epitopes. Christoph J. Binder, MD, PhD Extracellular Vesicles in Inflammation Krems, May 7 , 2015. Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis. End products of lipid peroxidation eg. MDA. Adduct

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Extracellular vesicles as carriers of oxidation-specific epitopes

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  1. Extracellularvesiclesascarriersofoxidation-specificepitopes Christoph J. Binder, MD, PhD ExtracellularVesicles in Inflammation Krems, May 7, 2015 Christian Doppler Laboratory for Innovative TherapyApproaches in Sepsis

  2. End products of lipid peroxidation eg. MDA Adduct formation Generation of Oxidation-specific Epitopes PUFAs 1 2 Oxidative stress PC Modified proteins Oxidtaion-specific Epitopes PC……..Phosphocholine of OxPL MDA…..Malondialdehyde OxCL….Oxidized Cardiolipin 4-HNE…4-Hydroxynonenal OxCL Apoptotic cell 4HNE MDA Malondialdehyde-acetaldehyde Oxidized phosphatidylcholine

  3. OxLDLandapoptoticcells promote inflammation OxLDL Apoptotic cells Modified from Glass & Witztum, Cell 2001

  4. Oxidation-specific epitopes are targets of innate immunity • Accumulation of OxLDL, apoptotic cells, cell debris • Contain lipid-peroxidation derived structures: • Malondialdehyde (MDA/MAA) • 4-Hydroxynonenal (4-HNE) • Oxidized Phosphatidylcholine (OxPC) • Oxidized Cardiolipin (OxCL) • Oxidation-specific epitopes are Danger Associated Molecular Patterns (DAMPs) • “Danger signals” recognized by Pattern Recognition Receptors (PRRs) of innate immunity (Scavenger Rec., TLRs, natural Abs,CRP, CFH)

  5. Altered Self Self DAMPs Innate Immunity Natural Antibodies T15/EO6 NA17 LRO1 Pattern Recognition Proteins PC MDA OxCL 4-HNE LDL OxLDL CRP CFH Pattern Recognition Receptors Apoptotic Cell Viable Cell CD36 SR-A PC……..Phosphocholine MDA…..Malondialdehyde OxCL….Oxidized Cardiolipin 4-HNE…4-Hydroxynonenal Shaw et al., J Clin Invest 2000 Chang et al., PNAS 2002 Chou et al., J Clin Invest 2009 Weismann et al., Nature 2011 Boullier et al., J Lipid Res 2005 Tuominen et al., ATVB 2006 Binder et al., Nature Med 2003 Chang et al., J Exp Med 2004

  6. Inverse association of anti-OxLDL IgM and MI Adjusted for: Age Gender Tobacco Hypertension Diabetes LDL-C HDL-C TG Pakistani Risk of Myocardial Infarction Study (~4,000 cases and ~4,000 controls; P<0.0001) *Taleb and Amir et al., mansucript in preparation

  7. Natural antibodies Germline encoded Limited repertoire Product of natural selection Bind microbial antigens 1. First line defense against bacterial & viral infections self-antigens (“auto-reactive”) 2. “House keeping” functions Clearance of self-antigens or stress-induced neo-self Naturally occurring Pre-existing antibodies Primarily IgM Secreted by B-1 cells

  8. T15/EO6 is a prototypic natural anti-PC IgM antibody • Natural germline-encoded Ab • Binds phosphorylcholine (PC) present on oxidized PL and on the capsular polysaccharide (CPS) of S. pneumoniae • The same natural antibody can be first line defense and have “house-keeping” functions • T15/EO6 IgM protect from atherosclerosis Binder & Silverman, SSI 2004 Binder et al., Nat Med 2003 Faria-Neto et al., Atherosclerosis 2006

  9. Binding properties of natural IgM antibodies A large part of B-1 cell derived natural IgM binds to oxidation epitopes Oxidation epitopes 35% Chou et al., J Clin Invest 2009

  10. Multiple Sclerosis Apoptotic cells Atherosclerosis Palinski et al. Arteriosclerosis 1996 Haider et al. Brain 2011 Acute lung injury Chang et al. PNAS 1999 Rheumatoid Arthritis Imai et al. Cell 2008 MDA is present in various pathological settings Alzheimer‘s disease Dei et al. Acta Neuropath. 2002 Oxidation Specific Epitopes Age-related Macular Degeneration Weismann et al. Nature 2011

  11. Tsiantoulas et al., Circ Res 2014

  12. Microvesicles are mediators of inflammation • 0.1 – 1 µm in size • carry different mediators • circulate in plasma • increased levels in several diseases MVs from C. Beyer & D.S. Pisetsky; 2010 Nature Reviews Rheumatology 6, 21-29 Inflammation Coagulation Vascular function

  13. Circulatingmicrovesicles carry oxidation-specific epitopes Binding of OxLDL-specific IgM mAb tocMVsby flow cytometry PC MDA MDA MDA Circulating microvesicles isolated from plasma of healthy volunteers (n=15) MP = MV Tsiantoulas et al., J Lipid Res 2015

  14. PC epitopesarenearlyexclusivelypresent on MV that carry MDA-epitopes MP = MV Tsiantoulas et al., J Lipid Res 2015

  15. MDA-epitopesarepresent on bothplateletandred-bloodcellmicrovesicles platelet MVs redbloodcell MVs endothelial MVs Circulating microvesicles isolated from plasma of healthy volunteers (n=15) MP = MV Tsiantoulas et al., J Lipid Res 2015

  16. Circulatingmicrovesicles carry IgM with specificity for MDA-LDL Circulating microvesiclesisolated from plasma of healthy volunteers MP = MV Tsiantoulas et al., J Lipid Res 2015

  17. MV-associatedIgMsareprimarilycomposedof IgM withspecificityfor MDA adducts Binding of MV-eluted IgM to coated MDA-LDL in the presence of soluble LDL, CuOx-LDL and MDA-LDL MP = MV Tsiantoulas et al., J Lipid Res 2015

  18. Malondialdehyde-acetaldehyde adducts upregulateexpression of pro-inflammatory genes Mouse bonemarrow-derivedmacrophagesstimualtedwith MAA-BSA MAA = Malondialdehyde-acetaldehyde Jana Busch

  19. MDA-epitopes mediate microvesicle-inducedIL-8 secretion by primary human monocytes platelet MV Ctrl LR04 counts IgM MV = MP = Microvesicles LRO4 = MAA-specific natural IgM Tsiantoulas et al., J Lipid Res 2015

  20. Increased numbers of MDA+ MVs in the coronary circulation of AMI patients MDA+ MV periphery coronary • * P < 0.05 14 AMI patients MP = MV Tsiantoulas et al., J Lipid Res 2015

  21. Altered Self Self DAMPs Innate Immunity Natural Antibodies PC MDA OxCL 4-HNE LDL OxLDL T15/EO6 NA17, LR04 LRO1 Clearance Neutralization Apoptotic Cell Microvesicles Viable Cell Atherosclerosis PC……..Phosphocholine MDA…..Malondialdehyde OxCL….Oxidized Cardiolipin 4-HNE…4-Hydroxynonenal

  22. Deficiency of secreted IgM increases atherosclerosis in mice Immunoglobulin M Is Required for Protection Against Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice, Lewis et al, Circulation, 2009 Ldlr -/- sIgM +/+ Ldlr -/- sIgM -/- Female mice fed a Western type diet for 16 weeks

  23. Altered Self Self DAMPs Innate Immunity Natural Antibodies PC MDA OxCL 4-HNE LDL OxLDL T15/EO6 NA17, LR04 LRO1 Clearance Neutralization Apoptotic Cell Microvesicles Viable Cell Atherosclerosis Thrombosis PC……..Phosphocholine MDA…..Malondialdehyde OxCL….Oxidized Cardiolipin 4-HNE…4-Hydroxynonenal

  24. Y MDA+ MVs mediate pro-inflammatoryeffectsofplatelet-derivedMV Immunodepletionof MDA+ MV fromPlateletMV Stimulation of THP-1 cellswith total anddepletedMVs Taras Afonyushkin

  25. MDA-specificnaturalIgMdelayMV-dependentcoagulationin plasma Effectofplatelet-derivedmicrovesicles (PMP) andMDA-specificIgM (LR04) on fibrinandthrombingeneration in plasma Georg Obermayer

  26. Austrian Study on Recurrent Venous Thromboembolism Kyrle, Eichinger, et al., in preparation

  27. Inverse association of anti-OxLDL IgM with DVT recurrence Kyrle, Eichinger, et al., in preparation

  28. Altered Self Self DAMPs Innate Immunity Natural Antibodies PC MDA OxCL 4-HNE LDL OxLDL IL-5 T15/EO6 NA17, LR04 LRO1 Siglec-G Clearance Neutralization Apoptotic Cell Microvesicles Viable Cell Atherosclerosis Thrombosis PC……..Phosphocholine MDA…..Malondialdehyde OxCL….Oxidized Cardiolipin 4-HNE…4-Hydroxynonenal

  29. Acknowledgements TarasAfonyushkin Jana Lui Busch Sabrina Gruber Laura Göderle Barbara Gritti Mate Kiss Maria Ozsvar Kozma Vesna Krajina Nikolina Papac-Milicevic Florian Puhm DimitrisTsiantoulas Georg Obermayer Univ. of Cambridge ZiadMallat Andrew Sage Medical Univ Vienna Irene Lang Andreas Mangold Alexander Kyrle Sabine Eichinger Thomas Perkmann UCSD Joseph L. Witztum Sam Tsimikas SFB SFB PARCC/INSERM Chantal Boulanger Donau Univ. Krems Michael B. Fischer In-Thro

  30. Oxidation-epitope specific IgM recognize distinct epitopes Binding of E06 and LR04 IgM mAb to modified BSA (ELISA)

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