Fgl2-MCOLN3-Autophagy Axis-Triggered Neutrophil Extracellular Traps Exacerbate Liver Injury In Fulminant Viral Hepatitis

Background&Aims:Neutrophil extracellular traps（NETs）were an unrecognized link between inflammation and coagulation,which are two main features of fulminant viral hepatitis（FVH）.Here,we aim to investigate the role and mechanism of NETs in the pathogenesis of FVH.Methods:A mice model of FVH was established by murine hepatitis virus strain-3（MHV-3）infection.Liver leukocytes of infected or uninfected mice were used for single cell RNA sequencing and whole transcriptome sequencing.NETs depletion was achieved using DNase 1.LY6G（clone 1A8）was used to deplete neutrophils.Neutrophils from the bone marrow of wild type（WT）mice or fgl2 knockout(fgl2^-/-)mice were used for in vitro studies.Acetaminophen was used to establish a mice model of non-virus caused acute liver failure.Clinically,NETs-related markers in liver,plasma and peripheral neutrophils were assessed in patients with HBV-related acute liver injury or ALF（HBV-ALI/ALF）.Results:Single-cell RNA sequencing showed that the number of neutrophils increased following MHV-3 infection.Increased hepatic NETs formation was observed in MHV-3-infected mice but not in acetaminophen-treated mice.NETs depletion improved the liver damage and survival rate（from 4%to 29%）in FVH by inhibiting hepatic fibrin deposition and inflammation.FGL2 expression was upregulated following MHV-3 infection in WT mice,fgl2^-/-mice showed significant decrease in NETs formation.Neutrophil from WT and fgl2^-/-mice were adoptive transferred to fgl2^-/-mice,and WT neutrophil adoption showed significant increase in liver damage and fibrin deposition compared to fgl2^-/-neutrophil adoption and PBS control,suggesting that neutrophil-specific FGL2 promoted NETs formation.FGL2 was found to directly interact with mucolipin3（MCOLN3）,which regulated calcium influx and initiated autophagy,leading to NETs formation.Clinically,elevated plasma NETs level was associated with coagulation dysfunction in patients with HBV-ALI.Colocalization of FGL2,NETs and fibrin in liver was observed in these patients.Conclusion:NETs aggravated liver injury in FVH by promoting fibrin deposition and inflammation.NETs formation was regulated by the FGL2-MCOLN3-autophagy axis.Targeting NETs may provide a new strategy for the treatment of FVH.