The Ability of Extracellular Vesicles to Induce a Pro-Inflammatory Host Response
Abstract
:1. Introduction
2. Inflammatory Effects of Extracellular Vesicles EVs
2.1. Extracellular Vesicle (EV)-Produced Inflammatory Mediators
2.2. EV-Mediated Pro-Inflammatory Responses of Effector Cells in the Circulation
2.3. EV-Mediated Inflammatory Responses of Endothelial Cells
2.4. EV-Mediated Inflammatory Responses in Effector Cells in Tissues
2.5. EV-Mediated Pro-Coagulant Response
2.6. EV-Mediated Host Response in Inflammatory Disease States
2.7. EVs also Have Anti-Inflammatory Effects
3. Conclusions
Author Contributions
Conflicts of Interest
References
- Van der Pol, E.; Boing, A.N.; Harrison, P.; Sturk, A.; Nieuwland, R. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol. Rev. 2012, 64, 676–705. [Google Scholar] [CrossRef] [PubMed]
- Coumans, F.A.W.; Brisson, A.R.; Buzas, E.I.; Dignat-George, F.; Drees, E.E.E.; El-Andaloussi, S.; Emanueli, C.; Gasecka, A.; Hendrix, A.; Hill, A.F.; et al. Methodological guidelines to study extracellular vesicles. Circ. Res. 2017, 120, 1632–1648. [Google Scholar] [CrossRef] [PubMed]
- Inamdar, S.; Nitiyanandan, R.; Rege, K. Emerging applications of exosomes in cancer therapeutics and diagnostics. Bioeng. Transl. Med. 2017, 2, 70–80. [Google Scholar] [CrossRef] [PubMed]
- Xitong, D.; Xiaorong, Z. Targeted therapeutic delivery using engineered exosomes and its applications in cardiovascular diseases. Gene 2016, 575 Pt 2, 377–384. [Google Scholar] [CrossRef] [PubMed]
- MacKenzie, A.; Wilson, H.L.; Kiss-Toth, E.; Dower, S.K.; North, R.A.; Surprenant, A. Rapid secretion of interleukin-1β by microvesicle shedding. Immunity 2001, 15, 825–835. [Google Scholar] [CrossRef]
- Wang, J.G.; Williams, J.C.; Davis, B.K.; Jacobson, K.; Doerschuk, C.M.; Ting, J.P.; Mackman, N. Monocytic microparticles activate endothelial cells in an IL-1β-dependent manner. Blood 2011, 118, 2366–2374. [Google Scholar] [CrossRef] [PubMed]
- Qu, Y.; Franchi, L.; Nunez, G.; Dubyak, G.R. Nonclassical IL-1β secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages. J. Immunol. 2007, 179, 1913–1925. [Google Scholar] [CrossRef] [PubMed]
- Watanabe, J.; Marathe, G.K.; Neilsen, P.O.; Weyrich, A.S.; Harrison, K.A.; Murphy, R.C.; Zimmerman, G.A.; McIntyre, T.M. Endotoxins stimulate neutrophil adhesion followed by synthesis and release of platelet-activating factor in microparticles. J. Biol. Chem. 2003, 278, 33161–33168. [Google Scholar] [CrossRef] [PubMed]
- Hawari, F.I.; Rouhani, F.N.; Cui, X.; Yu, Z.X.; Buckley, C.; Kaler, M.; Levine, S.J. Release of full-length 55-kDa TNF receptor 1 in exosome-like vesicles: A mechanism for generation of soluble cytokine receptors. Proc. Natl. Acad. Sci. USA 2004, 101, 1297–1302. [Google Scholar] [CrossRef] [PubMed]
- Straat, M.; Boing, A.N.; Tuip-De Boer, A.; Nieuwland, R.; Juffermans, N.P. Extracellular vesicles from red blood cell products induce a strong Pro-inflammatory host response, dependent on both numbers and storage duration. Transfus. Med. Hemother. 2016, 43, 302–305. [Google Scholar] [CrossRef] [PubMed]
- Belizaire, R.M.; Prakash, P.S.; Richter, J.R.; Robinson, B.R.; Edwards, M.J.; Caldwell, C.C.; Lentsch, A.B.; Pritts, T.A. Microparticles from stored red blood cells activate neutrophils and cause lung injury after hemorrhage and resuscitation. J. Am. Coll. Surg. 2012, 214, 648–655. [Google Scholar] [CrossRef] [PubMed]
- Zecher, D.; Cumpelik, A.; Schifferli, J.A. Erythrocyte-derived microvesicles amplify systemic inflammation by thrombin-dependent activation of complement. Arterioscler. Thromb. Vasc. Biol. 2014, 34, 313–320. [Google Scholar] [CrossRef] [PubMed]
- Straat, M.; van Hezel, M.E.; Boing, A.; Tuip-De Boer, A.; Weber, N.; Nieuwland, R.; van Bruggen, R.; Juffermans, N.P. Monocyte-mediated activation of endothelial cells occurs only after binding to extracellular vesicles from red blood cell products, a process mediated by β-integrin. Transfusion 2016, 56, 3012–3020. [Google Scholar] [CrossRef] [PubMed]
- Halim, A.T.; Ariffin, N.A.; Azlan, M. Review: The multiple roles of monocytic microparticles. Inflammation 2016, 39, 1277–1284. [Google Scholar] [CrossRef] [PubMed]
- Mastronardi, M.L.; Mostefai, H.A.; Soleti, R.; Agouni, A.; Martinez, M.C.; Andriantsitohaina, R. Microparticles from apoptotic monocytes enhance nitrosative stress in human endothelial cells. Fundam. Clin. Pharmacol. 2011, 25, 653–660. [Google Scholar] [CrossRef] [PubMed]
- Thomas, L.M.; Salter, R.D. Activation of macrophages by P2X7-induced microvesicles from myeloid cells is mediated by phospholipids and is partially dependent on TLR4. J. Immunol. 2010, 185, 3740–3749. [Google Scholar] [CrossRef] [PubMed]
- Esser, J.; Gehrmann, U.; D′Alexandri, F.L.; Hidalgo-Estevez, A.M.; Wheelock, C.E.; Scheynius, A.; Gabrielsson, S.; Radmark, O. Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration. J. Allergy. Clin. Immunol. 2010, 126, 1032–1040. [Google Scholar] [CrossRef] [PubMed]
- Teoh, N.C.; Ajamieh, H.; Wong, H.J.; Croft, K.; Mori, T.; Allison, A.C.; Farrell, G.C. Microparticles mediate hepatic ischemia-reperfusion injury and are the targets of Diannexin (ASP8597). PLoS ONE 2014, 9, e104376. [Google Scholar] [CrossRef] [PubMed]
- Mesri, M.; Altieri, D.C. Endothelial cell activation by leukocyte microparticles. J. Immunol. 1998, 161, 4382–4387. [Google Scholar] [PubMed]
- Gasser, O.; Schifferli, J.A. Microparticles released by human neutrophils adhere to erythrocytes in the presence of complement. Exp. Cell Res. 2005, 307, 381–387. [Google Scholar] [CrossRef] [PubMed]
- Scanu, A.; Molnarfi, N.; Brandt, K.J.; Gruaz, L.; Dayer, J.M.; Burger, D. Stimulated T cells generate microparticles, which mimic cellular contact activation of human monocytes: Differential regulation of pro- and anti-inflammatory cytokine production by high-density lipoproteins. J. Leukoc. Biol. 2008, 83, 921–927. [Google Scholar] [CrossRef] [PubMed]
- Martin, S.; Tesse, A.; Hugel, B.; Martinez, M.C.; Morel, O.; Freyssinet, J.M.; Andriantsitohaina, R. Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression. Circulation 2004, 109, 1653–1659. [Google Scholar] [CrossRef] [PubMed]
- Wolf, P.; Nghiem, D.X.; Walterscheid, J.P.; Byrne, S.; Matsumura, Y.; Matsumura, Y.; Bucana, C.; Ananthaswamy, H.N.; Ullrich, S.E. Platelet-activating factor is crucial in psoralen and ultraviolet A-induced immune suppression, inflammation, and apoptosis. Am. J. Pathol. 2006, 169, 795–805. [Google Scholar] [CrossRef] [PubMed]
- Xie, R.F.; Hu, P.; Wang, Z.C.; Yang, J.; Yang, Y.M.; Gao, L.; Fan, H.H.; Zhu, Y.M. Platelet-derived microparticles induce polymorphonuclear leukocyte-mediated damage of human pulmonary microvascular endothelial cells. Transfusion 2015, 55, 1051–1057. [Google Scholar] [CrossRef] [PubMed]
- Mooberry, M.J.; Bradford, R.; Hobl, E.L.; Lin, F.C.; Jilma, B.; Key, N.S. Procoagulant microparticles promote coagulation in a factor XI-dependent manner in human endotoxemia. J. Thromb. Haemost. 2016, 14, 1031–1042. [Google Scholar] [CrossRef] [PubMed]
- Balvers, K.; Curry, N.; Kleinveld, D.J.; Boing, A.N.; Nieuwland, R.; Goslings, J.C.; Juffermans, N.P. Endogenous microparticles drive the proinflammatory host immune response in severely injured trauma patients. Shock 2015, 43, 317–321. [Google Scholar] [CrossRef] [PubMed]
- Jansen, F.; Yang, X.; Hoelscher, M.; Cattelan, A.; Schmitz, T.; Proebsting, S.; Wenzel, D.; Vosen, S.; Franklin, B.S.; Fleischmann, B.K.; et al. Endothelial microparticle-mediated transfer of microRNA-126 promotes vascular endothelial cell repair via SPRED1 and is abrogated in glucose-damaged endothelial microparticles. Circulation 2013, 128, 2026–2038. [Google Scholar] [CrossRef] [PubMed]
- Lee, S.K.; Yang, S.H.; Kwon, I.; Lee, O.H.; Heo, J.H. Role of tumour necrosis factor receptor-1 and nuclear factor-κB in production of TNF-α-induced pro-inflammatory microparticles in endothelial cells. Thromb. Haemost. 2014, 112, 580–588. [Google Scholar] [CrossRef] [PubMed]
- Liu, Y.; Zhang, R.; Qu, H.; Wu, J.; Li, L.; Tang, Y. Endothelial microparticles activate endothelial cells to facilitate the inflammatory response. Mol. Med. Rep. 2017, 15, 1291–1296. [Google Scholar] [CrossRef] [PubMed]
- Xiong, Z.; Cavaretta, J.; Qu, L.; Stolz, D.B.; Triulzi, D.; Lee, J.S. Red blood cell microparticles show altered inflammatory chemokine binding and release ligand upon interaction with platelets. Transfusion 2011, 51, 610–621. [Google Scholar] [CrossRef] [PubMed]
- Kent, M.W.; Kelher, M.R.; West, F.B.; Silliman, C.C. The pro-inflammatory potential of microparticles in red blood cell units. Transfus. Med. 2014, 24, 176–181. [Google Scholar] [CrossRef] [PubMed]
- Eyre, J.; Burton, J.O.; Saleem, M.A.; Mathieson, P.W.; Topham, P.S.; Brunskill, N.J. Monocyte- and endothelial-derived microparticles induce an inflammatory phenotype in human podocytes. Nephron Exp. Nephrol. 2011, 119, e58–e66. [Google Scholar] [CrossRef] [PubMed]
- Smit, K.F.; Kerindongo, R.P.; Boing, A.; Nieuwland, R.; Hollmann, M.W.; Preckel, B.; Weber, N.C. Effects of helium on inflammatory and oxidative stress-induced endothelial cell damage. Exp. Cell Res. 2015, 337, 37–43. [Google Scholar] [CrossRef] [PubMed]
- Sarkar, A.; Mitra, S.; Mehta, S.; Raices, R.; Wewers, M.D. Monocyte derived microvesicles deliver a cell death message via encapsulated caspase-1. PLoS ONE 2009, 4, e7140. [Google Scholar] [CrossRef] [PubMed]
- Johnson, B.L., III; Midura, E.F.; Prakash, P.S.; Rice, T.C.; Kunz, N.; Kalies, K.; Caldwell, C.C. Neutrophil derived microparticles increase mortality and the counter-inflammatory response in a murine model of sepsis. Biochim. Biophys. Acta 2017. [Google Scholar] [CrossRef] [PubMed]
- Mazzeo, C.; Canas, J.A.; Zafra, M.P.; Marco, A.R.; Fernandez-Nieto, M.; Sanz, V.; Mittelbrunn, M.; Izquierdo, M.; Baixaulli, F.; Sastre, J.; et al. Exosome secretion by eosinophils: A possible role in asthma pathogenesis. J. Allergy Clin. Immunol. 2015, 135, 1603–1613. [Google Scholar] [CrossRef] [PubMed]
- Boilard, E.; Nigrovic, P.A.; Larabee, K.; Watts, G.F.; Coblyn, J.S.; Weinblatt, M.E.; Massarotti, E.M.; Remold-O’Donnell, E.; Farndale, R.W.; Ware, J.; et al. Platelets amplify inflammation in arthritis via collagen-dependent microparticle production. Science 2010, 327, 580–583. [Google Scholar] [CrossRef] [PubMed]
- Bei, J.J.; Liu, C.; Peng, S.; Liu, C.H.; Zhao, W.B.; Qu, X.L.; Chen, Q.; Zhou, Z.; Yu, Z.P.; Peter, K.; et al. Staphylococcal SSL5-induced platelet microparticles provoke proinflammatory responses via the CD40/TRAF6/NFκB signalling pathway in monocytes. Thromb. Haemost. 2016, 115, 632–645. [Google Scholar] [CrossRef] [PubMed]
- Sadallah, S.; Amicarella, F.; Eken, C.; Iezzi, G.; Schifferli, J.A. Ectosomes released by platelets induce differentiation of CD4+ T cells into T regulatory cells. Thromb. Haemost. 2014, 112, 1219–1229. [Google Scholar] [CrossRef] [PubMed]
- Edelstein, L.C. The role of platelet microvesicles in intercellular communication. Platelets 2017, 28, 222–227. [Google Scholar] [CrossRef] [PubMed]
- Meziani, F.; Delabranche, X.; Asfar, P.; Toti, F. Bench-to-bedside review: Circulating microparticles—A new player in sepsis? Crit. Care 2010, 14, 236. [Google Scholar] [CrossRef] [PubMed]
- Nomura, S.; Imamura, A.; Okuno, M.; Kamiyama, Y.; Fujimura, Y.; Ikeda, Y.; Fukuhara, S. Platelet-derived microparticles in patients with arteriosclerosis obliterans: Enhancement of high shear-induced microparticle generation by cytokines. Thromb. Res. 2000, 98, 257–268. [Google Scholar] [CrossRef]
- Berckmans, R.J.; Nieuwland, R.; Kraan, M.C.; Schaap, M.C.; Pots, D.; Smeets, T.J.; Sturk, A.; Tak, P.P. Synovial microparticles from arthritic patients modulate chemokine and cytokine release by synoviocytes. Arthritis Res. Ther. 2005, 7, R536–R544. [Google Scholar] [CrossRef] [PubMed]
- Renovato-Martins, M.; Matheus, M.E.; de Andrade, I.R.; Moraes, J.A.; da Silva, S.V.; dos Reis, M.C.; de Souza, A.A.; da Silva, C.C.; Bouskela, E.; Barja-Fidalgo, C. Microparticles derived from obese adipose tissue elicit a pro-inflammatory phenotype of CD16+, CCR5+ and TLR8+ monocytes. Biochim. Biophys. Acta 2017, 1863, 139–151. [Google Scholar] [CrossRef] [PubMed]
- Kumar, A.; Stoica, B.A.; Loane, D.J.; Yang, M.; Abulwerdi, G.; Khan, N.; Kumar, A.; Thom, S.R.; Faden, A.I. Microglial-derived microparticles mediate neuroinflammation after traumatic brain injury. J. Neuroinflammation 2017, 14, 47. [Google Scholar] [CrossRef] [PubMed]
- Chiva-Blanch, G.; Laake, K.; Myhre, P.; Bratseth, V.; Arnesen, H.; Solheim, S.; Badimon, L.; Seljeflot, I. Platelet-, monocyte-derived and tissue factor-carrying circulating microparticles are related to acute myocardial infarction severity. PLoS ONE 2017, 12, e0172558. [Google Scholar] [CrossRef] [PubMed]
- Gardiner, C.; Harrison, P.; Belting, M.; Boing, A.; Campello, E.; Carter, B.S.; Collier, M.E.; Coumans, F.; Ettelaie, C.; van Es, N.; et al. Extracellular vesicles, tissue factor, cancer and thrombosis—Discussion themes of the ISEV 2014 educational day. J. Extracell. Vesicles 2015, 4, 26901. [Google Scholar] [CrossRef] [PubMed]
- Ono, T.; Mimuro, J.; Madoiwa, S.; Soejima, K.; Kashiwakura, Y.; Ishiwata, A.; Takano, K.; Ohmori, T.; Sakata, Y. Severe secondary deficiency of von Willebrand factor—Cleaving protease (ADAMTS13) in patients with sepsis-induced disseminated intravascular coagulation: Its correlation with development of renal failure. Blood 2006, 107, 528–534. [Google Scholar] [CrossRef] [PubMed]
- Nieuwland, R.; Berckmans, R.J.; McGregor, S.; Boing, A.N.; Romijn, F.P.; Westendorp, R.G.; Hack, C.E.; Sturk, A. Cellular origin and procoagulant properties of microparticles in meningococcal sepsis. Blood 2000, 95, 930–935. [Google Scholar] [PubMed]
- Delabranche, X.; Boisrame-Helms, J.; Asfar, P.; Berger, A.; Mootien, Y.; Lavigne, T.; Grunebaum, L.; Lanza, F.; Gachet, C.; Freyssinet, J.M.; et al. Microparticles are new biomarkers of septic shock-induced disseminated intravascular coagulopathy. Intensive Care Med. 2013, 39, 1695–1703. [Google Scholar] [CrossRef] [PubMed]
- Bastarache, J.A.; Fremont, R.D.; Kropski, J.A.; Bossert, F.R.; Ware, L.B. Procoagulant alveolar microparticles in the lungs of patients with acute respiratory distress syndrome. Am. J. Physiol. Lung Cell. Mol. Physiol. 2009, 297, L1035–L1041. [Google Scholar] [CrossRef] [PubMed]
- Berckmans, R.J.; Nieuwland, R.; Tak, P.P.; Boing, A.N.; Romijn, F.P.; Kraan, M.C.; Breedveld, F.C.; Hack, C.E.; Sturk, A. Cell-derived microparticles in synovial fluid from inflamed arthritic joints support coagulation exclusively via a factor VII-dependent mechanism. Arthritis Rheum. 2002, 46, 2857–2866. [Google Scholar] [CrossRef] [PubMed]
- Matijevic, N.; Wang, Y.W.; Wade, C.E.; Holcomb, J.B.; Cotton, B.A.; Schreiber, M.A.; Muskat, P.; Fox, E.E.; del Junco, D.J.; Cardenas, J.C.; et al. Cellular microparticle and thrombogram phenotypes in the prospective observational multicenter major trauma transfusion (PROMMTT) study: Correlation with coagulopathy. Thromb. Res. 2014, 134, 652–658. [Google Scholar] [CrossRef] [PubMed]
- Midura, E.F.; Jernigan, P.L.; Kuethe, J.W.; Friend, L.A.; Veile, R.; Makley, A.T.; Caldwell, C.C.; Goodman, M.D. Microparticles impact coagulation after traumatic brain injury. J. Surg. Res. 2015, 197, 25–31. [Google Scholar] [CrossRef] [PubMed]
- Eken, C.; Martin, P.J.; Sadallah, S.; Treves, S.; Schaller, M.; Schifferli, J.A. Ectosomes released by polymorphonuclear neutrophils induce a MerTK-dependent anti-inflammatory pathway in macrophages. J. Biol. Chem. 2010, 285, 39914–39921. [Google Scholar] [CrossRef] [PubMed]
- Eken, C.; Sadallah, S.; Martin, P.J.; Treves, S.; Schifferli, J.A. Ectosomes of polymorphonuclear neutrophils activate multiple signaling pathways in macrophages. Immunobiology 2013, 218, 382–392. [Google Scholar] [CrossRef] [PubMed]
- Prakash, P.S.; Caldwell, C.C.; Lentsch, A.B.; Pritts, T.A.; Robinson, B.R. Human microparticles generated during sepsis in patients with critical illness are neutrophil-derived and modulate the immune response. J. Trauma Acute Care Surg. 2012, 73, 401–406. [Google Scholar] [CrossRef] [PubMed]
- Van Vught, L.A.; Wiewel, M.A.; Hoogendijk, A.J.; Frencken, J.F.; Scicluna, B.P.; Klein Klouwenberg, P.M.; Zwinderman, A.H.; Lutter, R.; Horn, J.; Schultz, M.J.; et al. The host response in sepsis patients developing intensive care unit-acquired secondary infections. Am. J. Respir. Crit. Care Med. 2017. [Google Scholar] [CrossRef] [PubMed]
Cellular EV Origin | Target Cell | Inflammatory Effect | Type of Study | Reference |
---|---|---|---|---|
RBC | Whole blood | Production of TNFα, IL-6, IL-8 | Ex vivo | Straat [10] |
RBC | Granulocytes | Respiratory burst | In vitro, in vivo | Belizaire [11] |
RBC | – | Leukocyte homing | In vivo | Zecher [12] |
RBC | Monocytes | Binding and phagocytosis | In vitro | Straat [13] |
RBC | Endothelial cells | Expression of ICAM-1, E-selectin | In vitro | Straat [13] |
Monocyte | Monocyte | IL-1β production | In vitro | McKenzie [5] |
Monocyte | Endothelial cells | IL-1β production | In vitro | Wang [6] |
Monocyte | Endothelial cells | Expression of ICAM-1, VCAM-1, E-selectin | In vitro | Wang [6], Halim [14] |
Monocytes | Endothelial cell | Induction nitrosative stress | In vitro | Mastronardi [15] |
Macrophages | Macrophages | Activate TLR-4, TNF production | In vitro | Thomas [16] |
Macrophages | – | IL-1, caspase-1 production | In vitro | Qu [7] |
Marcophages, Dendritic cells | – | Leukotrienes synthesis, Granulocyte migration | In vitro | Esser [17] |
Macrophages | Hepatocytes | TNF production | In vitro | Teoh [18] |
Granulocytes | – | PAF production | In vitro | Watanabe [8] Mesri [19] |
Granulocytes | Endothelial cells | TF and IL-6 production | In vitro | Mesri [19] |
Granulocytes | Red blood cells | Complement activation | In vitro | Gasser [20] |
T cells | Monocytes | TNF, IL-6 production | In vitro | Scanu [21] |
T cells | Endothelial cells | No synthase, COX-2 production | In vitro, in vivo | Martin [22] |
Platelets | Endothelial cells | COX-2 production | In vitro | Barry |
Platelets | Endothelial cells | PAF production | In vitro | Wolf [23] |
Platelets | Endothelial cells | CD11b expression | In vitro | Xie [24] |
Platelets | – | Thrombin generation | In vivo | Mooberry [25] |
Platelets | Whole blood | Production IL-6, TNFα | Ex vivo | Balvers [26] |
Endothelial cells | Endothelial cells | Transfer miRNA | In vitro | Jansen [27] |
Endothelial cells | Endothelial cells | Adherence monocytes, expression of ICAM-1 | In vitro | Lee [28] |
Endothelial cells | Endothelial cells | IP-10 production | In vitro | Liu [29] |
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Van Hezel, M.E.; Nieuwland, R.; Van Bruggen, R.; Juffermans, N.P. The Ability of Extracellular Vesicles to Induce a Pro-Inflammatory Host Response. Int. J. Mol. Sci. 2017, 18, 1285. https://doi.org/10.3390/ijms18061285
Van Hezel ME, Nieuwland R, Van Bruggen R, Juffermans NP. The Ability of Extracellular Vesicles to Induce a Pro-Inflammatory Host Response. International Journal of Molecular Sciences. 2017; 18(6):1285. https://doi.org/10.3390/ijms18061285
Chicago/Turabian StyleVan Hezel, Maike E., Rienk Nieuwland, Robin Van Bruggen, and Nicole P. Juffermans. 2017. "The Ability of Extracellular Vesicles to Induce a Pro-Inflammatory Host Response" International Journal of Molecular Sciences 18, no. 6: 1285. https://doi.org/10.3390/ijms18061285