Intact Toll-like receptor 9 signaling in neutrophils modulates normal thrombogenesis in mice

Abstract

Background Deletion of Toll-like receptor 9 (Tlr9) signaling, which is important for sterile inflammatory processes, results in impaired venous thrombosis (VT) resolution in mice. The purpose of this study was to determine if deletion of Tlr9 affected sterile necrosis, apoptosis, and neutrophil extracellular trap (NET) production in VT. Methods Stasis and non-stasis murine models of VT were used in wild type (WT and Tlr9−/− mice, with assessment of VT size, and determination of neutrophil extracellular traps (NETs), necrosis and apoptosis markers. Anti-PMN and anti-platelet antibody strategies were used to determine the cellular roles and their roles in WT and Tlr9−/− mice. Results At 2d, stasis thrombi in Tlr9−/− mice were 62% larger (n = 6–10) with 1.4 fold increased uric acid levels, 1.7 fold more apoptotic cells, 2 fold increased citrullinated histones (cit-H3), 2 fold increased peptidylarginine deiminase – 4 and 1.5 fold increased elastase, with a 2.4 fold reduction in tissue factor pathway inhibitor (TFPI) as compared with WT (all n = 4–7; P < .05). In contrast, non-stasis VT sizes were not significantly different in Tlr9−/− mice (n = 4–6), and did not have elevated necrosis or NET markers. Stasis VT size was not reduced at the 2d time-point in WT or TLR9−/− mice that received treatment with DNAse-I, or in PAD4−/− mice, which are incapable of forming NETs. Stasis VT size was reduced 18% inTlr9−/− mice undergoing PMN depletion (n = 8–10), and was associated with 29 fold decreased cit H3, 1.3 fold decreased elastase, and 1.5 fold increased TFPI (all n = 6; P < .05). Lastly, platelet depletion (>90% reduction) did not significantly reduce stasis VT inTlr9−/− mice. Conclusions These data suggest the thrombogenic model impacts Tlr9 thrombogenic mechanisms, and that functional Tlr9 signaling in PMN, but not platelets or NETs, is an important mechanism in early stasis experimental venous thrombogenesis.

In Vivo Role of Neutrophil Extracellular Traps in Antiphospholipid Antibody–Mediated Venous Thrombosis

“…This is known as a “stasis” model (30). Resulting thrombi (which form >97% of the time in control mice) are significantly enriched in RBCs (30).…”

“…This was done essentially as described (30). Briefly, mice underwent complete ligation of the IVC and all visible contributing vessels below the renal veins.…”

“…This produces full stasis. The stasis model is well characterized and consistently produces a thrombus (>97% of the time) (30). The stasis model differs from the stenosis model in that (i) the IVC is fully ligated and (ii) all visible side and back branches are also ligated.…”

The role of leukocytes in thrombosis

“…Although PAD4 deficiency was associated with impaired thrombus formation in the IVC stenosis model, 105 an electrolytic injury model reported no influence of PAD4 deficiency on DVT. 106 Additionally, neutrophil elastase deficiency has been shown to impair NET formation in response to microbial infection, 107,108 however, a sterile IVC stenosis model demonstrated elastase-deficient mice generate NETs and have normal venous thrombi. 109 Although overall these studies appear to support a role for leukocytes in the induction of thrombosis in response to venous stenosis, they also highlight how the variability of the models employed, including the extent of endothelial injury and underlying activation status of circulating leukocytes, can influence the induction of venous thrombosis.…”

Mouse models of cancer-associated thrombosis

“…For instance, vessel wall TF drives thrombosis in the IVC stasis model whereas leukocyte TF drives thrombosis in the stenosis model [34, 35]. In contrast, neutrophils and neutrophil extracellular traps (NETs) contribute to thrombosis in the IVC stenosis model but not in the stasis model [36]. Similarly, mice with a platelet-specific deletion of C-type lectin-like receptor 2 have reduced thrombosis in the stenosis but not the stasis model [37].…”