Background/Purpose: The leukocyte immunoglobulin-like receptor A3 (LILRA3) gene encodes the only soluble receptor within the LILR family. LILRA3 polymorphisms have been associated with both rheumatoid arthritis and lupus. Soluble LILRA3 appears to circulate at high levels in patients with rheumatoid arthritis, and has been suggested to have pro-inflammatory functions. Regarding its cellular source, a recent proteomic analysis of neutrophil granules detected LILRA3 as a key component of tertiary granules. To date, the role of LILRA3 in antiphospholipid syndrome (APS) has not been defined. Here, we hypothesized that LILRA3 might be delivered extracellularly via the release of neutrophil extracellular traps (NETs). We also considered whether LILRA3 might function as a NET-binding protein and whether it circulates at high levels in APS patients, where it may promote autoantibody formation or have thromboinflammatory functions.

Methods: LILRA3 expression was assessed in neutrophils of primary APS patients by real-time PCR. Circulating levels of soluble LILRA3 and anti-LILRA3 IgG were measured in primary APS patients and healthy controls by enzyme-linked immunosorbent assay. Electrophoretic mobility shifts assay (EMSA) was performed to determine whether LILRA3 binds neutrophil DNA (the dominant component of NETs). Human neutrophils were activated to release NETs, and co-localization of LILRA3 and NETs was assessed by immunofluorescence microscopy.

Results: Our previous RNA-sequencing of primary APS neutrophils (n=9) revealed ~3-fold upregulation of LILRA3 in APS patients as compared with matched controls. Here, we verified this overexpression by real-time PCR in an independent cohort of 20 primary APS patients and matched controls (mean 2 fold increase in APS). We also measured LILRA3 protein and anti-LILRA3 IgG in primary APS plasma, and found a positive correlation between LILRA3 protein levels and anti-beta-2 glycoprotein I IgM (P = 0.009). Furthermore, as compared with matched controls, primary APS was associated with higher circulating levels of both soluble LILRA3 (1.5 fold) and anti-LILRA3 IgG (1.6 fold), with a positive correlation between the two (P = 0.041). To investigate whether LILRA3 might directly interact with NET DNA, LILRA3 was incubated with purified neutrophil genomic DNA and analyzed by EMSA. The addition of 0.25 μM LILRA3 (partial effect) and 1.0 μM (full effect) significantly reduced the migration of genomic DNA (0.1mg/ml) through the agarose gel, suggested a direct interaction between LILRA3 and DNA. Furthermore, by immunofluorescence microscopy, we observed clear co-localization of LILRA3 and NET strands.

Conclusion: We demonstrate for the first time elevated levels of LILRA3 and anti-LILRA3 IgG in patients with primary APS. Furthermore, LILRA3 appears to be a NET-binding protein that can be delivered extracellularly in the context of NETs, where it has potential to play a role in the pathogenesis of APS. Further studies are underway in pursuit of potential thromboinflammatory functions of this NET-binding protein.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/elevated-levels-of-the-neutrophil-extracellular-trap-binding-protein-lilra3-in-primary-antiphospholipid-syndrome/