Antiphospholipid antibodies (Anti-β2GPI/B2GPI) prime neutrophil NLRP3 inflammasome activation via TLR2/4 signaling Free

Background: Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by the presence of antiphospholipid antibodies (APLA) in patients with thrombotic events or pregnancy-related complications. APLA, particularly anti-β2GPI antibodies, are known to promote neutrophil activation, neutrophil extracellular trap (NET) formation, and cytokine release, contributing to thrombo-inflammation. Published data show APLA-induced inflammasome activation in endothelial cells and monocytes, while their role in neutrophil inflammasome priming and activation remains unclear. Our previous studies identify neutrophils as critical mediators to APLA-induced arterial and venous thrombosis. Here, we show that APLA, in particular the anti-b2GPI/b2GPI complex, can prime neutrophil inflammasome activation, and this is mediated through TLR2/4 signaling. Objective: We investigated whether anti-β2GPI/β2GPI complexes (β2-APLA) can prime neutrophil NLRP3 inflammasome activation and assessed the involvement of TLR2 and TLR4 in this process.

Methods: Murine bone marrow neutrophils (BMNs) were isolated using immunomagnetic negative selection. For NLRP3 inflammasome priming, BMNs were stimulated with β2-APLA or isotype control IgG for 4 hours (h), followed by activation with nigericin (Nig) for 30 minutes (mins). As lipopolysaccharide (LPS) is the classical NLRP3 priming agent, in separate experiments, we pre-treated BMNs with β2-APLA for 1 h, followed by LPS for 3 h and Nig for 30 mins to investigate whether β2-APLA exaggerates LPS-mediated inflammasome activation. Inflammasome activation was evaluated by measuring cleavage (whole cell lysates) and extracellular release (supernatant) of IL-1β, IL-18, and caspase-1. TLR2, TLR4, NLRP3, and caspase-1 inhibitors were used to delineate the signaling pathway. C57BL/6 mice aged 8-10 weeks were treated intravenously with APLA obtained from patient samples as well as NLRP3 inhibitor MCC950 intraperitoneally (24 h and 2 h prior to thrombosis assay) and examined for carotid artery thrombosis using the Rose Bengal photochemical injury model.

Results: β2-APLA-treated BMNs demonstrated increased NLRP3 inflammasome activation following Nig treatment compared to isotype control IgG. Importantly, we noted that β2-APLA treatment alone is sufficient to prime BMNs, inducing significant IL-1β, IL-18, and cleaved caspase-1 release following Nig treatment, even in the absence of LPS priming. In LPS and Nig-treated cells, β2-APLA treatment was associated with greater IL-1β and IL-18 release as compared to LPS and Nig treatment alone, suggesting that β2-APLA enhances LPS-induced inflammasome activation. Inhibition of NLRP3 (MCC950) or caspase-1 (YVAD) significantly decreased inflammasome activation. Inhibition of either TLR2 or TLR4 receptors markedly reduced IL-1β secretion, indicating that signaling can occur via either of these receptors in β2-APLA-mediated inflammasome activation. In comparison to TLR2/4 inhibition, inhibition of NLRP3 or caspase-1 only partially suppressed IL-1β release, suggesting the involvement of alternate pathways of IL-1β processing. Finally, C57BL6J mice treated with APA demonstrated faster carotid artery thrombosis as compared to control IgG. This was significantly mitigated following inflammasome inhibition with MCC950, suggesting the importance of inflammasome activation in APA-mediated arterial thrombosis.

Conclusion: Our data demonstrate that anti-β2GPI/β2GPI complexes prime neutrophil inflammasome activation through TLR2/4 receptor signaling, mimicking microbial stimuli such as LPS. Our observation that anti-β2GPI/β2GPI complexes enhance LPS-Nig-mediated inflammasome activation demonstrates how APLA likely contributes to the thrombo-inflammatory effects during infections with enhanced IL-1β and IL-18 release. Our murine thrombosis studies identify inflammasome inhibition as a possible therapeutic approach in patients with APLA-mediated thrombosis. Ongoing studies are focused on understanding the downstream mediators of inflammasome-driven thrombosis, and the importance of inflammasome inhibition in APLS-mediated VTE. Our study elucidates the role of APLA in enhanced LPS-mediated inflammasome activation, thus provoking an exaggerated inflammatory response. These findings underscore a critical role for neutrophil NLRP3 inflammasome activation in the pathogenesis of APS and may inform therapeutic strategies targeting innate immune signaling in thrombo-inflammatory disease.