Targeting the HDAC6/Hint2/MICU1 axis to ameliorate acute liver failure via inhibiting NETosis

Aims: Acute liver failure (ALF) is marked by extensive inflammation and immune dysregulation, which are closely associated with neutrophil infiltration and NETosis. However, the specific mechanisms that drive NETosis in ALF remain poorly understood.

Materials and methods: We employed flow cytometry, western blot, qRT-PCR, and cf-DNA assay to investigate the link between NETosis and ALF. The role of HDAC6-mediated deacetylation of histidine triad nucleotide-binding protein 2 (Hint2) was assessed, along with the effects of lentiviral vector-based overexpression and knockdown of Hint2 on mitochondrial function and NETosis. Additionally, CO-IP, IF, protein docking analysis, mCa²⁺ uptake assay, and mtROS measurement were used to explore the interaction between Hint2 and mitochondrial calcium uniporter complex (MCUc). Finally, experimental neutrophil depletion in mice was conducted to confirm the protective effect of NETosis inhibition in ALF.

Key findings: Our study demonstrated that Hint2 undergoes HDAC6-mediated deacetylation, disrupting mitochondrial dynamics and triggering NETosis during ALF. Furthermore, MICU1 bridges Hint2 and NETosis by regulating mCa²⁺ homeostasis and mtROS production. Activation of Hint2, either through the HDAC6 Inhibitor ACY1215 or via overexpression, increased the level of MICU1 to suppress the opening of the MCUc and the associated mtROS release, thereby inhibiting NETosis. Conversely, Hint2 knockdown induced NETosis by surging mCa²⁺ overload and mtROS production, while the MCUc inhibitor RU265 mitigates NETosis by blocking mCa²⁺ influx.

Significance: Our findings recognized the HDAC6/Hint2/MICU1 axis as a novel pathway in neutrophils, the inhibition of which intercepts mCa²⁺ overload and mtROS accumulation, thereby reducing NETosis and facilitating liver recovery during ALF.

Acute liver failure; HDAC6; Histidine triad nucleotide-binding protein 2; Mitochondrial calcium uptake 1; Mitochondrial reactive oxygen species; NETosis.