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

Abstract

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 characterize the role of Tlr9 signaling 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 WT, corresponding with larger VT at 8 and 21d. Fewer M1 type (CCR2+) monocyte/macrophages (MØ) were present in Tlr9−/− thrombi than WT controls at 8d, 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 normalized VT resolution at 8d. Lastly, although the VT size was larger at 21d in Tlr9−/− mice and correlated with decreased endothelial antigen markers, no difference in fibrosis was found. These data suggest that Tlr9 signaling 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.

Association between TLR9 rs5743836 polymorphism and risk of recurrent venous thromboembolism

“…Gene knockout studies have shown a pivotal role of TLR9 in VTE resolution [22, 23]. Taken together with our findings, this suggests that TLR9 polymorphism may reduce the ability of VTE resolution and therefore may increase the risk of VTE recurrence.…”

The Emerging Role of NETs in Venous Thrombosis and Immunothrombosis

“…Further, TLR9 −/− mice had increased apoptosis, citrullinated histones, PAD4, and neutrophil elastase; and reduced TFPI ( 19 ), suggesting that TLR9 is important for normal thrombogenesis and thrombosis resolution. Lastly, M1-like (CCR2+) monocyte/macrophages were decreased in TLR9 −/− thrombi, consistent with impaired inflammatory cell influx, and this divergence was corrected with adoptive transfer of TLR9 +/+ bone marrow-derived monocytes with normalization of thrombus size ( 20 ). The new found composition of DNA-rich thrombi will likely have long-standing implications for future research in VT resolution.…”

“…Neutrophils, monocytes, and NETs have also been found to affect the clotting cascade in murine models of thrombosis ( 17 – 20 ). For example, myeloid cells roll along the venous endothelium in a P-selectin-dependent manner and produce thrombogenic tissue factor (TF) in the IVC stenosis model ( 17 ).…”

Immune Factors in Deep Vein Thrombosis Initiation

“…It has been shown that lack of this pattern-recognition receptor is associated with larger venous thrombi and increased levels of NETosis, necrosis, and apoptosis markers in the stasis, but not stenosis, model of DVT in mice [73] . It has also been shown that lack of TLR-9 leads to reduced monocyte recruitment to venous thrombi [81] .…”