Targeting Neutrophil CD14 Inhibits Deep Vein Thrombus Formation and Enhances Thrombus Resolution Following Inferior Vena Cava Stenosis in Mice

Background: Neutrophil-mediated persistent inflammation and neutrophil extracellular trap formation (NETosis) promote deep vein thrombosis (DVT). CD14, a co-receptor for toll-like receptor 4 (TLR4), is actively synthesized by neutrophils, and the CD14/TLR4 signaling pathway has been implicated in proinflammatory cytokine overproduction and several aspects of thromboinflammation. Using unbiased RNA sequencing and proteomics analyses, we observed significantly increased expression of CD14 in neutrophils, but not in monocytes, in mice with DVT. Both neutrophils and CD14 are implicated in inflammation and thrombotic processes; however, the molecular mechanisms and role of neutrophil-specific CD14 in the pathogenesis of DVT are unknown. Here, we hypothesized that CD14 on neutrophils promotes deep vein thrombus formation and impairs thrombus resolution.

Methods: DVT was induced in 12-14 weeks old C57BL/6 wild type mice by partial ligation of the inferior vena cava (IVC). Unbiased RNA sequencing and proteomic analyses were performed using isolated neutrophils following IVC stenosis. The plasma levels of granulocyte colony-stimulating factor (G-CSF), elastase, myeloperoxidase (MPO), and interleukin-10 (IL-10) were assessed using commercial ELISA. DVT outcomes (IVC thrombus weight and length, thrombosis incidence, neutrophil recruitment, and NETosis) were evaluated 48 hours after IVC stenosis in mice treated with a specific anti-CD14 antibody, 4C1 (4 mg/kg), or a control antibody. Neutrophil specific knockdown of Cd14 was performed by lentiviral vector expressing Cd14 targeting-miR30-shRNA (2.5X10⁷ transducing units) driven by human MRP8 promoter (for neutrophil specificity). Noninvasive ultrasound imaging was used to measure the extent of venous thrombosis up to day 14 post-DVT in mice with neutrophil-specific Cd14 knockdown and controls. To increase translational impact and clinical significance, primary human neutrophils were stimulated by oxidized PAPC (oxPAPC), an ednogenous and proinflammatory CD14 ligand. We evaluated apoptosis using a caspase 3 activity assay kit and NETs using immunofluorescence staining of citrullinated histone H3 (H3Cit) in human neutrophils stimulated with oxPAPC and treated with anti-CD14 antibody (15 µg/ml) or control antibody.

Results: Mice with IVC stenosis exhibited increased plasma levels of granulocyte colony-stimulating factor (G-CSF) along with a higher neutrophil-to-lymphocyte ratio, and increased plasma levels of cell-free DNA, elastase, and myeloperoxidase. Quantitative measurement of total neutrophil mRNA and protein expression revealed distinct profiles in mice with IVC stenosis compared with mice subjected to sham surgery. Neutrophils of mice with IVC stenosis exhibit increased inflammatory transcriptional and proteomic responses, along with increased expression of CD14. The flow cytometry assay confirmed the significant increase in CD14 on post-DVT neutrophils; however, monocyte CD14 levels remained unchanged. Intriguingly, CD14 blockage did not reduce DVT severity in mice treated with anti-CD14 antibody, possibly due to the significant reduction in circulating IL-10 (anti-inflammatory cytokine). Next, to evaluate the role of neutrophil-specific CD14 inhibition, we performed in vivo shRNA-based neutrophil-specific Cd14 knockdown and evaluated DVT outcomes. Ultrasound imaging results revealed that mice treated with Cd14 targeted shRNA exhibited significantly reduced DVT severity (reduced thrombus area and thrombosis incidence on day 2) and enhanced thrombus resolution (on day 14) compared to control mice. In vitro mechanistic studies revealed that oxPAPC-stimulated primary human neutrophils exhibited significantly reduced caspase 3 activity (suggesting reduced apoptosis) and increased NETosis, which was partially but significantly restored by anti-CD14 antibody treatment.

Conclusion: Collectively, the present study highlights the critical and previously unrecognized role of neutrophil CD14 in the pathogenesis of DVT and suggests that targeting neutrophil CD14 inhibits deep vein thrombus formation and enhances thrombus resolution.