Background High-mobility group box 1 (HMGB1) is a highly conserved ubiquitous nuclear protein with key functions including regulation of DNA binding, transcription and repair. Extracellular HMGB1 elicits chemotaxis  cytokine induction and has pro-inflammatory effects . Due to the diversity of its immunological properties, interest has surrounded its role in chronic inflammatory and autoimmune conditions with increased expression being shown in Rheumatoid Arthritis and SLE . Adult and juvenile patients with SLE have been shown to have a type I Interferon (IFN) genetic signature . Studies have shown an important role of JAK/STAT1 signaling activation via IFNs in mediating HMGB1 release [5-7]. HMGB1 contributes to Neutrophil Extracellular Trap (NET) formation  a process that is potentially thought to lead to the breaking of tolerance and the promotion of autoimmunity  in SLE. The interaction of IFN alpha and HMGB1 in JSLE neutrophils therefore requires investigation.
Objectives To investigate the role of IFN alpha signalling on the release of HMGB1 and how it may contribute to NET formation.
Methods Neutrophils from control (n=3) or JSLE patients (n=3) were either unstimulated or incubated with 10% JSLE serum or 10ng IFN alpha +/- a JAK inhibitor (to prevent IFN signalling) for 2 hours. NET formation and HMGB1 expression and location was observed following immunofluorescent staining and confocal microscopy. As activation of the JAK/STAT1 pathway is important in mediating HMGB1 release phosphorylated STAT1 (pSTAT1) was measured using western blotting in neutrophils isolated from control (n=8) and JSLE patients (n=10).
Results Increased NET formation and nuclear translocation of HMGB1 (Figure 1; white arrows) into the cytoplasm was observed in JSLE compared to control neutrophils which was further increased following incubation with JSLE serum or IFN alpha (See figure 1A-C). Pre-incubation with a JAK inhibitor reduced NET formation and HMGB1 nuclear translocation and release (See figure 1D). Increased pSTAT1 expression (mean ± SEM) was also observed in JSLE neutrophils (0.9±0.05) compared to controls (0.39±0.07; p<0.01), demonstrating increased IFN signalling.
Conclusions The pathogenic effects of HMGB1 have been documented in several diseases. Type I IFN's are also thought to contribute to the pathogenesis of SLE. Here we show that freshly isolated JSLE neutrophils (potentially due to an IFN environment in vivo) have increased HMGB1 in the cytoplasm compared to controls and that IFN signalling is involved in increased nuclear translocation (and potential release) of HMGB1. JAK inhibitors could be beneficial for JSLE patients by reducing NET formation and nuclear translocation /release of HMGB1 in it pro inflammatory isoform.
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Disclosure of Interest None declared