Accepted Article
Article Type: Commentary Commentary to Maugeri et al., JTH-2014-00190.R2
Moonlighting proteins dictate the cross-talk between thrombosis and innate immunity
Mona Saffarzadeh (1), Klaus T. Preissner (2)*
(1) Center for Thrombosis and Hemostasis (CTH), University Medical Center, 55131, Mainz, Germany (2) Department of Biochemistry, Medical School, Justus-Liebig-University, 35392 Giessen, Germany
* Corresponding author
Corresponding address: Klaus T. Preissner, Ph.D. Department of Biochemistry, Medical School Justus-Liebig-University Friedrichstrasse 24 D-35392 Giessen Germany Tel. +49-641-994-7500 Fax. +49-641-994-7509 e.mail: [email protected]
Keywords HMGB1 Protein , Innate immunity , Neutrophil , Platelet , Thrombosis Upon exposure of our body towards infectious or damaging factors, an immediate host response is mobilized, including toll-like receptor (TLR)-mediated pathogen recognition, to provoke the release of alarming molecules, followed by a spatiotemporal cytokine storm (1). This is succeeded by the This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jth.12754 This article is protected by copyright. All rights reserved.
Accepted Article
recruitment of leukocytes to the site of inflammation and culminates in the catching and killing of microbial invaders, particularly by neutrophil granulocytes. The required effective tissue repair and regeneration process is achieved by the hemostasis and blood coagulation machinery, resulting in wound sealing by aggregated platelets and a stable fibrin network, which also prevents further entry of microorganisms (2). The molecular cooperation between the inflammatory cascade and the hemostasis system has long been recognized and is evident from e.g. inflammatory functions of coagulation factors that are transmitted via “protease-activated receptors” and related signal transduction into immune or vascular cells (3). Only recently however, several groundbreaking studies have uncovered principle mechanisms of the inflammation-hemostasis cross-talk, designated as “immunothrombosis”, that involves some unexpected factors like nuclear histones and DNA to be operative in the extracellular space (4). Thus, it appears that these formerly intracellular components have found new jobs outside cells in the context of immune defense, rendering them “moonlighting” factors (5). Another example for a “moonlighting protein”, which has different functions inside and outside cells, is the non-histone, chromatin protein “High mobility group box-1” (HMGB1 or amphoterin), which is expressed in almost all cell types: In the nucleus, HMGB1 interacts with nucleosomes, transcription factors, and histones, organizes DNA compaction by remodeling the chromatin and regulates transcription (6-8). Posttranslational acetylation of HMGB1 causes its translocation into the cytosol, from where it may become released under stress, injury or inflammation (such as in sepsis) to serve as an endogenous danger signal. In general, HMGB1 can be sensed by cells via the “Receptor for advanced glycation endproducts” (RAGE) as well as by Toll-like receptors TLR2 and TLR4 (9). Interestingly enough, once activated by exogenous and endogenous stimulants, neutrophils are provoked to eject their entire nucleosome material in the form of ultra-large DNA-histone scaffolds, designated “neutrophil extracellular traps” (NETs), which facilitate the recognition and destruction of pathogens with the help of neutrophil enzymes and anti-microbial proteins (10). Surprisingly, activated platelets, which are known to form cellular conjugates with neutrophils, where found to promote NETosis as well. The generated NETs thereby provide an appropriate surface in the blood stream or at the vessel wall to initiate coagulation and thrombus formation but also foster thrombotic events, if this process remains uncontrolled (11-15). In fact, this cross-talk between neutrophils and platelets appears to play a key role in regulating inflammation and vascular tone, and the presence of NETs together with platelet-neutrophil conjugates has been documented in sepsis or lung injury as well (16-18). However, the mechanisms involved in the initial mutual interactions between platelets and neutrophils to provoke NETosis are hardly uncovered. In this issue of JTH, Maugeri et al. (19) reported that coronary thrombi of patients with acute myocardial infarction contained activated platelets and HMGB1, which may trigger neutrophils to commit autophagy and NET generation. Although such a co-localization may not necessarily correspond with a functional relation, the authors could show that platelets release HMGB1 to promote NETosis via RAGE but not via TLRs on neutrophils. While these molecular interactions could provide the basis for the proposed early triggering of neutrophil responses, resulting in subsequent NET formation and development of thrombosis, diverging findings from other groups need to be taken in consideration to understand the underlying pathomechanisms.
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For example, Tadie et al. recently documented that HMGB1 promotes NETosis as well as intracellular histone 3-citrullination, a signaling event that precedes chromatin decondensation, predominantly via TLR4 but not RAGE (20). Also, together with the classical TLR4-ligand lipopolysaccharide (LPS), HMGB1 (which binds to LPS) enhanced the formation of NET and thereby may contribute to the severity of neutrophil-associated inflammation (9, 21). While a cross-talk between RAGE and TLR4 and their shared downstream cellular signaling via MyD88 may be considered to explain these findings (22), Maugeri et al. (19) have shown that MyD88-deficient neutrophils can still produce NETs following exposure towards HMGB1, whereas RAGE-deficient neutrophils cannot. Although they concluded that HMGB1 acts via RAGE and not TLR4, one should not ignore the MyD88-independent pathway of TLR4 signaling which may be involved as well (23). Yet, could other platelet secretory products influence the interactions of HMGB1 with neutrophils? Upon activation, platelets degranulate and release multiple low molecular agonists and proteins, including HMGB1, as well as procoagulant microparticles (7, 24) and inorganic polyphosphates (polyP) that have recently been characterized as potent inducers of blood clotting and modulators of vascular tone and complement in inflammation (25). These polyanionic inorganic molecules (60 to 100 phosphate units) not only increase thrombin generation via contact phase activation and to promote factor XIa generation (26, 27), but polyP may amplify HMGB1- and histone-mediated inflammatory signaling in human endothelial cells via interaction with RAGE and the purinergic receptor P2Y1. Binding of polyP to basic proteins like histones and HMGB1 thereby enhances the interaction of both ligands to RAGE, whereby polyP serve as bridging molecules for interactions with the P2Y1 receptor. As a consequence, clustering of RAGE may promote selective downstream signaling in target cells (28), and such interactions could explain the current findings by Maugeri et al. (19) as well. The authors, however, did not explore whether polyP and HMGB1 are secreted as a complex by activated platelets and whether the indicated molecular bridging mechanism involving polyP would favor RAGE-mediated signaling but prevent HMGB1 from engaging TLR4. In addition to histones as major components of NETs (29), HMGB1 has also been identified as NETassociated protein (30), and it is plausible to assume that these “moonlighting proteins” can trigger a feed-forward loop by activating platelets, promoting NETosis and thereby enhancing inflammatory responses and thrombus growth (Figure 1). Mechanistically, histones directly activate platelets via TLR2 and TLR4 (31), in turn releasing HMGB1 and polyP, which together engage RAGE on endothelial cells and on neutrophils to foster a strong cellular induction with subsequent amplification of inflammatory responses and NETosis. Interestingly, during bacterial infection, complement-mediated bacterial lysis results in the occurrence of microbial polyP with chain length >100 phosphate units (32) that may complex appreciable amounts of HMGB1 and histones, much stronger than plateletderived polyP would do. Consequently, receptor bridging und clustering would provoke a robust inflammatory and cytotoxic response in endothelial cells (28), such that this exaggerated immune response can lead to devastating pathological consequences as in sepsis. Although the indicated cellular responses towards isolated histones and HMGB1 have been characterized in the cited publication, association with DNA and NETs may substantially modulate the functional properties of these “moonlighting proteins” with respect to affinity, receptor occupancy or ligand bridging. In fact, hyper-citrullination of histones (33) as well hyper-acetylation of HMGB1 appear to be required for liberation of these proteins from cells, but the impact of such post-
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Accepted Article
translational modifications on ligand binding and activation of TLRs and RAGE has not been considered in detail. While Maugeri et al. (19) have shown that association of isolated mammalian DNA with HMGB1 did not influence its ability to promote NET-formation, such data may not necessarily reflect the authentic situation of NET-associated HMGB1. Although Maugeri et al. (19) have suggested an HMGB1-inhibitor to counteract such complications, and anti-HMGB1 antibodies in LPS-treated mice partially prevented NETosis in lung neutrophils (20), targeting HMGB1 may not be an ideal regimen due to its important role in autophagy. Anti-histone antibodies that have been applied in several inflammatory models promoted a beneficial outcome to dampen the side effect of hyper-inflammation or cytotoxicity (34). Another natural, histonecomplexing compound, polysialic acid, exhibited a significant protective effect against isolated as well as NET-associated histone-mediated cytotoxicity and was superior to than anti-histone antibodies, at least under in vitro conditions (35). As an alternative, activated protein C (APC), was found to inhibit polyP-mediated proinflammatory activities of HMGB1 and histones by degrading these basic “moonlighting proteins” (29). Likewise, APC efficiently cleaves isolated histones and reduces their inflammatory potential in an experimental sepsis model (36). However, high concentrations of APC are required to achieve such protective effects, and NET-associated histones appear to be resistant against APC (37). Finally, soluble RAGE, which lacks the transmembrane and signaling domains of RAGE (38), may serve as decoy receptor to inhibit HMGB1- and histone-mediated signaling, particularly in combination with polyP, which binds to soluble RAGE with high affinity (28). In essence, the work by Maugeri et al. (19) adds another relevant player to the system of immunothrombosis that not only has a major impact on the cross-talk between hemostasis and inflammation in body defense, but may derail into severe inflammatory disease and thrombosis if not counteracted properly. Here, the new data provide another challenge for searching novel therapeutic strategies to fight against the dark side of NETs (38).
Addendum Both authors were responsible for research, writing and editing of the manuscript.
Acknowledgements The work of the authors cited in this article was supported by the Federal Ministry of Education and Research (BMBF 01EO1003) (Virchow Fellowship) as well as by the Deutsche Forschungsgemeinschaft (Bonn, Germany) within the "Excellence-cluster Cardio-pulmonary System" (ECCPS).
Disclosure Authors report no conflicts of interest.
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References 1 Tisoncik JR, Korth MJ, Simmons CP, Farrar J, Martin TR, Katze MG. Into the eye of the cytokine storm. Microbiol Mol Biol Rev. 2012; 76: 16-32. 2 Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol. 2013; 13: 159-175. 3 Rothmeier AS, Ruf W. Protease-activated receptor 2 signaling in inflammation. Sem Immunopathol. 2012; 34: 133-149. 4 Engelmann B, Massberg S. Thrombosis as an intravascular effector of innate immunity. Nat Rev Immunol. 2013; 13: 34-45. 5 Huberts DH, van der Klei IJ. Moonlighting proteins: an intriguing mode of multitasking. Biochim Biophys Acta. 2010;1803: 520-525. 6 Andersson U, Tracey KJ. HMGB1 is a therapeutic target for sterile inflammation and infection. Annu Rev Immunol. 2011;29: 139-162 7 Rouhiainen A, Imai S, Rauvala H, Parkkinen J. Occurrence of amphoterin (HMG1) as an endogenous protein of human platelets that is exported to the cell surface upon platelet activation. Thromb Haemost. 2000; 84: 1087-1094. 8 Tsung A, Tohme S, Billiar TR. High mobility group box-1 in sterile inflammation. Journal of internal medicine. 2014. 10.1111/joim.12276. 9 Ibrahim ZA, Armour CL, Phipps S, Sukkar MB. RAGE and TLRs: Relatives, friends or neighbors? Mol Immunol 2013;56:739-744. 10 Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A. Neutrophil extracellular traps kill bacteria. Science. 2004; 303: 1532-1535. 11 Massberg S, Grahl L, von Bruehl ML, Manukyan D, Pfeiler S, Goosmann C, Brinkmann V, Lorenz M, Bidzhekov K, Khandagale AB, Konrad I, Kennerknecht E, Reges K, Holdenrieder S, Braun S, Reinhardt C, Spannagl M, Preissner KT, Engelmann B. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. Nat Med. 2010;16:887-896. 12 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 USA. 2010; 107: 15880-15885. 13 Maugeri N, Baldini M, Ramirez GA, Rovere-Querini P, Manfredi AA. Platelet-leukocyte deregulated interactions foster sterile inflammation and tissue damage in immune-mediated vessel diseases. Thromb Res. 2012; 129: 267-273. 14 von Bruhl ML, Stark K, Steinhart A, Chandraratne S, Konrad I, Lorenz M, Khandoga A, Tirniceriu A, Coletti R, Kollnberger M, Byrne RA, Laitinen I, Walch A, Brill A, Pfeiler S, Manukyan D, Braun S, Lange P, Riegger J, Ware J, Eckart A, Haidari S, Rudelius M, Schulz C, Echtler K, Brinkmann V, Schwaiger M, Preissner KT, Wagner DD, Mackman N, Engelmann B, Massberg S. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med. 2012; 209: 819-835. 15 Schulz C, Engelmann B, Massberg S. Crossroads of coagulation and innate immunity: the case of deep vein thrombosis. J Thromb Hemost. 2013; 11 Suppl 1: 233-241. 16 Clark SR, Ma AC, Tavener SA, McDonald B, Goodarzi Z, Kelly MM, Patel KD, Chakrabarti S, McAvoy E, Sinclair GD, Keys EM, Allen-Vercoe E, Devinney R, Doig CJ, Green FH, Kubes P. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med. 2007; 13: 463469.
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17 Caudrillier A, Kessenbrock K, Gilliss BM, Nguyen JX, Marques MB, Monestier M, Toy P, Werb Z, Looney MR. Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. J Clin Invest. 2012; 122: 2661-2671. 18 Gardiner EE, Andrews RK. Neutrophil extracellular traps (NETs) and infection-related vascular dysfunction. Blood Rev. 2012; 26: 255-259. 19 Maugeri N, Campana L, Gavina M, Covino C, De Metrio M, Panciroli C, Maiuri L, Maseri A, D'Angelo A, Bianchi ME, Rovere-Querini P, Manfredi AA. Activated platelets present High Mobility Group Box 1 to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps. J Thromb Hemost. 2014. (in press) 10.1111/jth.12710. 20 Tadie JM, Bae HB, Jiang S, Park DW, Bell CP, Yang H, Pittet JF, Tracey K, Thannickal VJ, Abraham E, Zmijewski JW. HMGB1 promotes neutrophil extracellular trap formation through interactions with Toll-like receptor 4. Am J Physiol Lung Cell Mol Physiol. 2013; 304: L342-349. 21 Sims GP, Rowe DC, Rietdijk ST, Herbst R, Coyle AJ. HMGB1 and RAGE in inflammation and cancer. Annu Rev Immunol. 2010; 28: 367-388. 22 Sakaguchi M, Murata H, Yamamoto K, Ono T, Sakaguchi Y, Motoyama A, Hibino T, Kataoka K, Huh NH. TIRAP, an adaptor protein for TLR2/4, transduces a signal from RAGE phosphorylated upon ligand binding. PloS ONE. 2011; 6: e23132. 23 Wieland CW, Florquin S, Maris NA, Hoebe K, Beutler B, Takeda K, Akira S, van der Poll T. The MyD88-dependent, but not the MyD88-independent, pathway of TLR4 signaling is important in clearing nontypeable Haemophilus influenzae from the mouse lung. J Immunol. 2005;175:6042-6049. 24 Maugeri N, Rovere-Querini P, Baldini M, Baldissera E, Sabbadini MG, Bianchi ME, Manfredi AA. Oxidative stress elicits platelet/leukocyte inflammatory interactions via HMGB1: a candidate for microvessel injury in sytemic sclerosis. Antioxid Redox Signal. 2014; 20: 1060-1074. 25 Smith SA, Morrissey JH. Polyphosphate: a new player in the field of hemostasis. Curr Opin Hematol. 2014; 21: 388-394. 26 Smith SA, Mutch NJ, Baskar D, Rohloff P, Docampo R, Morrissey JH. Polyphosphate modulates blood coagulation and fibrinolysis. Proc Natl Acad Sci USA. 2006; 103: 903-908. 27 Choi SH, Smith SA, Morrissey JH. Polyphosphate is a cofactor for the activation of factor XI by thrombin. Blood. 2011; 118: 6963-6970. 28 Dinarvand P, Hassanian SM, Qureshi SH, Manithody C, Eissenberg JC, Yang L, Rezaie AR. Polyphosphate amplifies proinflammatory responses of nuclear proteins through interaction with receptor for advanced glycation end products and P2Y1 purinergic receptor. Blood. 2014; 123: 935945. 29 Urban CF, Ermert D, Schmid M, Abu-Abed U, Goosmann C, Nacken W, Brinkmann V, Jungblut PR, Zychlinsky A. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS Pathogens. 2009; 5: e1000639. 30 Mitroulis I, Kambas K, Chrysanthopoulou A, Skendros P, Apostolidou E, Kourtzelis I, Drosos GI, Boumpas DT, Ritis K. Neutrophil extracellular trap formation is associated with IL-1ß and autophagyrelated signaling in gout. PloS ONE. 2011; 6: e29318. 31 Semeraro F, Ammollo CT, Morrissey JH, Dale GL, Friese P, Esmon NL, Esmon CT. Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4. Blood. 2011; 118: 1952-1961. 32 Brown MR, Kornberg A. Inorganic polyphosphate in the origin and survival of species. Proc Natl Acad Sci USA. 2004; 101: 16085-16087. 33 Wang Y, Li M, Stadler S, Correll S, Li P, Wang D, Hayama R, Leonelli L, Han H, Grigoryev SA,
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Allis CD, Coonrod SA. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol. 2009; 184: 205-213. 34 Chen R, Kang R, Fan XG, Tang D. Release and activity of histone in diseases. Cell Death Dis. 2014; 5: e1370. 35 Saffarzadeh M, Juenemann C, Queisser MA, Lochnit G, Barreto G, Galuska SP, Lohmeyer J, Preissner KT. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PloS ONE. 2012; 7: e32366. 36 Xu J, Zhang X, Pelayo R, Monestier M, Ammollo CT, Semeraro F, Taylor FB, Esmon NL, Lupu F, Esmon CT. Extracellular histones are major mediators of death in sepsis. Nat Med. 2009; 15: 13181321. 37 Raucci A, Cugusi S, Antonelli A, Barabino SM, Monti L, Bierhaus A, Reiss K, Saftig P, Bianchi ME. A soluble form of the receptor for advanced glycation endproducts (RAGE) is produced by proteolytic cleavage of the membrane-bound form by the sheddase a disintegrin and metalloprotease 10 (ADAM10). FASEB J 2008; 22: 3716-3727. 38 Saffarzadeh M, Preissner KT. Fighting against the dark side of neutrophil extracellular traps in disease: manoeuvres for host protection. Curr Opin Hematol. 2013; 20: 3-9.
Legend to Figure 1: Upon interactions between (activated) platelets and neutrophils, which are reinforced by cell-cell adhesion like P-selectin and P-selectin-glycoligand-1 (PSGL-1) as indicated (A), platelet degranulation products, including HMGB1, are released that promote neutrophil activation. Subsequent signal transduction events triggered by the “Receptor for advanced glycation endproducts” (RAGE) as well as by the Toll-like receptors TLR2 and TLR4 result in immediate DNA decompaction, ejection of nucleosomes and the formation of “Neutrophil extracellular traps” (NET) (B). These DNA-histone scaffolds, to which HMGB1, neutrophil proteases and antimicrobial peptides attach, not only serve to combat infection but may also provide the seed for intravascular thrombus formation, resulting in thrombotic events. These processes, which are not only initiated but also amplified by the “moonlighting proteins” HMGB1 and histones may lead to cytotoxic events as well with the progression of an inflammatory and/or vascular disease. Counteracting strategies such as DNase or inhibitors of HMGB1 and histones (C) appear to provide new strategies to control or block the dark side of NETs.
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Accepted Article This article is protected by copyright. All rights reserved.
Article Type: Commentary Commentary to Maugeri et al., JTH-2014-00190.R2
Moonlighting proteins dictate the cross-talk between thrombosis and innate immunity
Mona Saffarzadeh (1), Klaus T. Preissner (2)*
(1) Center for Thrombosis and Hemostasis (CTH), University Medical Center, 55131, Mainz, Germany (2) Department of Biochemistry, Medical School, Justus-Liebig-University, 35392 Giessen, Germany
* Corresponding author
Corresponding address: Klaus T. Preissner, Ph.D. Department of Biochemistry, Medical School Justus-Liebig-University Friedrichstrasse 24 D-35392 Giessen Germany Tel. +49-641-994-7500 Fax. +49-641-994-7509 e.mail: [email protected]
Keywords HMGB1 Protein , Innate immunity , Neutrophil , Platelet , Thrombosis Upon exposure of our body towards infectious or damaging factors, an immediate host response is mobilized, including toll-like receptor (TLR)-mediated pathogen recognition, to provoke the release of alarming molecules, followed by a spatiotemporal cytokine storm (1). This is succeeded by the This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jth.12754 This article is protected by copyright. All rights reserved.
Accepted Article
recruitment of leukocytes to the site of inflammation and culminates in the catching and killing of microbial invaders, particularly by neutrophil granulocytes. The required effective tissue repair and regeneration process is achieved by the hemostasis and blood coagulation machinery, resulting in wound sealing by aggregated platelets and a stable fibrin network, which also prevents further entry of microorganisms (2). The molecular cooperation between the inflammatory cascade and the hemostasis system has long been recognized and is evident from e.g. inflammatory functions of coagulation factors that are transmitted via “protease-activated receptors” and related signal transduction into immune or vascular cells (3). Only recently however, several groundbreaking studies have uncovered principle mechanisms of the inflammation-hemostasis cross-talk, designated as “immunothrombosis”, that involves some unexpected factors like nuclear histones and DNA to be operative in the extracellular space (4). Thus, it appears that these formerly intracellular components have found new jobs outside cells in the context of immune defense, rendering them “moonlighting” factors (5). Another example for a “moonlighting protein”, which has different functions inside and outside cells, is the non-histone, chromatin protein “High mobility group box-1” (HMGB1 or amphoterin), which is expressed in almost all cell types: In the nucleus, HMGB1 interacts with nucleosomes, transcription factors, and histones, organizes DNA compaction by remodeling the chromatin and regulates transcription (6-8). Posttranslational acetylation of HMGB1 causes its translocation into the cytosol, from where it may become released under stress, injury or inflammation (such as in sepsis) to serve as an endogenous danger signal. In general, HMGB1 can be sensed by cells via the “Receptor for advanced glycation endproducts” (RAGE) as well as by Toll-like receptors TLR2 and TLR4 (9). Interestingly enough, once activated by exogenous and endogenous stimulants, neutrophils are provoked to eject their entire nucleosome material in the form of ultra-large DNA-histone scaffolds, designated “neutrophil extracellular traps” (NETs), which facilitate the recognition and destruction of pathogens with the help of neutrophil enzymes and anti-microbial proteins (10). Surprisingly, activated platelets, which are known to form cellular conjugates with neutrophils, where found to promote NETosis as well. The generated NETs thereby provide an appropriate surface in the blood stream or at the vessel wall to initiate coagulation and thrombus formation but also foster thrombotic events, if this process remains uncontrolled (11-15). In fact, this cross-talk between neutrophils and platelets appears to play a key role in regulating inflammation and vascular tone, and the presence of NETs together with platelet-neutrophil conjugates has been documented in sepsis or lung injury as well (16-18). However, the mechanisms involved in the initial mutual interactions between platelets and neutrophils to provoke NETosis are hardly uncovered. In this issue of JTH, Maugeri et al. (19) reported that coronary thrombi of patients with acute myocardial infarction contained activated platelets and HMGB1, which may trigger neutrophils to commit autophagy and NET generation. Although such a co-localization may not necessarily correspond with a functional relation, the authors could show that platelets release HMGB1 to promote NETosis via RAGE but not via TLRs on neutrophils. While these molecular interactions could provide the basis for the proposed early triggering of neutrophil responses, resulting in subsequent NET formation and development of thrombosis, diverging findings from other groups need to be taken in consideration to understand the underlying pathomechanisms.
This article is protected by copyright. All rights reserved.
Accepted Article
For example, Tadie et al. recently documented that HMGB1 promotes NETosis as well as intracellular histone 3-citrullination, a signaling event that precedes chromatin decondensation, predominantly via TLR4 but not RAGE (20). Also, together with the classical TLR4-ligand lipopolysaccharide (LPS), HMGB1 (which binds to LPS) enhanced the formation of NET and thereby may contribute to the severity of neutrophil-associated inflammation (9, 21). While a cross-talk between RAGE and TLR4 and their shared downstream cellular signaling via MyD88 may be considered to explain these findings (22), Maugeri et al. (19) have shown that MyD88-deficient neutrophils can still produce NETs following exposure towards HMGB1, whereas RAGE-deficient neutrophils cannot. Although they concluded that HMGB1 acts via RAGE and not TLR4, one should not ignore the MyD88-independent pathway of TLR4 signaling which may be involved as well (23). Yet, could other platelet secretory products influence the interactions of HMGB1 with neutrophils? Upon activation, platelets degranulate and release multiple low molecular agonists and proteins, including HMGB1, as well as procoagulant microparticles (7, 24) and inorganic polyphosphates (polyP) that have recently been characterized as potent inducers of blood clotting and modulators of vascular tone and complement in inflammation (25). These polyanionic inorganic molecules (60 to 100 phosphate units) not only increase thrombin generation via contact phase activation and to promote factor XIa generation (26, 27), but polyP may amplify HMGB1- and histone-mediated inflammatory signaling in human endothelial cells via interaction with RAGE and the purinergic receptor P2Y1. Binding of polyP to basic proteins like histones and HMGB1 thereby enhances the interaction of both ligands to RAGE, whereby polyP serve as bridging molecules for interactions with the P2Y1 receptor. As a consequence, clustering of RAGE may promote selective downstream signaling in target cells (28), and such interactions could explain the current findings by Maugeri et al. (19) as well. The authors, however, did not explore whether polyP and HMGB1 are secreted as a complex by activated platelets and whether the indicated molecular bridging mechanism involving polyP would favor RAGE-mediated signaling but prevent HMGB1 from engaging TLR4. In addition to histones as major components of NETs (29), HMGB1 has also been identified as NETassociated protein (30), and it is plausible to assume that these “moonlighting proteins” can trigger a feed-forward loop by activating platelets, promoting NETosis and thereby enhancing inflammatory responses and thrombus growth (Figure 1). Mechanistically, histones directly activate platelets via TLR2 and TLR4 (31), in turn releasing HMGB1 and polyP, which together engage RAGE on endothelial cells and on neutrophils to foster a strong cellular induction with subsequent amplification of inflammatory responses and NETosis. Interestingly, during bacterial infection, complement-mediated bacterial lysis results in the occurrence of microbial polyP with chain length >100 phosphate units (32) that may complex appreciable amounts of HMGB1 and histones, much stronger than plateletderived polyP would do. Consequently, receptor bridging und clustering would provoke a robust inflammatory and cytotoxic response in endothelial cells (28), such that this exaggerated immune response can lead to devastating pathological consequences as in sepsis. Although the indicated cellular responses towards isolated histones and HMGB1 have been characterized in the cited publication, association with DNA and NETs may substantially modulate the functional properties of these “moonlighting proteins” with respect to affinity, receptor occupancy or ligand bridging. In fact, hyper-citrullination of histones (33) as well hyper-acetylation of HMGB1 appear to be required for liberation of these proteins from cells, but the impact of such post-
This article is protected by copyright. All rights reserved.
Accepted Article
translational modifications on ligand binding and activation of TLRs and RAGE has not been considered in detail. While Maugeri et al. (19) have shown that association of isolated mammalian DNA with HMGB1 did not influence its ability to promote NET-formation, such data may not necessarily reflect the authentic situation of NET-associated HMGB1. Although Maugeri et al. (19) have suggested an HMGB1-inhibitor to counteract such complications, and anti-HMGB1 antibodies in LPS-treated mice partially prevented NETosis in lung neutrophils (20), targeting HMGB1 may not be an ideal regimen due to its important role in autophagy. Anti-histone antibodies that have been applied in several inflammatory models promoted a beneficial outcome to dampen the side effect of hyper-inflammation or cytotoxicity (34). Another natural, histonecomplexing compound, polysialic acid, exhibited a significant protective effect against isolated as well as NET-associated histone-mediated cytotoxicity and was superior to than anti-histone antibodies, at least under in vitro conditions (35). As an alternative, activated protein C (APC), was found to inhibit polyP-mediated proinflammatory activities of HMGB1 and histones by degrading these basic “moonlighting proteins” (29). Likewise, APC efficiently cleaves isolated histones and reduces their inflammatory potential in an experimental sepsis model (36). However, high concentrations of APC are required to achieve such protective effects, and NET-associated histones appear to be resistant against APC (37). Finally, soluble RAGE, which lacks the transmembrane and signaling domains of RAGE (38), may serve as decoy receptor to inhibit HMGB1- and histone-mediated signaling, particularly in combination with polyP, which binds to soluble RAGE with high affinity (28). In essence, the work by Maugeri et al. (19) adds another relevant player to the system of immunothrombosis that not only has a major impact on the cross-talk between hemostasis and inflammation in body defense, but may derail into severe inflammatory disease and thrombosis if not counteracted properly. Here, the new data provide another challenge for searching novel therapeutic strategies to fight against the dark side of NETs (38).
Addendum Both authors were responsible for research, writing and editing of the manuscript.
Acknowledgements The work of the authors cited in this article was supported by the Federal Ministry of Education and Research (BMBF 01EO1003) (Virchow Fellowship) as well as by the Deutsche Forschungsgemeinschaft (Bonn, Germany) within the "Excellence-cluster Cardio-pulmonary System" (ECCPS).
Disclosure Authors report no conflicts of interest.
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Legend to Figure 1: Upon interactions between (activated) platelets and neutrophils, which are reinforced by cell-cell adhesion like P-selectin and P-selectin-glycoligand-1 (PSGL-1) as indicated (A), platelet degranulation products, including HMGB1, are released that promote neutrophil activation. Subsequent signal transduction events triggered by the “Receptor for advanced glycation endproducts” (RAGE) as well as by the Toll-like receptors TLR2 and TLR4 result in immediate DNA decompaction, ejection of nucleosomes and the formation of “Neutrophil extracellular traps” (NET) (B). These DNA-histone scaffolds, to which HMGB1, neutrophil proteases and antimicrobial peptides attach, not only serve to combat infection but may also provide the seed for intravascular thrombus formation, resulting in thrombotic events. These processes, which are not only initiated but also amplified by the “moonlighting proteins” HMGB1 and histones may lead to cytotoxic events as well with the progression of an inflammatory and/or vascular disease. Counteracting strategies such as DNase or inhibitors of HMGB1 and histones (C) appear to provide new strategies to control or block the dark side of NETs.
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