Intracellular HMGB1 mediating neutrophil extracellular trap formation during liver sterile inflammation

Abstract Neutrophil extracellular traps (NETs) has been recently found exacerbate sterile inflammatory injury during liver ischemia/reperfusion (I/R). High mobility group box-1 (HMGB1) released from damaged cells, mediates liver damage and inflammatory responses after I/R. However, the intracellular role of HMGB1 in NET formation remains unknown. We sought to determine the role of intracellular HMGB1 in NET formation. Myloid cells HMGB1 knockout (LysM-HMGB1 KO) and HMGB1-flox mice were subjected to a non-lethal warm liver I/R. Less NETs was identified in ischemic liver lobes of LysM-HMGB1 KO mice compared with flox mice by confocal immunofluorescence (IF) imaging. This was associated with significantly less NET markers in LysM-HMGB1 KO mice, serum level of myeloperoxidase (MPO)-DNA complexes, and tissue level of citrullinated histone H3. Absence of HMGB1 in myloid cells protected the live from I/R injury indicated by significantly decreased serum transaminases (sALT), less necrosis and inflammatory cytokines production compared to flox mice. In vitro, no NET formation was observed in HMGB1 KO neutrophils under stimulation of PMA (positive control) or extracellular HMGB1 or histones confirmed by IF imaging. Chromatin in HMGB1 KO neutrophils failed to de-condensate in response to PMA which is the key step of NET formation. Additionally, gene deletion of HMGB1 in neutrophils completely blocked citrullination of histone H3 as a key step of NET formation, compared with control neutrophils in response to various stimulations. Our study demonstrates the dominant role of intracellular HMGB1 in NET formation. Lack of HMGB1 in neutrophils leads to diminished NET formation protect the liver from organ damage and cells death after liver I/R.