Background/Purpose: Antibodies targeting beta-2-glycoprotein I (anti-β2GPI) promote inflammation and thrombosis in antiphospholipid syndrome (APS). It has been shown that anti-β2GPI activate neutrophils through Toll-like receptor 4 (TLR4) to promote the release of pathogenic neutrophil extracellular traps (NETs), which promote a thrombo-inflammatory milieu. It is also known that APS patient neutrophils upregulate TLR4-associated genes, including those encoding CD14 and p38 MAP kinase (MAPK). Here, we sought to elucidate the potential role of CD14-dependent MAPK signaling in the neutrophil response to anti-β2GPI.

Methods: For patient profiling, peripheral blood was collected from healthy donors (n=9) or primary APS patients (n=9, meeting the 2023 ACR/EULAR Criteria), and neutrophil expression of CD14 and activation markers was assessed by flow cytometry. For mechanistic studies, affinity-purified anti-β2GPI IgG (10 µg/mL) was added to isolated primary human neutrophils or whole blood for 1-5 hours (depending on the assay) in the presence or absence of the CD14 blocking antibody atibuclimab (10 µg/mL). CD14 and activation marker expression was assessed by flow cytometry. NETs were visualized by immunofluorescence microscopy, and p38 phosphorylation was quantified by immunoblotting. Lastly, C57BL/6J mice were pretreated with the p38 inhibitor adezmapimod (25 mg/kg) or an appropriate vehicle control 1 hour prior to inducing thrombosis in the inferior vena cava. At the time of thrombosis induction, mice also received APS or control IgG (500 µg). Thrombi were collected and weighed 24 hours after surgery.

Results: Neutrophil surface CD14 showed a positive association with the activation markers CD11b and CD18 in APS patients (R2=0.7318, p=0.0033 and R2=0.4702, p=0.0415) but not in healthy controls (R2=0.2448, p=0.1757 and R2=0.2237, p=0.1985). Furthermore, healthy neutrophils treated with anti-β2GPI displayed increased surface CD14 expression (1.7-fold change, p=0.0078). 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 attenuated anti-β2GPI-induced neutrophil CD11b expression (0.7-fold change, p=0.0319), p38 phosphorylation (0.5-fold change), and NET formation. Having seen that p38 phosphorylation downstream of anti-β₂GPI was CD14 dependent, we assessed whether p38 inhibition with adezmapimod could modulate APS-associated thrombosis in vivo. In mice, APS IgG potentiated thrombus weight (mean 5.5 mg vs 2.6 mg, p=0.002), which was significantly attenuated with adezmapimod treatment (mean 2.1 mg vs 5.5 mg, p=0.0004).

Conclusion: Neutrophil CD14 appears to be required for the p38 MAPK-mediated neutrophil activation and NETosis reliably triggered by anti-β2GPI in APS. This work identifies neutrophil surface CD14 and downstream p38 MAPK signaling as novel therapeutic targets with the potential to restrain not only thrombosis, but also the upstream inflammation that underlies APS.

Schematic illustration of the proposed roles for CD14 and p38 MAPK in pathogenic anti-β₂GPI antibody-mediated neutrophil activation and NETosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cd14-dependent-map-kinase-signaling-is-required-for-pathogenic-neutrophil-extracellular-trap-formation-in-aps/