Neutrophils Cast NETs in Atherosclerosis: Employing Peptidylarginine Deiminase as a Therapeutic Target Yvonne Döring, Oliver Soehnlein and Christian Weber Circ Res. 2014;114:931-934 doi: 10.1161/CIRCRESAHA.114.303479 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/114/6/931
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/
Downloaded from http://circres.ahajournals.org/ at Tulane University on September 5, 2014
Editorial Neutrophils Cast NETs in Atherosclerosis Employing Peptidylarginine Deiminase as a Therapeutic Target Yvonne Döring, Oliver Soehnlein, Christian Weber
A
therosclerosis, an inflammatory response in large arteries starting with a subclinical endothelial dysfunction, can turn into a life-threatening disease manifesting as myocardial infarction or stroke. Throughout the past decades, it has become apparent that atherosclerosis is not solely based on an imbalanced lipid metabolism but is mainly driven by a chronic inflammation of the vessel wall involving numerous cell types.1 Regardless of intensive research in the past 30 years, gold standard atherosclerosis-targeted therapies are still restricted to the control of primary risk factors such as hypertension and hyperlipidemia. Despite the proven efficacy of these drugs, cardiovascular diseases remain the leading cause of mortality in Western societies, underlining the need for new therapeutic approaches.1 In this issue of Circulation Research, Knight et al2 show that the inhibition of peptidylarginine deiminase (PAD) by Cl-amidine treatment prevents neutrophil extracellular trap (NET) formation and thereby decreases atherosclerotic lesion size and delays carotid artery thrombosis in Apoe−/− mice receiving a cholesterol-rich diet. Specific inhibition of NETosis was accompanied by a decrease in arterial interferon (IFN)-α expression, less neutrophil recruitment to the arterial wall, and a decline in intimal macrophages. Notably, all these findings could not be repeated when Cl-amidine was administered into Apoe−/− mice treated with a neutrophil-depleting antibody or into mice lacking a functional type I interferon receptor. These data suggest an important role for NET formation and the subsequently instigated type I interferon response in atherogenesis. In contrast to studies on early lesion formation, a treatment regimen targeting advanced stages of atherosclerosis was without success, underscoring the importance of neutrophil-driven proatherosclerotic processes during atherogenesis only.3
Article, see p 947
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From Institute for Cardiovascular Prevention, L udwig-MaximiliansUniversity Munich, Germany (Y.D., O.S., C.W.); Academic Medical Center, Department of Pathology, Amsterdam University, the Netherlands (O.S.); and DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Germany (O.S., C.W.). Correspondence to Drs Yvonne Döring, Oliver Soehnlein, or Christian Weber, Institute for Cardiovascular Prevention, LudwigMaximilians-University Munich, Pettenkoferstr 9, 80336 Munich, Germany. E-mail [email protected], oliver.soehnlein@gmail. com, or [email protected] (Circ Res. 2014;114:931-934.) © 2014 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.114.303479
NETs and Type I Interferons in Cardiovascular Disease Neutrophils have just recently emerged as important contributors to atherosclerosis.4 Depletion studies revealed that neutrophils primarily orchestrate the early stages of atherosclerosis by mechanisms involving the release of alarmins such as cathelicidin (LL37 in humans, CRAMP in mice), which promotes arterial recruitment of classical monocytes.3,5–7 In addition, NETs were identified in luminal location in murine and human atherosclerotic lesions, but their pathophysiological relevance in atherosclerosis remained initially unclear.8,9 The importance of these structures has been described in various diseases, including infection-associated thrombosis,10 small vessel vasculitis,11 systemic lupus erythematosus (SLE),12–14 and skin inflammation15 (Table). A current study has shown that NET fragments may serve as indicators of cardiovascular inflammation,16 while we could elucidate a mechanism of NET-driven atherogenesis involving the autoimmune activation of plasmacytoid dendritic cells.9,17 Mechanistically, complexes of self-DNA (presumably NET-borne DNA, but also self-DNA from dying cells) and neutrophil-derived granule proteins (eg, cathelicidin) stimulate plasmacytoid dendritic cells in the vessel wall, resulting in a strong type I interferon response, which drives atherogenesis.17 In line, the depletion of plasmacytoid dendritic cells has been shown to reduce plaque burden and type I interferon response.17,18 Moreover, NETs have also been implicated in direct activation and damage of endothelial cells (Figure).19 The importance of a type I interferon response in atherosclerosis has been reported earlier, when long-term treatment of Ldlr−/− mice with IFN-α was found to significantly increase atherosclerotic lesion development.17,20 Moreover, in human plaques, IFN-α was shown to trigger the activation of IFN-γ producing CD4+ T-cells, which kill stressed vascular smooth muscle cells in a tumor necrosis factor–related apoptosis-inducing ligand–dependent way, thereby decreasing plaque stability.21 The same group extended these findings by demonstrating that IFN-α upregulates Toll-like receptor 4 expression on and increases proinflammatory cytokine production of conventional dendritic cells (Figure).22 In addition, a central role for IFN-β in aggravating lesion formation in Apoe−/− and Ldlr−/− mice on high-fat diet has more recently been identified. Herein, IFN-β initiated a chemokine-dependent augmented endothelial cell activation and leukocyte attraction to atherosclerosis-prone sites along with an increased macrophage accumulation within plaques.23 Notably, patients with SLE exhibit chronically enhanced type I interferon levels and show a strong tendency to develop atherosclerosis.24 Generally, it is proposed that type I IFNs (and in particular IFN-α) alter the phenotype and function of endothelial progenitor cells resulting in endothelial dysfunction and defective vascular repair, thereby explaining an increased cardiovascular risk
Downloaded from http://circres.ahajournals.org/ 931 at Tulane University on September 5, 2014
932 Circulation Research March 14, 2014 Table. Pathophysiological Relevance of Neutrophil Extracellular Traps (NETs) and Inhibition of NETosis in Disease Diseases With a Pathophysiological Relevance of NETs Atherosclerosis Systemic lupus erythematosus Infection-associated thrombosis
Reference
PAD4 Inhibition Reduces Disease Severity
2,9,17 12–14 10
Venous thrombosis
29,30,35
Arterial thrombosis
2,12
Reference
Yes
2
Yes
12
Not investigated PAD4−/− Yes
35 2,12
Small vessel vasculitis
11
Not investigated
Rheumatoid arthritis
33
Yes
33 34
Colitis
34
Yes
Skin inflammation (psoriasis)
15
Not investigated
PAD indicates peptidylarginine deiminase.
and premature atherosclerosis in SLE patients.25,26 Additionally, IFN-α enhances the expression of scavenger receptor on monocytes/macrophages in SLE.27 At later stages of atherosclerosis, a NET-instructed vicious circle involving platelets and neutrophils may lead to atherothrombosis. Heteromers of platelet-derived chemokines activate neutrophils to release NETs.28 These NETs may, in turn,
further propagate platelet activation and induce thrombus formation as was shown in the models of venous thrombosis29,30 or arterial thrombosis.12
Reducing Hypercitrullination by PAD Inhibition—A Therapeutic Option in Chronic Diseases? Although mechanisms underlying the pathophysiology of NET-driven atherogenesis and atherothrombosis have been elucidated, the present study provides a possible therapeutic link. Chromatin decondensation plays an important role in NET formation and is highly associated with histone citrullination. The latter is catalyzed by PAD4, a neutrophil-enriched nuclear enzyme. Li et al31 showed that PAD4 is crucial for NET formation and bacterial killing because PAD4-knockout mice were not able to generate NETs and were strongly susceptible to bacterial infections. The authors concluded that NET formation depends on histone hypercitrullination mediated via PAD4. Dysregulation of PAD activity has been described for several chronic (autoimmune) diseases, including rheumatoid arthritis (see the Table). The emergence of PAD4 as a possible therapeutic target has led to the development of specific inhibitors of which Cl-amidine, which was used in the present study, has been shown to be the most potent one.32 Citrullinated synovial proteins, generated by PAD, are known to develop in murine collagen-induced arthritis, a model of inflammatory arthritis. Here, Cl-amidine treatment
Figure. Pathophysiological alliance of neutrophil extracellular traps (NETs) and type I interferon response in atherogenesis. Counter clockwise: Luminally netting neutrophils activate leukocytes, platelets, and endothelial cells (ECs) creating a proinflammatory milieu, presumably resulting in endothelial dysfunction, an early trigger of lesion development. Lesional NETs initiate a plasmacytoid dendritic cell (pDC)-dependent type I interferon response, leading to the activation of macrophages, neutrophils, DCs, and lymphocytes. Eventually, NET-driven proinflammatory responses will cause an inflammatory environment that favors plaque destabilization. In atherothrombosis, NETs may initiate the activation of the coagulation cascade, thus orchestrating arterial occlusion. IL12 indicates interleukin 12; MMP, matrix metalloproteinase; MPO, myeloperoxidase; ROS, reactive oxygen species; TLR4, Toll-like receptor 4; TNFα, tumor necrosis factor-α; TRAIL, TNF-related apoptosis-inducing ligand; and VSMC, vascular smooth muscle cell.
Downloaded from http://circres.ahajournals.org/ at Tulane University on September 5, 2014
Döring et al NET PAD in Atherosclerosis 933 resulted in a decreased generation of synovial citrulline, a diminished antibody response to citrullinated proteins and other autoantigens, and a substantially reduced arthritis severity.33 Furthermore, elevated PAD levels can also be measured in human and mouse colitis, and Cl-amidine treatment reduces the clinical signs of colitis along with increased apoptosis of inflammatory cells in a dextran sulfate sodium mouse model.34 In addition, the previous work of Knight et al12 revealed enhanced NET formation in New Zealand mixed mice, a model of SLE driven by type I IFNs and characterized by accelerated vascular dysfunction and prothrombotic risk. In contrast, Clamidine–treated New Zealand mixed mice in this study showed reduced NET formation, improved endothelium-dependent vasorelaxation, and prominently delayed time to arterial thrombosis. Interestingly, PAD4 and netting neutrophils also play an important role in deep vein thrombosis. The absence of NETosis in PAD4-knockout mice resulted in the formation of fewer thrombi, and PAD4 deficiency did not influence initial vessel wall activation and platelet–leukocyte adhesion. Moreover, PAD4−/− platelets were fully functional and able to produce platelet plugs comparable with wild-type platelets. The authors concluded that a low incidence of deep vein thrombosis in PAD4−/− mice is largely caused by a lack of PAD4 in neutrophils, resulting in decreased NET formation. Other functions of neutrophils seem not be affected because PAD4−/− neutrophils interact properly with the vessel wall and are present in the rare thrombi that form in PAD4−/− veins.35
Concluding Remarks Taken together, the ill alliance of NET formation and a type I interferon response seems to be a pathophysiologically relevant concept in many chronic (autoimmune) diseases, and preventive or therapeutic targeting seems as a promising approach. Interfering with hypercitrullination by PAD inhibition, resulting in the inhibition of NETosis, has recently emerged as a therapeutic tool with surprisingly low side effects over long time of treatment and even at high doses.34 However, not many studies have been published to date, and all were in mice. Moreover, the efficacy of PAD4 inhibition and also the impact of NETosis seems to be d isease-dependent. Cl-amidine treatment reduced colitis both before and after the onset of disease,34 whereas atheroprogression could not be prevented.2 One should also keep in mind that NETs represent an important first defense against invading bacteria,31 and their (long-term) inhibition may come at the cost of debilitated host defense.
Sources of Funding The authors’ research is supported by the NWO (VIDI project 91712303), the DFG (SO876/3-1, SO876/6-1, FOR809, SFB914-B08, SFB1054-B04), the Leducq Transatlantic Network of Excellence CVGeneF(x), the Else Kröner Fresenius Stiftung, and the FöFoLe program of the Ludwig-Maximilians-University Munich.
Disclosures None.
References 1. Weber C, Noels H. Atherosclerosis: current pathogenesis and therapeutic options. Nat Med. 2011;17:1410–1422.
2. Knight JS, Luo W, O’Dell AA, Yalavarthi S, Zhao W, Subramanian V, Guo C, Grenn RC, Thompson PR, Eitzman DT, Kaplan MJ. Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis. Circ Res. 2014;114:947–956. 3. Drechsler M, Megens RT, van Zandvoort M, Weber C, Soehnlein O. Hyperlipidemia-triggered neutrophilia promotes early atherosclerosis. Circulation. 2010;122:1837–1845. 4. Soehnlein O. Multiple roles for neutrophils in atherosclerosis. Circ Res. 2012;110:875–888. 5. Döring Y, Drechsler M, Wantha S, Kemmerich K, Lievens D, Vijayan S, Gallo RL, Weber C, Soehnlein O. Lack of neutrophil-derived CRAMP reduces atherosclerosis in mice. Circ Res. 2012;110:1052–1056. 6. Döring Y, Soehnlein O, Drechsler M, Shagdarsuren E, Chaudhari SM, Meiler S, Hartwig H, Hristov M, Koenen RR, Hieronymus T, Zenke M, Weber C, Zernecke A. Hematopoietic interferon regulatory factor 8-deficiency accelerates atherosclerosis in mice. Arterioscler Thromb Vasc Biol. 2012;32:1613–1623. 7. Wantha S, Alard JE, Megens RT, van der Does AM, Döring Y, Drechsler M, Pham CT, Wang MW, Wang JM, Gallo RL, von Hundelshausen P, Lindbom L, Hackeng T, Weber C, Soehnlein O. Neutrophil-derived cathelicidin promotes adhesion of classical monocytes. Circ Res. 2013;112:792–801. 8. de Boer OJ, Li X, Teeling P, Mackaay C, Ploegmakers HJ, van der Loos CM, Daemen MJ, de Winter RJ, van der Wal AC. Neutrophils, neutrophil extracellular traps and interleukin-17 associate with the organisation of thrombi in acute myocardial infarction. Thromb Haemost. 2013;109:290–297. 9. Megens RT, Vijayan S, Lievens D, Döring Y, van Zandvoort MA, Grommes J, Weber C, Soehnlein O. Presence of luminal neutrophil extracellular traps in atherosclerosis. Thromb Haemost. 2012;107: 597–598. 10. Fuchs TA, Brill A, Duerschmied D, Schatzberg D, Monestier M, Myers DD Jr, Wrobleski SK, Wakefield TW, Hartwig JH, Wagner DD. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A. 2010;107:15880–15885. 11. Kessenbrock K, Krumbholz M, Schönermarck U, Back W, Gross WL, Werb Z, Gröne HJ, Brinkmann V, Jenne DE. Netting neutrophils in autoimmune small-vessel vasculitis. Nat Med. 2009;15:623–625. 12. Knight JS, Zhao W, Luo W, Subramanian V, O’Dell AA, Yalavarthi S, Hodgin JB, Eitzman DT, Thompson PR, Kaplan MJ. Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus. J Clin Invest. 2013;123:2981–2993. 13. Garcia-Romo GS, Caielli S, Vega B, Connolly J, Allantaz F, Xu Z, Punaro M, Baisch J, Guiducci C, Coffman RL, Barrat FJ, Banchereau J, Pascual V. Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci Transl Med. 2011;3:73ra20. 14. Lande R, Ganguly D, Facchinetti V, et al. Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. Sci Transl Med. 2011;3:73ra19. 15. Keijsers RR, Hendriks AG, van Erp PE, van Cranenbroek B, van de Kerkhof PC, Koenen HJ, Joosten I. In vivo induction of cutaneous inflammation results in the accumulation of extracellular trap-forming neutrophils expressing rorgammat and il-17 [published online ahead of print 9 January 2014]. J Invest Dermatol. doi:10.1038/jid.2013.526. 16. Borissoff JI, Joosen IA, Versteylen MO, Brill A, Fuchs TA, Savchenko AS, Gallant M, Martinod K, Ten Cate H, Hofstra L, Crijns HJ, Wagner DD, Kietselaer BL. Elevated levels of circulating DNA and chromatin are independently associated with severe coronary atherosclerosis and a prothrombotic state. Arterioscler Thromb Vasc Biol. 2013;33:2032–2040. 17. Döring Y, Manthey HD, Drechsler M, et al. Auto-antigenic protein-DNA complexes stimulate plasmacytoid dendritic cells to promote atherosclerosis. Circulation. 2012;125:1673–1683. 18. Macritchie N, Grassia G, Sabir SR, Maddaluno M, Welsh P, Sattar N, Ialenti A, Kurowska-Stolarska M, McInnes IB, Brewer JM, Garside P, Maffia P. Plasmacytoid dendritic cells play a key role in promoting atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2012;32:2569–2579. 19. Villanueva E, Yalavarthi S, Berthier CC, et al. Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus. J Immunol. 2011;187:538–552.
Downloaded from http://circres.ahajournals.org/ at Tulane University on September 5, 2014
934 Circulation Research March 14, 2014 20. Levy Z, Rachmani R, Trestman S, Dvir A, Shaish A, Ravid M, Harats D. Low-dose interferon-alpha accelerates atherosclerosis in an LDL receptor-deficient mouse model. Eur J Intern Med. 2003;14:479–483. 21. Niessner A, Sato K, Chaikof EL, Colmegna I, Goronzy JJ, Weyand CM. Pathogen-sensing plasmacytoid dendritic cells stimulate cytotoxic T-cell function in the atherosclerotic plaque through interferon-alpha. Circulation. 2006;114:2482–2489. 22. Niessner A, Shin MS, Pryshchep O, Goronzy JJ, Chaikof EL, Weyand CM. Synergistic proinflammatory effects of the antiviral cytokine interferon-alpha and Toll-like receptor 4 ligands in the atherosclerotic plaque. Circulation. 2007;116:2043–2052. 23. Goossens P, Gijbels MJ, Zernecke A, Eijgelaar W, Vergouwe MN, van der Made I, Vanderlocht J, Beckers L, Buurman WA, Daemen MJ, Kalinke U, Weber C, Lutgens E, de Winther MP. Myeloid type I interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions. Cell Metab. 2010;12:142–153. 24. Rönnblom L. The type I interferon system in the etiopathogenesis of autoimmune diseases. Ups J Med Sci. 2011;116:227–237. 25. Denny MF, Thacker S, Mehta H, Somers EC, Dodick T, Barrat FJ, McCune WJ, Kaplan MJ. Interferon-alpha promotes abnormal vasculogenesis in lupus: a potential pathway for premature atherosclerosis. Blood. 2007;110:2907–2915. 26. Thacker SG, Zhao W, Smith CK, Luo W, Wang H, Vivekanandan-Giri A, Rabquer BJ, Koch AE, Pennathur S, Davidson A, Eitzman DT, Kaplan MJ. Type I interferons modulate vascular function, repair, thrombosis, and plaque progression in murine models of lupus and atherosclerosis. Arthritis Rheum. 2012;64:2975–2985. 27. Li J, Fu Q, Cui H, Qu B, Pan W, Shen N, Bao C. Interferon-alpha priming promotes lipid uptake and macrophage-derived foam cell formation: a novel link between interferon-alpha and atherosclerosis in lupus. Arthritis Rheum. 2011;63:492–502. 28. Rossaint J, Herter JM, Van Aken H, Doring Y, Weber C, Soehnlein O, Zarbock A. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap mediated sterile inflammation
[published online ahead of print 12 December 2013]. Blood. doi:10.1182/ blood-2013-07-516484. 29. Brill A, Fuchs TA, Savchenko AS, Thomas GM, Martinod K, De Meyer SF, Bhandari AA, Wagner DD. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost. 2012;10: 136–144. 30. von Brühl ML, Stark K, Steinhart A, et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med. 2012;209:819–835. 31. Li P, Li M, Lindberg MR, Kennett MJ, Xiong N, Wang Y. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med. 2010;207:1853–1862. 32. Knuckley B, Causey CP, Pellechia PJ, Cook PF, Thompson PR. Haloacetamidine-based inactivators of protein arginine deiminase 4 (PAD4): evidence that general acid catalysis promotes efficient inactivation. Chembiochem. 2010;11:161–165. 33. Willis VC, Gizinski AM, Banda NK, Causey CP, Knuckley B, Cordova KN, Luo Y, Levitt B, Glogowska M, Chandra P, Kulik L, Robinson WH, Arend WP, Thompson PR, Holers VM. N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide, a protein arginine deiminase inhibitor, reduces the severity of murine collagen-induced arthritis. J Immunol. 2011;186:4396–4404. 34. Chumanevich AA, Causey CP, Knuckley BA, Jones JE, Poudyal D, Chumanevich AP, Davis T, Matesic LE, Thompson PR, Hofseth LJ. Suppression of colitis in mice by Cl-amidine: a novel peptidylarginine deiminase inhibitor. Am J Physiol Gastrointest Liver Physiol. 2011;300:G929–G938. 35. Martinod K, Demers M, Fuchs TA, Wong SL, Brill A, Gallant M, Hu J, Wang Y, Wagner DD. Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice. Proc Natl Acad Sci U S A. 2013;110:8674–8679. Key Words: atherosclerosis ■ N-alpha-benzoyl-N5-(2-chloro-1-iminoethyl)L-ornithine amide ■ protein-arginine deiminase
Downloaded from http://circres.ahajournals.org/ at Tulane University on September 5, 2014
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/114/6/931
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/
Downloaded from http://circres.ahajournals.org/ at Tulane University on September 5, 2014
Editorial Neutrophils Cast NETs in Atherosclerosis Employing Peptidylarginine Deiminase as a Therapeutic Target Yvonne Döring, Oliver Soehnlein, Christian Weber
A
therosclerosis, an inflammatory response in large arteries starting with a subclinical endothelial dysfunction, can turn into a life-threatening disease manifesting as myocardial infarction or stroke. Throughout the past decades, it has become apparent that atherosclerosis is not solely based on an imbalanced lipid metabolism but is mainly driven by a chronic inflammation of the vessel wall involving numerous cell types.1 Regardless of intensive research in the past 30 years, gold standard atherosclerosis-targeted therapies are still restricted to the control of primary risk factors such as hypertension and hyperlipidemia. Despite the proven efficacy of these drugs, cardiovascular diseases remain the leading cause of mortality in Western societies, underlining the need for new therapeutic approaches.1 In this issue of Circulation Research, Knight et al2 show that the inhibition of peptidylarginine deiminase (PAD) by Cl-amidine treatment prevents neutrophil extracellular trap (NET) formation and thereby decreases atherosclerotic lesion size and delays carotid artery thrombosis in Apoe−/− mice receiving a cholesterol-rich diet. Specific inhibition of NETosis was accompanied by a decrease in arterial interferon (IFN)-α expression, less neutrophil recruitment to the arterial wall, and a decline in intimal macrophages. Notably, all these findings could not be repeated when Cl-amidine was administered into Apoe−/− mice treated with a neutrophil-depleting antibody or into mice lacking a functional type I interferon receptor. These data suggest an important role for NET formation and the subsequently instigated type I interferon response in atherogenesis. In contrast to studies on early lesion formation, a treatment regimen targeting advanced stages of atherosclerosis was without success, underscoring the importance of neutrophil-driven proatherosclerotic processes during atherogenesis only.3
Article, see p 947
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From Institute for Cardiovascular Prevention, L udwig-MaximiliansUniversity Munich, Germany (Y.D., O.S., C.W.); Academic Medical Center, Department of Pathology, Amsterdam University, the Netherlands (O.S.); and DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Germany (O.S., C.W.). Correspondence to Drs Yvonne Döring, Oliver Soehnlein, or Christian Weber, Institute for Cardiovascular Prevention, LudwigMaximilians-University Munich, Pettenkoferstr 9, 80336 Munich, Germany. E-mail [email protected], oliver.soehnlein@gmail. com, or [email protected] (Circ Res. 2014;114:931-934.) © 2014 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.114.303479
NETs and Type I Interferons in Cardiovascular Disease Neutrophils have just recently emerged as important contributors to atherosclerosis.4 Depletion studies revealed that neutrophils primarily orchestrate the early stages of atherosclerosis by mechanisms involving the release of alarmins such as cathelicidin (LL37 in humans, CRAMP in mice), which promotes arterial recruitment of classical monocytes.3,5–7 In addition, NETs were identified in luminal location in murine and human atherosclerotic lesions, but their pathophysiological relevance in atherosclerosis remained initially unclear.8,9 The importance of these structures has been described in various diseases, including infection-associated thrombosis,10 small vessel vasculitis,11 systemic lupus erythematosus (SLE),12–14 and skin inflammation15 (Table). A current study has shown that NET fragments may serve as indicators of cardiovascular inflammation,16 while we could elucidate a mechanism of NET-driven atherogenesis involving the autoimmune activation of plasmacytoid dendritic cells.9,17 Mechanistically, complexes of self-DNA (presumably NET-borne DNA, but also self-DNA from dying cells) and neutrophil-derived granule proteins (eg, cathelicidin) stimulate plasmacytoid dendritic cells in the vessel wall, resulting in a strong type I interferon response, which drives atherogenesis.17 In line, the depletion of plasmacytoid dendritic cells has been shown to reduce plaque burden and type I interferon response.17,18 Moreover, NETs have also been implicated in direct activation and damage of endothelial cells (Figure).19 The importance of a type I interferon response in atherosclerosis has been reported earlier, when long-term treatment of Ldlr−/− mice with IFN-α was found to significantly increase atherosclerotic lesion development.17,20 Moreover, in human plaques, IFN-α was shown to trigger the activation of IFN-γ producing CD4+ T-cells, which kill stressed vascular smooth muscle cells in a tumor necrosis factor–related apoptosis-inducing ligand–dependent way, thereby decreasing plaque stability.21 The same group extended these findings by demonstrating that IFN-α upregulates Toll-like receptor 4 expression on and increases proinflammatory cytokine production of conventional dendritic cells (Figure).22 In addition, a central role for IFN-β in aggravating lesion formation in Apoe−/− and Ldlr−/− mice on high-fat diet has more recently been identified. Herein, IFN-β initiated a chemokine-dependent augmented endothelial cell activation and leukocyte attraction to atherosclerosis-prone sites along with an increased macrophage accumulation within plaques.23 Notably, patients with SLE exhibit chronically enhanced type I interferon levels and show a strong tendency to develop atherosclerosis.24 Generally, it is proposed that type I IFNs (and in particular IFN-α) alter the phenotype and function of endothelial progenitor cells resulting in endothelial dysfunction and defective vascular repair, thereby explaining an increased cardiovascular risk
Downloaded from http://circres.ahajournals.org/ 931 at Tulane University on September 5, 2014
932 Circulation Research March 14, 2014 Table. Pathophysiological Relevance of Neutrophil Extracellular Traps (NETs) and Inhibition of NETosis in Disease Diseases With a Pathophysiological Relevance of NETs Atherosclerosis Systemic lupus erythematosus Infection-associated thrombosis
Reference
PAD4 Inhibition Reduces Disease Severity
2,9,17 12–14 10
Venous thrombosis
29,30,35
Arterial thrombosis
2,12
Reference
Yes
2
Yes
12
Not investigated PAD4−/− Yes
35 2,12
Small vessel vasculitis
11
Not investigated
Rheumatoid arthritis
33
Yes
33 34
Colitis
34
Yes
Skin inflammation (psoriasis)
15
Not investigated
PAD indicates peptidylarginine deiminase.
and premature atherosclerosis in SLE patients.25,26 Additionally, IFN-α enhances the expression of scavenger receptor on monocytes/macrophages in SLE.27 At later stages of atherosclerosis, a NET-instructed vicious circle involving platelets and neutrophils may lead to atherothrombosis. Heteromers of platelet-derived chemokines activate neutrophils to release NETs.28 These NETs may, in turn,
further propagate platelet activation and induce thrombus formation as was shown in the models of venous thrombosis29,30 or arterial thrombosis.12
Reducing Hypercitrullination by PAD Inhibition—A Therapeutic Option in Chronic Diseases? Although mechanisms underlying the pathophysiology of NET-driven atherogenesis and atherothrombosis have been elucidated, the present study provides a possible therapeutic link. Chromatin decondensation plays an important role in NET formation and is highly associated with histone citrullination. The latter is catalyzed by PAD4, a neutrophil-enriched nuclear enzyme. Li et al31 showed that PAD4 is crucial for NET formation and bacterial killing because PAD4-knockout mice were not able to generate NETs and were strongly susceptible to bacterial infections. The authors concluded that NET formation depends on histone hypercitrullination mediated via PAD4. Dysregulation of PAD activity has been described for several chronic (autoimmune) diseases, including rheumatoid arthritis (see the Table). The emergence of PAD4 as a possible therapeutic target has led to the development of specific inhibitors of which Cl-amidine, which was used in the present study, has been shown to be the most potent one.32 Citrullinated synovial proteins, generated by PAD, are known to develop in murine collagen-induced arthritis, a model of inflammatory arthritis. Here, Cl-amidine treatment
Figure. Pathophysiological alliance of neutrophil extracellular traps (NETs) and type I interferon response in atherogenesis. Counter clockwise: Luminally netting neutrophils activate leukocytes, platelets, and endothelial cells (ECs) creating a proinflammatory milieu, presumably resulting in endothelial dysfunction, an early trigger of lesion development. Lesional NETs initiate a plasmacytoid dendritic cell (pDC)-dependent type I interferon response, leading to the activation of macrophages, neutrophils, DCs, and lymphocytes. Eventually, NET-driven proinflammatory responses will cause an inflammatory environment that favors plaque destabilization. In atherothrombosis, NETs may initiate the activation of the coagulation cascade, thus orchestrating arterial occlusion. IL12 indicates interleukin 12; MMP, matrix metalloproteinase; MPO, myeloperoxidase; ROS, reactive oxygen species; TLR4, Toll-like receptor 4; TNFα, tumor necrosis factor-α; TRAIL, TNF-related apoptosis-inducing ligand; and VSMC, vascular smooth muscle cell.
Downloaded from http://circres.ahajournals.org/ at Tulane University on September 5, 2014
Döring et al NET PAD in Atherosclerosis 933 resulted in a decreased generation of synovial citrulline, a diminished antibody response to citrullinated proteins and other autoantigens, and a substantially reduced arthritis severity.33 Furthermore, elevated PAD levels can also be measured in human and mouse colitis, and Cl-amidine treatment reduces the clinical signs of colitis along with increased apoptosis of inflammatory cells in a dextran sulfate sodium mouse model.34 In addition, the previous work of Knight et al12 revealed enhanced NET formation in New Zealand mixed mice, a model of SLE driven by type I IFNs and characterized by accelerated vascular dysfunction and prothrombotic risk. In contrast, Clamidine–treated New Zealand mixed mice in this study showed reduced NET formation, improved endothelium-dependent vasorelaxation, and prominently delayed time to arterial thrombosis. Interestingly, PAD4 and netting neutrophils also play an important role in deep vein thrombosis. The absence of NETosis in PAD4-knockout mice resulted in the formation of fewer thrombi, and PAD4 deficiency did not influence initial vessel wall activation and platelet–leukocyte adhesion. Moreover, PAD4−/− platelets were fully functional and able to produce platelet plugs comparable with wild-type platelets. The authors concluded that a low incidence of deep vein thrombosis in PAD4−/− mice is largely caused by a lack of PAD4 in neutrophils, resulting in decreased NET formation. Other functions of neutrophils seem not be affected because PAD4−/− neutrophils interact properly with the vessel wall and are present in the rare thrombi that form in PAD4−/− veins.35
Concluding Remarks Taken together, the ill alliance of NET formation and a type I interferon response seems to be a pathophysiologically relevant concept in many chronic (autoimmune) diseases, and preventive or therapeutic targeting seems as a promising approach. Interfering with hypercitrullination by PAD inhibition, resulting in the inhibition of NETosis, has recently emerged as a therapeutic tool with surprisingly low side effects over long time of treatment and even at high doses.34 However, not many studies have been published to date, and all were in mice. Moreover, the efficacy of PAD4 inhibition and also the impact of NETosis seems to be d isease-dependent. Cl-amidine treatment reduced colitis both before and after the onset of disease,34 whereas atheroprogression could not be prevented.2 One should also keep in mind that NETs represent an important first defense against invading bacteria,31 and their (long-term) inhibition may come at the cost of debilitated host defense.
Sources of Funding The authors’ research is supported by the NWO (VIDI project 91712303), the DFG (SO876/3-1, SO876/6-1, FOR809, SFB914-B08, SFB1054-B04), the Leducq Transatlantic Network of Excellence CVGeneF(x), the Else Kröner Fresenius Stiftung, and the FöFoLe program of the Ludwig-Maximilians-University Munich.
Disclosures None.
References 1. Weber C, Noels H. Atherosclerosis: current pathogenesis and therapeutic options. Nat Med. 2011;17:1410–1422.
2. Knight JS, Luo W, O’Dell AA, Yalavarthi S, Zhao W, Subramanian V, Guo C, Grenn RC, Thompson PR, Eitzman DT, Kaplan MJ. Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis. Circ Res. 2014;114:947–956. 3. Drechsler M, Megens RT, van Zandvoort M, Weber C, Soehnlein O. Hyperlipidemia-triggered neutrophilia promotes early atherosclerosis. Circulation. 2010;122:1837–1845. 4. Soehnlein O. Multiple roles for neutrophils in atherosclerosis. Circ Res. 2012;110:875–888. 5. Döring Y, Drechsler M, Wantha S, Kemmerich K, Lievens D, Vijayan S, Gallo RL, Weber C, Soehnlein O. Lack of neutrophil-derived CRAMP reduces atherosclerosis in mice. Circ Res. 2012;110:1052–1056. 6. Döring Y, Soehnlein O, Drechsler M, Shagdarsuren E, Chaudhari SM, Meiler S, Hartwig H, Hristov M, Koenen RR, Hieronymus T, Zenke M, Weber C, Zernecke A. Hematopoietic interferon regulatory factor 8-deficiency accelerates atherosclerosis in mice. Arterioscler Thromb Vasc Biol. 2012;32:1613–1623. 7. Wantha S, Alard JE, Megens RT, van der Does AM, Döring Y, Drechsler M, Pham CT, Wang MW, Wang JM, Gallo RL, von Hundelshausen P, Lindbom L, Hackeng T, Weber C, Soehnlein O. Neutrophil-derived cathelicidin promotes adhesion of classical monocytes. Circ Res. 2013;112:792–801. 8. de Boer OJ, Li X, Teeling P, Mackaay C, Ploegmakers HJ, van der Loos CM, Daemen MJ, de Winter RJ, van der Wal AC. Neutrophils, neutrophil extracellular traps and interleukin-17 associate with the organisation of thrombi in acute myocardial infarction. Thromb Haemost. 2013;109:290–297. 9. Megens RT, Vijayan S, Lievens D, Döring Y, van Zandvoort MA, Grommes J, Weber C, Soehnlein O. Presence of luminal neutrophil extracellular traps in atherosclerosis. Thromb Haemost. 2012;107: 597–598. 10. Fuchs TA, Brill A, Duerschmied D, Schatzberg D, Monestier M, Myers DD Jr, Wrobleski SK, Wakefield TW, Hartwig JH, Wagner DD. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A. 2010;107:15880–15885. 11. Kessenbrock K, Krumbholz M, Schönermarck U, Back W, Gross WL, Werb Z, Gröne HJ, Brinkmann V, Jenne DE. Netting neutrophils in autoimmune small-vessel vasculitis. Nat Med. 2009;15:623–625. 12. Knight JS, Zhao W, Luo W, Subramanian V, O’Dell AA, Yalavarthi S, Hodgin JB, Eitzman DT, Thompson PR, Kaplan MJ. Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus. J Clin Invest. 2013;123:2981–2993. 13. Garcia-Romo GS, Caielli S, Vega B, Connolly J, Allantaz F, Xu Z, Punaro M, Baisch J, Guiducci C, Coffman RL, Barrat FJ, Banchereau J, Pascual V. Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci Transl Med. 2011;3:73ra20. 14. Lande R, Ganguly D, Facchinetti V, et al. Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. Sci Transl Med. 2011;3:73ra19. 15. Keijsers RR, Hendriks AG, van Erp PE, van Cranenbroek B, van de Kerkhof PC, Koenen HJ, Joosten I. In vivo induction of cutaneous inflammation results in the accumulation of extracellular trap-forming neutrophils expressing rorgammat and il-17 [published online ahead of print 9 January 2014]. J Invest Dermatol. doi:10.1038/jid.2013.526. 16. Borissoff JI, Joosen IA, Versteylen MO, Brill A, Fuchs TA, Savchenko AS, Gallant M, Martinod K, Ten Cate H, Hofstra L, Crijns HJ, Wagner DD, Kietselaer BL. Elevated levels of circulating DNA and chromatin are independently associated with severe coronary atherosclerosis and a prothrombotic state. Arterioscler Thromb Vasc Biol. 2013;33:2032–2040. 17. Döring Y, Manthey HD, Drechsler M, et al. Auto-antigenic protein-DNA complexes stimulate plasmacytoid dendritic cells to promote atherosclerosis. Circulation. 2012;125:1673–1683. 18. Macritchie N, Grassia G, Sabir SR, Maddaluno M, Welsh P, Sattar N, Ialenti A, Kurowska-Stolarska M, McInnes IB, Brewer JM, Garside P, Maffia P. Plasmacytoid dendritic cells play a key role in promoting atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2012;32:2569–2579. 19. Villanueva E, Yalavarthi S, Berthier CC, et al. Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus. J Immunol. 2011;187:538–552.
Downloaded from http://circres.ahajournals.org/ at Tulane University on September 5, 2014
934 Circulation Research March 14, 2014 20. Levy Z, Rachmani R, Trestman S, Dvir A, Shaish A, Ravid M, Harats D. Low-dose interferon-alpha accelerates atherosclerosis in an LDL receptor-deficient mouse model. Eur J Intern Med. 2003;14:479–483. 21. Niessner A, Sato K, Chaikof EL, Colmegna I, Goronzy JJ, Weyand CM. Pathogen-sensing plasmacytoid dendritic cells stimulate cytotoxic T-cell function in the atherosclerotic plaque through interferon-alpha. Circulation. 2006;114:2482–2489. 22. Niessner A, Shin MS, Pryshchep O, Goronzy JJ, Chaikof EL, Weyand CM. Synergistic proinflammatory effects of the antiviral cytokine interferon-alpha and Toll-like receptor 4 ligands in the atherosclerotic plaque. Circulation. 2007;116:2043–2052. 23. Goossens P, Gijbels MJ, Zernecke A, Eijgelaar W, Vergouwe MN, van der Made I, Vanderlocht J, Beckers L, Buurman WA, Daemen MJ, Kalinke U, Weber C, Lutgens E, de Winther MP. Myeloid type I interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions. Cell Metab. 2010;12:142–153. 24. Rönnblom L. The type I interferon system in the etiopathogenesis of autoimmune diseases. Ups J Med Sci. 2011;116:227–237. 25. Denny MF, Thacker S, Mehta H, Somers EC, Dodick T, Barrat FJ, McCune WJ, Kaplan MJ. Interferon-alpha promotes abnormal vasculogenesis in lupus: a potential pathway for premature atherosclerosis. Blood. 2007;110:2907–2915. 26. Thacker SG, Zhao W, Smith CK, Luo W, Wang H, Vivekanandan-Giri A, Rabquer BJ, Koch AE, Pennathur S, Davidson A, Eitzman DT, Kaplan MJ. Type I interferons modulate vascular function, repair, thrombosis, and plaque progression in murine models of lupus and atherosclerosis. Arthritis Rheum. 2012;64:2975–2985. 27. Li J, Fu Q, Cui H, Qu B, Pan W, Shen N, Bao C. Interferon-alpha priming promotes lipid uptake and macrophage-derived foam cell formation: a novel link between interferon-alpha and atherosclerosis in lupus. Arthritis Rheum. 2011;63:492–502. 28. Rossaint J, Herter JM, Van Aken H, Doring Y, Weber C, Soehnlein O, Zarbock A. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap mediated sterile inflammation
[published online ahead of print 12 December 2013]. Blood. doi:10.1182/ blood-2013-07-516484. 29. Brill A, Fuchs TA, Savchenko AS, Thomas GM, Martinod K, De Meyer SF, Bhandari AA, Wagner DD. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost. 2012;10: 136–144. 30. von Brühl ML, Stark K, Steinhart A, et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med. 2012;209:819–835. 31. Li P, Li M, Lindberg MR, Kennett MJ, Xiong N, Wang Y. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med. 2010;207:1853–1862. 32. Knuckley B, Causey CP, Pellechia PJ, Cook PF, Thompson PR. Haloacetamidine-based inactivators of protein arginine deiminase 4 (PAD4): evidence that general acid catalysis promotes efficient inactivation. Chembiochem. 2010;11:161–165. 33. Willis VC, Gizinski AM, Banda NK, Causey CP, Knuckley B, Cordova KN, Luo Y, Levitt B, Glogowska M, Chandra P, Kulik L, Robinson WH, Arend WP, Thompson PR, Holers VM. N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide, a protein arginine deiminase inhibitor, reduces the severity of murine collagen-induced arthritis. J Immunol. 2011;186:4396–4404. 34. Chumanevich AA, Causey CP, Knuckley BA, Jones JE, Poudyal D, Chumanevich AP, Davis T, Matesic LE, Thompson PR, Hofseth LJ. Suppression of colitis in mice by Cl-amidine: a novel peptidylarginine deiminase inhibitor. Am J Physiol Gastrointest Liver Physiol. 2011;300:G929–G938. 35. Martinod K, Demers M, Fuchs TA, Wong SL, Brill A, Gallant M, Hu J, Wang Y, Wagner DD. Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice. Proc Natl Acad Sci U S A. 2013;110:8674–8679. Key Words: atherosclerosis ■ N-alpha-benzoyl-N5-(2-chloro-1-iminoethyl)L-ornithine amide ■ protein-arginine deiminase
Downloaded from http://circres.ahajournals.org/ at Tulane University on September 5, 2014
