35 Investigating CD14 as a novel therapeutic target in antiphospholipid syndrome

Objectives/Goals: Autoantibodies targeting beta-2-glycoprotein I (anti-β2GPI) are found in many patients with antiphospholipid syndrome (APS), where they trigger the release of pathogenic neutrophil extracellular traps (NETs). Here, we investigated the relevance of the cell signaling protein CD14 in this process to assess its potential as a therapeutic target. Methods/Study Population: Peripheral blood was collected from healthy donors or primary APS patients (all meeting the most recent ACR/EULAR Criteria). Isolated neutrophils were subjected to bulk RNAseq analysis. Neutrophil expression of CD14 and activation markers was validated by immunoblotting and flow cytometry. For mechanistic studies, affinity-purified anti-β2GPI IgG was added to isolated primary human neutrophils or whole blood in the presence or absence of the CD14 blocking antibody atibuclimab. CD14 and activation marker expression were assessed by flow cytometry, and NETs were visualized by immunofluorescence microscopy and quantified via SYTOX green assay. Ongoing work will involve CD14 blockade in mice subjected to the electrolytic inferior vena cava model of thrombosis, accompanied by APS or control IgG injection. Results/Anticipated Results: Neutrophils from APS patients overexpressed CD14 when compared to healthy controls, and patient neutrophil surface CD14 showed a strong positive association with the activation markers CD11b, CD18, and CD66b. Further, healthy neutrophils activated with patient-derived anti-β2GPI increased surface expression of CD14. Given the association between CD14 and APS-associated neutrophil activity, we asked whether CD14 blockade with atibuclimab could mitigate the effects of anti-β2GPI on neutrophils. Indeed, atibuclimab treatment attenuated anti-β2GPI-induced expression of CD11b, CD18, and CD66b, and NET formation. In vivo, we anticipate that CD14 blockade will reduce APS IgG-enhanced thrombi size and plasma levels of NET biomarkers and inflammatory cytokines. Discussion/Significance of Impact: Neutrophil CD14 appears to be required for neutrophil activation and NET formation triggered by anti-β2GPI in patients with APS. This work identified neutrophil surface CD14 and its associated downstream pathways as novel therapeutic targets with the potential to restrain not only thrombosis but also the upstream inflammation that underlies APS.