Circulating histones are major mediators of systemic inflammation and cellular injury in patients with acute liver failure

Acute liver failure (ALF) is a life-threatening systemic disorder. Here we investigated the impact of circulating histones, recently identified inflammatory mediators, on systemic inflammation and liver injury in murine models and patients with ALF. We analyzed histone levels in blood samples from 62 patients with ALF, 60 patients with chronic liver disease, and 30 healthy volunteers. We incubated patients'' sera with human L02 hepatocytes and monocytic U937 cells to assess cellular damage and cytokine production. d-galactosamine plus lipopolysaccharide (GalN/LPS), concanavalin A (ConA), and acetaminophen (APAP) were given to C57BL/6N mice to induce liver injury, respectively, and the pathogenic role of circulating histones was studied. Besides, the protective effect of nonanticoagulant heparin, which can bind histones, was evaluated with in vivo and ex vivo investigations. We observed that circulating histones were significantly increased in patients with ALF, and correlated with disease severity and mortality. Significant systemic inflammation was also pronounced in ALF patients, which were associated with histone levels. ALF patients'' sera induced significant L02 cell death and stimulated U937 cells to produce cytokines, which were abrogated by nonanticoagulant heparin. Furthermore, circulating histones were all released remarkably in GalN/LPS, ConA, and APAP-treated mice, and associated with high levels of inflammatory cytokines. Heparin reduced systemic inflammation and liver damage in mice, suggesting that it could interfere with histone-associated liver injury. Collectively, these findings demonstrate that circulating histones are critical mediators of systemic inflammation and cellular damage in ALF, which may be potentially translatable for clinical use.Acute liver failure (ALF) is a complex condition wherein rapid-onset liver insult results in coagulopathy, hepatic encephalopathy (HE), and even multiple organ failure and death in a patient without previously recognized liver disease.^{1, 2} The causes of ALF vary greatly by geographical region. In the Western countries, acetaminophen (APAP) constitutes nearly 60–70% of the cases, whereas the primary cause of ALF in China is viral hepatitis B, which accounts for 70–80% of the cases.^{3, 4} Despite recent therapeutic advances, ALF remains a serious clinical condition associated with a high mortality rate. The underlying mechanisms of ALF are multifactorial and incompletely understood. Emerging evidence suggests that, regardless of various etiologies of ALF, its acute onset is generally associated with significant and uncontrolled activation of systemic inflammation, which may consequently lead to multiple organ dysfunction and a poorer prognosis in ALF.^{5, 6} It has been accepted that systemic inflammation may have a crucial role in the initiation and progression of ALF, but the key factors or mechanisms that are responsible for its activation in ALF are largely unclear. Therefore, identification of key mediators that may modulate ALF-associated inflammation is highly desirable.Extracellular histones in the circulation are recently identified as the pivotal mediators in systemic inflammatory diseases.^{7, 8, 9, 10} It reveals that circulating histones have numerous toxic effects including direct cytotoxicity, induction of vascular permeability, coagulation activation, platelet aggregation, and cytokine production,^{11, 12, 13} all of which are possible mechanisms related to the development of inflammatory organ injuries. Furthermore, histone-targeted therapy has promising potentials for the treatment of various inflammatory injuries. We, therefore, investigated circulating histone levels in patients with ALF, with a hypothesis that histones present in the circulation of ALF patients could serve as inflammatory mediators mediating cellular damage and interfering with disease progression and mortality. We further examined whether histone-mediated toxicity could be neutralized by nonanticoagulant heparin, which can bind histones,¹⁴ through in vivo and ex vivo translational studies, with an aim to providing an interventional strategy for ALF in clinical practice.