Toll-like receptors 2 and 7 mediate coagulation activation and coagulopathy in murine sepsis

Abstract

Background: Sepsis is a life-threatening condition often manifested as marked inflammation and severe coagulopathy. Toll-like receptors (TLRs) play a pivotal role in inflammation, organ dysfunction and mortality in animal sepsis.

Objectives: To investigate the role of TLR signaling in mediating sepsis-induced coagulopathy (SIC) in a mouse model.

Methods: Polymicrobial sepsis was created by cecal ligation and puncture (CLP) or fecal slurry peritoneal injection. To quantify global clotting function, two viscoelastic assays were performed with rotational thromboelastometry, and the results were presented as maximum clot firmness (MCF): (a) EXTEM to test tissue factor (TF)-initiated clot formation; and (b) FIBTEM to test EXTEM in the presence of a platelet inhibitor, cytochalasin D. Plasma coagulation factors were quantified with ELISA. TF gene expression and protein expression were determined with real-time quantitative reverse transcription PCR and flow cytometry, respectively.

Results: Between 4 and 24 hours after CLP surgery, wild-type mice showed significant MCF reduction in both EXTEM and FIBTEM tests. This was accompanied by marked thrombocytopenia and a significant increase in the levels of plasminogen activator inhibitor-1, plasma TF, and D-dimer. In comparison, TLR2^-/- and TLR7^-/- CLP mice showed preserved MCF and platelet counts, and near-normal plasma TF levels. Bone marrow-derived macrophages treated with a TLR2 agonist Pam3cys-Ser-(Lys)4 (Pam3cys) or a TLR7 agonist (R837) showed marked increases in TF gene expression and protein expression. MicroRNA-146a, a newly identified proinflammatory mediator that is upregulated during sepsis, induced TF production via a TLR7-dependent mechanism.

Conclusions: Murine sepsis leads to an increased procoagulant response, thrombocytopenia, and global coagulopathy. TLR2 and TLR7 play an important role in procoagulant production and in SIC.

Keywords: ROTEM; Toll-like receptor; coagulopathy; microRNA; sepsis.

Figure 1.. Hepatic injury of CLP mice.

Twenty-four hours after sham or CLP surgery, liver tissue was collected, fixed in formalin for 48 hours, followed by 70% ethanol. Liver sections were stained with H&E staining and assessed by a pathologist. Hepatic microvesicular steatosis and vacuolization are indicated by the white arrows in the CLP samples.

Figure 2.. Septic mice develop global coagulopathy.

WT C57BL/6 mice were subjected to CLP surgery and sacrificed at indicated time points for blood collection. Sham mice were sacrificed at 24 hours. A, Representative pictures of ROTEM tracings. Both representative EXTEM and FIBTEM tracings from sham and CLP mice are shown. MCF is marked in each tracing at 30 min. B-C, Time course of MCF values in EXTEM and FIBTEM assays following CLP. *P<0.05, **P<0.01, ***P<0.001. D-F, Time course of clotting time (CT), alpha angle, and clotting formation time (CFT) following CLP procedure. CLP = cecal ligation and puncture, MCF = maximum clot firmness, TLR = Toll-like receptor.

Figure 3.. Clotting factors in sham and septic mice.

Plasma was prepared at 24 hours after sham and CLP procedures. A-F, Platelets, plasma fibrinogen, PAI-1, TF, ATIII, and D-dimer were measured as indicated. *P<0.05, **P<0.01, ****P<0.0001. CLP = cecal ligation and puncture, PAI-1 = plasminogen activator inhibitor-1, TF = tissue factor, ATIII = antithrombin III.

Figure 4.. Fecal slurry (FS) model of polymicrobial sepsis.

A. Diagram of the fecal slurry peritonitis model. B. Core temperature. Fecal slurry was collected from WT, TLR7^−/−, and TLR2^−/− mice and injected i.p. to WT mice. Control mice received i.p. injection of 5% dextrose in water (D5). Twenty-four hours later, rectal temperature was measured. C. EXTEM assay. Blood was collected for E-MCF assay. ^ΔP = 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.. Toll-like receptor (TLR) ligands and miR-146a induce tissue factor (TF) production in mouse macrophages via TLR7-dependent mechanism.

A. TF mRNA production in BMDMs treated with TLR2 agonist, Pam3Cys (P3C) 10 μg/ml and TLR7 agonist, Imiquimod (R837) 1 μg/ml for 4 hours. n = 3/group. B and C. Cell surface expression of TF in BMDMs. Cells were treated with P3C (10 μg/ml) and R837 (1 μg/ml) for 16 hours. Cells were gated for TF using flow cytometry (B) and expressed as % of total cells (C). N=4/group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. D. Cells were stimulated with single-stranded miR-146a-5p mimic or its U→A mutant at 50 nM for 4 hours. miRNA mimics were complexed with lipofectmaine 3000 prior to transfection. N=3/group. **P<0.01. E-F. TF expression as measured by flow cytometry. WT or TLR7^−/− BMDMs were treated with miR-146a and its U→A mutant at 50 nM for 16 hours. N=3/group. *P<0.05. BMDM = bone marrow derived macrophages. TF = tissue factor. TLR = Toll-like receptor