Divergent effects of Tlr9 deletion in experimental late venous thrombosis resolution and vein wall injury

Nicholas A. Dewyer; Osama M. El-Sayed; Catherine E. Luke; Megan Elfline; Nicolai Kittan; Ron Allen; Adriana Laser; Carson Oostra; Anthony Comerota; Cory Hogaboam; Steven L. Kunkel; Peter K. Henke

doi:10.1160/TH14-12-1031

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2015; 114(05): 1028-1037
DOI: 10.1160/TH14-12-1031

Blood Cells, Inflammation and Infection

Schattauer GmbH

Divergent effects of Tlr9 deletion in experimental late venous thrombosis resolution and vein wall injury

Nicholas A. Dewyer^*

¹Department of Vascular Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA

,

Osama M. El-Sayed^*

¹Department of Vascular Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA

,

Catherine E. Luke

¹Department of Vascular Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA

,

Megan Elfline

¹Department of Vascular Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA

,

Nicolai Kittan

²Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA

,

Ron Allen

²Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA

,

Adriana Laser

¹Department of Vascular Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA

,

Carson Oostra

³Jobst Vascular Institute, Toledo Hospital, Toledo, Michigan, USA

,

Anthony Comerota

³Jobst Vascular Institute, Toledo Hospital, Toledo, Michigan, USA

,

Cory Hogaboam

²Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA

,

Steven L. Kunkel

²Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA

,

Peter K. Henke

¹Department of Vascular Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA

› Author Affiliations

Abstract

Summary

Deep-vein thrombosis (DVT) resolves via a sterile inflammatory response. Defining the inflammatory response of DVT may allow for new therapies that do not involve anticoagulation. Previously, we have shown that Toll-like receptor 9 (Tlr9) gene deleted mice had impaired venous thrombosis (VT) resolution. Here, we further characterise the role of Tlr9 signalling and sterile inflammation in chronic VT and vein wall responses. First, we found a human precedent exists with Tlr9 ⁺ cells present in chronic post thrombotic intraluminal tissue. Second, in a stasis VT mouse model, endogenous danger signal mediators of uric acid, HMGB-1, and neutrophil extracellular traps marker of citrullinated histone-3 (and extracellular DNA) were greater in Tlr9^-/- thrombi as compared with wild-type (WT), corresponding with larger VT at 8 and 21 days. Fewer M1 type (CCR2+) monocyte/macrophages (MØ) were present in Tlr9^-/- thrombi than WT controls at 8 days, suggesting an impaired inflammatory cell influx. Using bone marrow-derived monocyte (BMMØ) cell culture, we found decreased fibrinolytic gene expression with exposure to several endogenous danger signals. Next, adoptive transfer of cultured Tlr9^+/+ BMMØ to Tlr9^-/- mice normalised VT resolution at 8 days. Lastly, although the VT size was larger at 21 days in Tlr9^-/- mice and correlated with decreased endothelial antigen markers, no difference in fibrosis was found. These data suggest that Tlr9 signalling in MØ is critical for later VT resolution, is associated with necrosis clearance, but does not affect later vein wall fibrosis. These findings provide insight into the Tlr9 MØ mechanisms of sterile inflammation in this disease process.

Keywords

Animal models - deep-vein thrombosis - inflammation - venous thrombosis

Full Text

References

References
1 Heit JA. The epidemiology of venous thromboembolism in the community. Arterioscler Thromb Vasc Biol 2008; 28: 370-372.
2 Henke PK, Comerota AJ. An update on etiology, prevention, and therapy of postthrombotic syndrome. J Vasc Surg 2011; 53: 500-509.
3 Kahn SR, Lim W, Dunn AS. et al. American College of Chest Physicians. Prevention of VTE in nonsurgical patients: Antithrombotic therapy and prevention of thrombosis, 9th ed: American college of chest physicians evidence-based clinical practice guidellines. Chest 2012; 141: e195S-226S.
4 Wakefield TW, Myers DD, Henke PK. Mechanisms of venous thrombosis and resolution. Arterioscler Thromb Vasc Biol 2008; 28: 387-391.
5 Kono H, Rock KL. How dying cells alert the immune system to danger. Nat Rev Immunol 2008; 8: 279-289.
6 Shi Y, Evans JE, Rock KL. Molecular identification of a danger signal that alerts the immune system to dying cells. Nature 2003; 425: 516-521.
7 Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein hmgb1 by necrotic cells triggers inflammation. Nature 2002; 418: 191-195.
8 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 USA 2013; 110: 8674-8679.
9 Brill A, Fuchs TA, Savchenko AS. et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost 2012; 10: 136-144.
10 Saha P, Humphries J, Modarai B. et al. Leukocytes and the natural history of deep vein thrombosis: Current concepts and future directions. Arterioscler Thromb Vasc Biol 2011; 31: 506-512.
11 Sindrilaru A, Peters T, Wieschalka S. et al. An unrestrained proinflammatory m1 macrophage population induced by iron impairs wound healing in humans and mice. J Clin Invest 2011; 121: 985-997.
12 Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 2003; 19: 71-82.
13 Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 2011; 11: 723-737.
14 Chen GY, Nunez G. Sterile inflammation: Sensing and reacting to damage. Nat Rev Immunol 2010; 10: 826-837.
15 Henke PK, Pearce CG, Moaveni DM. et al. Targeted deletion of ccr2 impairs deep vein thombosis resolution in a mouse model. J Immunol 2006; 177: 3388-3397.
16 Henke PK, Varma MR, Moaveni DK. et al. Fibrotic injury after experimental deep vein thrombosis is determined by the mechanism of thrombogenesis. Thromb Haemost 2007; 98: 1045-1055.
17 Laser A, Elfline M, Luke C. et al. Deletion of cysteine-cysteine receptor 7 promotes fibrotic injury in experimental post-thrombotic vein wall remodelling. Arterioscler Thromb Vasc Biol 2014; 34: 377-385.
18 Deatrick KB, Luke CE, Elfline MA. et al. The effect of matrix metalloproteinase 2 and matrix metalloproteinase 2/9 deletion in experimental post-thrombotic vein wall remodelling. J Vasc Surg 2013; 58: 1375-1384 e1372.
19 Chen CJ, Kono H, Golenbock D. et al. Identification of a key pathway required for the sterile inflammatory response triggered by dying cells. Nat Med 2007; 13: 851-856.
20 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.
21 Blasius AL, Beutler B. Intracellular toll-like receptors. Immunity 2010; 32: 305-315.
22 Henke PK, Mitsuya M, Luke CE. et al. Toll-like receptor 9 signalling is critical for early experimental deep vein thrombosis resolution. Arterioscler Thromb Vasc Biol 2011; 31: 43-49.
23 Diaz JA, Obi AT, Myers Jr. DD. et al. Critical review of mouse models of venous thrombosis. Arterioscler Thromb Vasc Biol 2012; 32: 556-562.
24 Deatrick KB, Eliason JL, Lynch EM. et al. Vein wall remodelling after deep vein thrombosis involves matrix metalloproteinases and late fibrosis in a mouse model. J Vasc Surg 2005; 42: 140-148.
25 Henke PK, Varga A, De S. et al. Deep vein thrombosis resolution is modulated by monocyte cxcr2-mediated activity in a mouse model. Arterioscler Thromb Vasc Biol 2004; 24: 1130-1137.
26 Diaz JA, Hawley AE, Alvarado CM. et al. Thrombogenesis with continuous blood flow in the inferior vena cava. A novel mouse model. Thromb Haemost 2010; 104: 366-375.
27 Deatrick KB, Obi A, Luke CE. et al. Matrix metalloproteinase-9 deletion is associated with decreased mid-term vein wall fibrosis in experimental stasis DVT. Thromb Res 2013; 132: 360-366.
28 Ito T, Schaller M, Hogaboam CM. et al. Tlr9 regulates the mycobacteria-elicited pulmonary granulomatous immune response in mice through dc-derived notch ligand delta-like 4. J Clin Invest 2009; 119: 33-46.
29 Joshi AD, Raymond T, Coelho AL. et al. A systemic granulomatous response to schistosoma mansoni eggs alters responsiveness of bone-marrow-derived macrophages to toll-like receptor agonists. J Leukoc Biol 2008; 83: 314-324.
30 Sood V, Luke CE, Deatrick KB. et al. Urokinase plasminogen activator independent early experimental thrombus resolution: Mmp2 as an alternative mechanism. Thromb Haemost 2014; 1174-1183.
31 Comerota AJ, Grewal NK, Thakur S. et al. Endovenectomy of the common femoral vein and intraoperative iliac vein recanalisation for chronic iliofemoral venous occlusion. J Vasc Surg 2010; 52: 243-247.
32 Varma MR, Varga AJ, Knipp BS. et al. Neutropenia impairs venous thrombosis resolution in the rat. J Vasc Surg 2003; 38: 1090-1098.
33 Fuchs TA, Brill A, Duerschmied D. et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci USA 2010; 107: 15880-15885.
34 Pannucci CJ, Laird S, Dimick JB. et al. A validated risk model to predict 90-day vte events in postsurgical patients. Chest 2014; 145: 567-573.
35 Massberg S, Grahl L, von Bruehl ML. et al. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. Nat Med 2010; 16: 887-896.
36 Meissner MH, Caps MT, Bergelin RO. et al. Propagation, rethrombosis and new thrombus formation after acute deep venous thrombosis. J Vasc Surg 1995; 22: 558-567.
37 Obi AT, Diaz JA, Ballard-Lipka NL. et al. Plasminogen activator-1 overexpression decreases experimental postthrombotic vein wall fibrosis by a non-vitronectin-dependent mechanism. J Thromb Haemost 2014; 12: 1353-1363.
38 Deatrick KB, Elfline M, Baker N. et al. Postthrombotic vein wall remodelling: Preliminary observations. J Vasc Surg 2011; 53: 139-146.
39 Brill A, Fuchs TA, Savchenko A. et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost 2012; 10: 136-144.
40 Garcia-Romo GS, Caielli S, Vega B. et al. Netting neutrophils are major inducers of type i ifn production in pediatric systemic lupus erythematosus. Sci Transl Med 2011; 3: 73ra20.
41 Guiducci C, Tripodo C, Gong M. et al. Autoimmune skin inflammation is dependent on plasmacytoid dendritic cell activation by nucleic acids via tlr7 and tlr9. J Exp Med 2010; 207: 2931-2942.
42 Faraday N, Schunke K, Saleem S. et al. Cathepsin g-dependent modulation of platelet thrombus formation in vivo by blood neutrophils. PloS one 2013; 8: e71447.
43 Gould TJ, Vu TT, Swystun LL. et al. Neutrophil extracellular traps promote thrombin generation through platelet-dependent and platelet-independent mechanisms. Arterioscler Thromb Vasc Biol 2014; 34: 1977-1984.
44 Ammollo CT, Semeraro F, Xu J. et al. Extracellular histones increase plasma thrombin generation by impairing thrombomodulin-dependent protein c activation. J Thromb Haemost 2011; 9: 1795-1803.
45 von Bruhl 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.
46 Dewyer NA, Sood V, Lynch EM. et al. Plasmin inhibition increases mmp-9 activity and decreases vein wall stiffness during venous thrombosis resolution. J Surg Res 2007; 142: 357-363.
47 Meissner MH, Manzo RA, Bergelin RO, Markel A, Strandness Jr. DE. Deep venous insufficiency: The relationship between lysis and subsequent reflux. J Vasc Surg. 1993 18. 596-605 discussion 606–608.
48 Baldwin JF, Sood V, Elfline MA. et al. The role of urokinase plasminogen activator and plasmin activator inhibitor-1 on vein wall remodelling in experimental deep vein thrombosis. J Vasc Surg 2012; 56: 1089-1097.
49 Imaeda AB, Watanabe A, Sohail MA. et al. Acetaminophen-induced hepatotoxicity in mice is dependent on tlr9 and the nalp3 inflammasome. J Clin Invest 2009; 119: 305-314.

Supplementary Material

Online Supplementary Material (PDF)