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

• Published in: Blood 2024-11, Vol.144, p.137-137
• Main Authors: Pandey, Nilesh, Kaur, Harpreet, Chandaluri, Lakshmi, Anand, Sumit Kumar, Mohamed, Tarek Magdy, Stokes, Karen, Beedupalli, Kavitha, Orr, A Wayne, Rom, Oren, Dhanesha, Nirav
• Format: Article
• Language: English
• ISSN: 0006-4971
• DOI: 10.1182/blood-2024-208713

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 DN