Abstract
Background:Neutrophil-released neutrophil extracellular traps (NETs) are upregulated and promote autoinflammation and thrombosis in Behçet’s disease (BD), a multi-system inflammatory disease with unknown etiology1,2. However, whether NETs promote macrophage activation in BD remains unclear.Objectives:To investigate the potential role of NETs in promoting aberrant macrophage activation in BD.Methods:We quantified NETs by measuring dsDNA using ELISA and immunofluorescence. Macrophages were stimulated with BD- and healthy controls (HC)-derived NETs, and IL-8 and TNF-α production were measured by ELISA. NETs-stimulated macrophages were incubated with naive CD4+T cells, and Th1 cell differentiation was examined on day 7 by flow cytometry. Histones H1, H2A, H2B, H3, H4, S100A8 and neutrophil elastase in NETs were analyzed by western blot. Macrophages were stimulated with anti-Histone 4 antibody-treated NETs, and IL-8 production was measured by ELISA.Results:Circulating NETs (2336±534 ng/ml vs. 1472±549 ng/ml,P=0.0008) and neutrophil-derived NETs (909.2±485.2 ng/ml vs. 582.4±199.2 ng/ml,P=0.0108) were significantly higher in BD patients compared with those in HC. BD NETs stimulated macrophages to produce a higher level of IL-8 (17±4 ng/ml vs. 13±4 ng/ml,P=0.0474) and TNF-α (166±61 pg/ml vs. 102±48 pg/ml,P=0.0132) than HC NETs. Moreover, BD NETs promoted macrophages to facilitate Th1 differentiation than HC NETs (33±10% vs. 24±7%,P=0.0398). Western blot analysis revealed more Histone H4 (289076 (144365, 544038) IOD values vs. 42121 (6958, 129625) IOD values,P=0.0286), but not Histones H1, H2A, H2B, H3, S100A8 or neutrophil elastase in BD NETs compared to HC NETs. Importantly, neutralizing Histone H4 abrogated the BD NETs-stimulated IL-8 overproduction by macrophages (9.99±2.07 ng/ml vs. 13.95±2.91 ng/ml,P=0.021).Conclusion:BD NETs promoted macrophages activation, which might be mediated by a higher level of Histone H4.
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