Thromb Haemost 2021; 121(11): 1448-1463
DOI: 10.1055/s-0041-1726093
Cellular Signalling and Proteolysis

Protective Role of Activated Protein C against Viral Mimetic Poly(I:C)-Induced Inflammation

Xiaofeng Cai
1   Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States
,
1   Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States
,
Indranil Biswas
1   Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States
,
Hemant Giri
1   Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States
,
Alireza R. Rezaie
1   Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States
2   Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
› Author Affiliations
Funding This work was supported by grants awarded by the National Heart, Lung, and Blood Institute of the National Institute of Health (HL 101917 and HL 62565 to A.R.R.).

Abstract

Activated protein C (APC) is an anticoagulant plasma serine protease which exhibits potent cytoprotective and anti-inflammatory activities. Here, we studied protective effects of APC on the proinflammatory function of polyinosinic:polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, in cellular and animal models. Poly(I:C) induced histone H3 extranuclear translocation via interaction with toll-like receptor 3 in two established endothelial cell lines. Furthermore, poly(I:C) induced histone H3 extranuclear translocation in J774A.1 macrophages and human neutrophils and formation of macrophage and neutrophil extracellular traps (ETs). Mechanistically, poly(I:C) was found to upregulate expression of peptidylarginine deiminase 4 and enhance its interaction with histone H3, thereby leading to increased histone citrullination and neutrophil ET formation. Poly(I:C) elicited proinflammatory signaling responses by inducing nuclear factor kappa B activation and disrupting endothelial cell permeability. In vivo, poly(I:C) enhanced cell surface expression of Mac-1 on neutrophils in mice and facilitated their infiltration to lung tissues. Poly(I:C) also downregulated thrombomodulin expression in mouse tissues and reduced its circulating soluble level in plasma. We demonstrate in this study that APC and a signaling-selective mutant of APC effectively inhibit proinflammatory signaling effects of poly(I:C) in both cellular and animal models. We further demonstrate that unlike the requirement for endothelial protein C receptor on endothelial cells, the integrin Mac-1 is involved in the protease-activated receptor 1-dependent APC inhibition of macrophage ET formation in J774A.1 cells. Taken together, these results support a key role for APC signaling in inhibiting the viral mimetic-induced proinflammatory signaling responses and histone translocation-associated formation of ETs by innate immune cells.

Author Contributions

X.C. designed the experiments, performed the research, and analyzed the data; S.R.P designed and performed the mouse and flow cytometry experiments and analyzed the data; I.B. designed the experiments and performed the cell permeability assay research; H.G. performed the soluble thrombomodulin ELISA experiments; A.R.R. designed the experiments, analyzed the data, and wrote the manuscript.


Supplementary Material



Publication History

Received: 08 October 2020

Accepted: 29 January 2021

Article published online:
11 March 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 
  • References

  • 1 Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science 2002; 296 (5574): 1880-1882
  • 2 Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109 (08) 3161-3172
  • 3 Mohan Rao LV, Esmon CT, Pendurthi UR. Endothelial cell protein C receptor: a multiliganded and multifunctional receptor. Blood 2014; 124 (10) 1553-1562
  • 4 Rezaie AR. Protease-activated receptor signalling by coagulation proteases in endothelial cells. Thromb Haemost 2014; 112 (05) 876-882
  • 5 Cai X, Biswas I, Panicker SR, Giri H, Rezaie AR. Activated protein C inhibits lipopolysaccharide-mediated acetylation and secretion of high-mobility group box 1 in endothelial cells. J Thromb Haemost 2019; 17 (05) 803-817
  • 6 Bae JS, Rezaie AR. Activated protein C inhibits high mobility group box 1 signaling in endothelial cells. Blood 2011; 118 (14) 3952-3959
  • 7 Gould TJ, Lysov Z, Liaw PC. Extracellular DNA and histones: double-edged swords in immunothrombosis. J Thromb Haemost 2015; 13 (Suppl. 01) S82-S91
  • 8 Xu J, Zhang X, Pelayo R. et al. Extracellular histones are major mediators of death in sepsis. Nat Med 2009; 15 (11) 1318-1321
  • 9 Fritz G. RAGE: a single receptor fits multiple ligands. Trends Biochem Sci 2011; 36 (12) 625-632
  • 10 Ibrahim ZA, Armour CL, Phipps S, Sukkar MB. RAGE and TLRs: relatives, friends or neighbours?. Mol Immunol 2013; 56 (04) 739-744
  • 11 Allam R, Kumar SV, Darisipudi MN, Anders HJ. Extracellular histones in tissue injury and inflammation. J Mol Med (Berl) 2014; 92 (05) 465-472
  • 12 Paues Göranson S, Thålin C, Lundström A. et al. Circulating H3Cit is elevated in a human model of endotoxemia and can be detected bound to microvesicles. Sci Rep 2018; 8 (01) 12641
  • 13 Grilz E, Mauracher LM, Posch F. et al. Citrullinated histone H3, a biomarker for neutrophil extracellular trap formation, predicts the risk of mortality in patients with cancer. Br J Haematol 2019; 186 (02) 311-320
  • 14 Dinarvand P, Hassanian SM, Qureshi SH. et al. Polyphosphate amplifies proinflammatory responses of nuclear proteins through interaction with receptor for advanced glycation end products and P2Y1 purinergic receptor. Blood 2014; 123 (06) 935-945
  • 15 Nazir S, Gadi I, Al-Dabet MM. et al. Cytoprotective activated protein C averts Nlrp3 inflammasome-induced ischemia-reperfusion injury via mTORC1 inhibition. Blood 2017; 130 (24) 2664-2677
  • 16 Brinkmann V, Reichard U, Goosmann C. et al. Neutrophil extracellular traps kill bacteria. Science 2004; 303 (5663): 1532-1535
  • 17 Chow OA, von Köckritz-Blickwede M, Bright AT. et al. Statins enhance formation of phagocyte extracellular traps. Cell Host Microbe 2010; 8 (05) 445-454
  • 18 Okubo K, Kurosawa M, Kamiya M. et al. Macrophage extracellular trap formation promoted by platelet activation is a key mediator of rhabdomyolysis-induced acute kidney injury. Nat Med 2018; 24 (02) 232-238
  • 19 Liu P, Wu X, Liao C. et al. Escherichia coli and Candida albicans induced macrophage extracellular trap-like structures with limited microbicidal activity. PLoS One 2014; 9 (02) e90042
  • 20 Wang Y, Li M, Stadler S. et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol 2009; 184 (02) 205-213
  • 21 Leshner M, Wang S, Lewis C. et al. PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures. Front Immunol 2012; 3: 307
  • 22 Martinod K, Demers M, Fuchs TA. et al. Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice. Proc Natl Acad Sci U S A 2013; 110 (21) 8674-8679
  • 23 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 (09) 1853-1862
  • 24 Behnen M, Leschczyk C, Möller S. et al. Immobilized immune complexes induce neutrophil extracellular trap release by human neutrophil granulocytes via FcγRIIIB and Mac-1. J Immunol 2014; 193 (04) 1954-1965
  • 25 Rossaint J, Herter JM, Van Aken H. et al. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation. Blood 2014; 123 (16) 2573-2584
  • 26 Wardini AB, Guimarães-Costa AB, Nascimento MT. et al. Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus. J Gen Virol 2010; 91 (Pt 1): 259-264
  • 27 Narasaraju T, Yang E, Samy RP. et al. Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis. Am J Pathol 2011; 179 (01) 199-210
  • 28 Saitoh T, Komano J, Saitoh Y. et al. Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1. Cell Host Microbe 2012; 12 (01) 109-116
  • 29 Schönrich G, Raftery MJ. Neutrophil extracellular traps go viral. Front Immunol 2016; 7: 366
  • 30 Zuo Y, Yalavarthi S, Shi H. et al. Neutrophil extracellular traps in COVID-19. JCI Insight 2020; 5 (11) 138999
  • 31 Barnes BJ, Adrover JM, Baxter-Stoltzfus A. et al. Targeting potential drivers of COVID-19: neutrophil extracellular traps. J Exp Med 2020; 217 (06) e20200652
  • 32 Healy LD, Puy C, Fernández JA. et al. Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo . J Biol Chem 2017; 292 (21) 8616-8629
  • 33 Antoniak S, Mackman N. Multiple roles of the coagulation protease cascade during virus infection. Blood 2014; 123 (17) 2605-2613
  • 34 Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappaB by toll-like receptor 3. Nature 2001; 413 (6857): 732-738
  • 35 Zimmer S, Steinmetz M, Asdonk T. et al. Activation of endothelial toll-like receptor 3 impairs endothelial function. Circ Res 2011; 108 (11) 1358-1366
  • 36 Kawai T, Akira S. Toll-like receptor and RIG-I-like receptor signaling. Ann N Y Acad Sci 2008; 1143: 1-20
  • 37 Gan T, Yang Y, Hu F. et al. TLR3 regulated poly I:C-induced neutrophil extracellular traps and acute lung injury partly through p38 MAP kinase. Front Microbiol 2018; 9: 3174
  • 38 Bae JS, Yang L, Manithody C, Rezaie AR. Engineering a disulfide bond to stabilize the calcium-binding loop of activated protein C eliminates its anticoagulant but not its protective signaling properties. J Biol Chem 2007; 282 (12) 9251-9259
  • 39 Cao C, Gao Y, Li Y, Antalis TM, Castellino FJ, Zhang L. The efficacy of activated protein C in murine endotoxemia is dependent on integrin CD11b. J Clin Invest 2010; 120 (06) 1971-1980
  • 40 Esmon CT. Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface. FASEB J 1995; 9 (10) 946-955
  • 41 Conway EM. Thrombomodulin and its role in inflammation. Semin Immunopathol 2012; 34 (01) 107-125
  • 42 Giri H, Cai X, Panicker SR, Biswas I, Rezaie AR. Thrombomodulin regulation of mitogen-activated protein kinases. Int J Mol Sci 2019; 20 (08) E1851
  • 43 Shibamiya A, Hersemeyer K, Schmidt Wöll T. et al. A key role for toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells. Blood 2009; 113 (03) 714-722
  • 44 Anzilotti C, Pratesi F, Tommasi C, Migliorini P. Peptidylarginine deiminase 4 and citrullination in health and disease. Autoimmun Rev 2010; 9 (03) 158-160
  • 45 Funchal GA, Jaeger N, Czepielewski RS. et al. Respiratory syncytial virus fusion protein promotes TLR-4-dependent neutrophil extracellular trap formation by human neutrophils. PLoS One 2015; 10 (04) e0124082
  • 46 El Shikh MEM, El Sayed R, Nerviani A. et al. Extracellular traps and PAD4 released by macrophages induce citrullination and auto-antibody production in autoimmune arthritis. J Autoimmun 2019; 105: 102297
  • 47 Radermecker C, Sabatel C, Vanwinge C. et al. Locally instructed CXCR4hi neutrophils trigger environment-driven allergic asthma through the release of neutrophil extracellular traps. Nat Immunol 2019; 20 (11) 1444-1455
  • 48 McDonald B, Davis RP, Kim SJ. et al. Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice. Blood 2017; 129 (10) 1357-1367
  • 49 Bock F, Shahzad K, Wang H. et al. Activated protein C ameliorates diabetic nephropathy by epigenetically inhibiting the redox enzyme p66Shc. Proc Natl Acad Sci U S A 2013; 110 (02) 648-653
  • 50 Raftery MJ, Lalwani P, Krautkrämer E. et al. β2 integrin mediates hantavirus-induced release of neutrophil extracellular traps. J Exp Med 2014; 211 (07) 1485-1497