Acute liver failure (ALF) is a severe, life-threatening condition triggered by drug toxicity, viral hepatitis, autoimmune disorders, or hepatic ischemia [1]. It is characterized by rapid progression of massive hepatocyte necrosis, liver dysfunction, and frequent complications such as coagulopathy and encephalopathy [2]. Despite its high morbidity and mortality, treatment options for ALF remain extremely limited, with liver transplantation being the only definitive therapy—highlighting the urgent need for mechanistic insights into ALF pathogenesis.

During ALF, dysregulated immune cell activation and infiltration play a central role in driving liver injury. In acetaminophen (APAP)-induced ALF models, hepatocyte necrosis leads to the release of mitochondrial DNA (mtDNA), which recruits neutrophils to the liver, and blocking this process significantly reduces hepatotoxicity [3]. Activated neutrophils exacerbate tissue damage by releasing neutrophil extracellular traps (NETs)—unfolded chromatin scaffolds decorated with granule-derived enzymes such as neutrophil elastase (NE) and myeloperoxidase (MPO)—that serve to immobilize and eliminate pathogens [4]. Although NETosis is critical for host defense, clinical studies have reported elevated plasma NETs markers in ALF patients, which correlate with mortality and the need for emergency liver transplantation [5]. Once infiltrated into the liver parenchyma, neutrophils engage in crosstalk with hepatocytes and Kupffer cells, amplifying oxidative stress and accelerating bystander hepatocyte death [6]. Notably, NE KO mice display reduced NETosis, attenuated liver injury, and improved hepatic function [7], suggesting that targeting neutrophil infiltration and NETosis could offer a promising therapeutic approach in ALF. However, the definitive molecular targets that regulate neutrophil recruitment and NETosis remain to be established.

The histidine triad (HIT) protein superfamily comprises three subgroups: histidine triad nucleotide-binding protein (HINT), fragile histidine triad (Fhit), and galactose-1-phosphate uridylyltransferase (GALT) [8]. Among these, the HINT subfamily—including Hint1 [9], Hint2 [10], and Hint3 [11]—has been extensively studied for its tumor-suppressive properties. Emerging evidence highlights the regulatory role of Hint2 in mitochondrial homeostasis. In metabolic dysfunction-associated steatotic liver disease (MASLD) mice, Hint2 enhanced mitochondrial protein deacetylation, thereby improving mitochondrial function and lipid metabolism [12]. Similarly, in acute pancreatitis, Hint2 restored oxidative phosphorylation and reduced reactive oxygen species (ROS) accumulation [13]. Intriguingly, Hint2 is also closely linked to mitochondrial calcium homeostasis—a critical factor in neutrophil activation and NETosis, where both ROS and calcium flux play pivotal roles. Our previous proteomic analysis of mouse liver tissues revealed a downregulation of Hint2 in the ALF group. However, whether Hint2 modulates neutrophil recruitment and NETosis during ALF has yet to be clarified.

Acetylation is one of the most common post-translational modifications, involving both histone and non-histone proteins [14]. Under physiological conditions, a dynamic equilibrium is maintained between acetylation and deacetylation, which are primarily catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively [15]. This reversible modification regulates a wide range of pathophysiological processes—including metabolism, DNA damage repair, and inflammatory responses—by modulating protein-protein interactions, enzymatic activity, and subcellular localization [16]. Among HDACs, HDAC6 is a unique class IIb isoform that primarily acts on cytoplasmic, non-histone substrates remodeling, mitochondrial dynamics, and protein homeostasis [17]. Among the various post-translational modifications, lysine acetylation is reversible and druggable [18]. Pharmacological HDAC6 inhibitors—such as ACY1215—have shown therapeutic potential by restoring acetylation levels and alleviating liver injury during ALF [19]. Although Hint2 harbors multiple lysine residues, it remains unknown whether HDAC6-mediated deacetylation influences Hint2 activity.

In this study, we demonstrate that HDAC6-mediated deacetylation negatively regulates the protein level of Hint2, which could be reversed by pharmacological inhibition of HDAC6. We further show that the K119R mutation, which substitutes lysine 119 with arginine, abolishes Hint2 acetylation and impairs its biological function. In addition, we investigate the role of Hint2 in regulating NETosis and hepatic neutrophil recruitment during ALF and identify its downstream molecular targets involved in mitochondrial calcium homeostasis and proteostasis. Hint2 OE and acetylation limit mitochondrial calcium influx and protein dysregulation, thereby suppressing NETosis and neutrophil chemotaxis. These findings highlight Hint2 as a potential therapeutic target for mitigating the progression of ALF.