Role of hepatic resident and infiltrating macrophages in liver repair after acute injury.

Treatment of liver disease, caused by hepatotoxins, viral infections, alcohol ingestion, or autoimmune conditions, remains challenging and costly. The liver has a powerful capacity to repair and regenerate, thus a thorough understanding of this tightly orchestrated process will undoubtedly improve clinical means of restoring liver function after injury. Using a murine model of acute liver injury caused by overdose of acetaminophen (APAP), our studies demonstrated that the combined absence of liver resident macrophages (Kupffer cells, KCs), and infiltrating macrophages (IMs) resulted in a marked delay in liver repair, even though the initiation and extent of peak liver injury was not impacted. This delay was not due to impaired hepatocyte proliferation but rather prolonged vascular leakage, which is caused by APAP-induced liver sinusoidal endothelial cell (LSEC) injury. We also found that KCs and IMs express an array of angiogenic factors and induce LSEC proliferation and migration. Our mechanistic studies suggest that hypoxia-inducible factor (HIF) may be involved in regulating the angiogenic effect of hepatic macrophages (Macs), as we found that APAP challenge resulted in hypoxia and stabilization of HIF in the liver and hepatic Macs. Together, these data indicate an important role for hepatic Macs in liver blood vessel repair, thereby contributing to tissue recovery from acute injury.

You Q ，Holt M ，Yin H ，Li G ，Hu CJ ，Ju C ... -  《-》

Hypoxia-Inducible Factor-2α Reprograms Liver Macrophages to Protect Against Acute Liver Injury Through the Production of Interleukin-6.

Acetaminophen (APAP) overdose represents the most frequent cause of acute liver failure, resulting in death or liver transplantation in more than one third of patients in the United States. The effectiveness of the only antidote, N-acetylcysteine, declines rapidly after APAP ingestion, long before patients are admitted to the clinic with symptoms of severe liver injury. The direct hepatotoxicity of APAP triggers a cascade of innate immune responses that may exacerbate or limit the progression of tissue damage. A better understanding of this complex mechanism will help uncover targets for therapeutic interventions. We observed that APAP challenge caused stabilization of hypoxia-inducible factors (HIFs) in the liver and hepatic macrophages (MΦs), particularly HIF-2α. Genetic deletion of the HIF-2α gene in myeloid cells (HIF-2αmye/- ) markedly exacerbated APAP-induced liver injury (AILI) without affecting APAP bioactivation and detoxification. In contrast, hepatic and serum levels of the hepatoprotective cytokine interleukin 6 (IL-6), its downstream signal transducer and transcription factor 3 activation in hepatocytes, as well as hepatic MΦ IL-6 expression were markedly reduced in HIF-2αmye/- mice compared to wild-type mice post-APAP challenge. In vitro experiments revealed that hypoxia induced IL-6 production in hepatic MΦs and that such induction was abolished in HIF-2α-deleted hepatic MΦs. Restoration of IL-6 by administration of exogenous IL-6 ameliorated AILI in HIF-2αmye/- mice. Finally, IL-6-mediated hepatoprotection against AILI was abolished in hepatocyte-specific IL-6 receptor knockout mice. The data demonstrate that APAP treatment leads to HIF-2α stabilization in hepatic MΦs and that HIF-2α subsequently reprograms hepatic MΦs to produce the hepatoprotective cytokine IL-6, thereby ameliorating AILI.

Gao RY ，Wang M ，Liu Q ，Feng D ，Wen Y ，Xia Y ，Colgan SP ，Eltzschig HK ，Ju C ... -  《-》

Exacerbation of acetaminophen-induced disturbances of liver sinusoidal endothelial cells in the absence of Kupffer cells in mice.

Although there has been considerable research in terms of liver sinusoidal endothelial cells (LSECs) and hepatic resident macrophages (Kupffer cells, KCs) during the overall pathogenesis of acetaminophen (APAP)-induced liver injury, little is known about their potential interaction and relationship. In the present study, employing the use of liposome/clodronate to deplete KCs, we were able to confirm the previously demonstrated hepato-protective role for KCs. Such a protective role may be mediated, in part, via regulation of LSEC homeostasis and integrity. The further aggravation of APAP-induced LSEC disturbance upon depletion of KCs correlated with increased hepatic vascular permeability and red blood cell accumulation. The depletion of KCs prior to APAP challenge also resulted in the increased expression of cellular adhesion molecules on LSECs. Such increased disturbance in the hepatic endothelial may represent a contributing factor in the exacerbation of APAP-induced liver injury in the absence of KCs. However, these disturbances may also be a result of the increased hepatic damage, and as such, does not rule out the potential for additional mechanisms of KC-mediated hepato-protection during the pathogenesis of APAP-induced hepatotoxicity.

Holt MP ，Yin H ，Ju C 《-》

Fibrin(ogen) drives repair after acetaminophen-induced liver injury via leukocyte α(M)β(2) integrin-dependent upregulation of Mmp12.

Acetaminophen (APAP)-induced liver injury is coupled with activation of the blood coagulation cascade and fibrin(ogen) accumulation within APAP-injured livers of experimental mice. We sought to define the role of fibrin(ogen) deposition in APAP-induced liver injury and repair. Wild-type, fibrinogen-deficient mice, mutant mice with fibrin(ogen) incapable of binding leukocyte αMβ2 integrin (Fibγ390-396A mice) and matrix metalloproteinase 12 (Mmp12)-deficient mice were fasted, injected with 300mg/kg APAP i.p. and evaluated at a range of time-points. Plasma and liver tissue were analyzed. Rescue of Fibγ390-396A mice was carried out with exogenous Mmp12. To examine the effect of the allosteric leukocyte integrin αMβ2 activator leukadherin-1 (LA-1), APAP-treated mice were injected with LA-1. In wild-type mice, APAP overdose increased intrahepatic levels of high molecular weight cross-linked fibrin(ogen). Anticoagulation reduced early APAP hepatotoxicity (6h), but increased hepatic injury at 24h, implying a protective role for coagulation at the onset of repair. Complete fibrin(ogen) deficiency delayed liver repair after APAP overdose, evidenced by a reduction of proliferating hepatocytes (24h) and unresolved hepatocellular necrosis (48 and 72h). Fibγ390-396A mice had decreased hepatocyte proliferation and increased multiple indices of liver injury, suggesting a mechanism related to fibrin(ogen)-leukocyte interaction. Induction of Mmp12, was dramatically reduced in APAP-treated Fibγ390-396A mice. Mice lacking Mmp12 displayed exacerbated APAP-induced liver injury, resembling Fibγ390-396A mice. In contrast, administration of LA-1 enhanced hepatic Mmp12 mRNA and reduced necrosis in APAP-treated mice. Further, administration of recombinant Mmp12 protein to APAP-treated Fibγ390-396A mice restored hepatocyte proliferation. These studies highlight a novel pathway of liver repair after APAP overdose, mediated by fibrin(ogen)-αMβ2 integrin engagement, and demonstrate a protective role of Mmp12 expression after APAP overdose. Acetaminophen overdose leads to activation of coagulation cascade and deposition of high molecular weight cross-linked fibrin(ogen) species in the liver. Fibrin(ogen) is required for stimulating liver repair after acetaminophen overdose. The mechanism whereby fibrin(ogen) drives liver repair after acetaminophen overdose requires engagement of leukocyte αMβ2 integrin and subsequent induction of matrix metalloproteinase 12.

Kopec AK ，Joshi N ，Cline-Fedewa H ，Wojcicki AV ，Ray JL ，Sullivan BP ，Froehlich JE ，Johnson BF ，Flick MJ ，Luyendyk JP ... -  《-》

Macrophage-Inducible C-Type Lectin Signaling Exacerbates Acetaminophen-Induced Liver Injury by Promoting Kupffer Cell Activation in Mice.

Overdose of acetaminophen (APAP) has become one of the most frequent causes of acute liver failure. Macrophage-inducible C-type lectin (Mincle) acts as a key moderator in immune responses by recognizing spliceosome-associated protein 130 (SAP130), which is an endogenous ligand released by necrotic cells. This study aims to explore the function of Mincle in APAP-induced hepatotoxicity. Wild-type (WT) and Mincle knockout (KO) mice were used to induce acute liver injury by injection of APAP. The hepatic expressions of Mincle, SAP130, and Mincle signaling intermediate (Syk) were markedly upregulated after the APAP challenge. Mincle KO mice showed attenuated injury in the liver, as shown by reduced pathologic lesions, decreased alanine aminotransferase and aspartate aminotransferase levels, downregulated levels of inflammatory cytokines, and decreased neutrophil infiltration. Consistently, inhibition of Syk signaling by GS9973 alleviated APAP hepatotoxicity. Most importantly, Kupffer cells (KCs) were found as the major cellular source of Mincle. The depletion of KCs abolished the detrimental role of Mincle, and the adoptive transfer of WT KC to Mincle KO mice partially reversed the hyporesponsiveness to hepatotoxicity induced by APAP. Furthermore, the expression levels of interleukin (IL)-1β and neutrophil-attractant CXC chemokines were substantially lower in KCs isolated from APAP-treated Mincle KO mice compared with those from WT mice. Similar results were found in primary Mincle KO KCs treated with a ligand of Mincle (trehalose-6,6-dibehenate) or in conditioned media obtained from APAP-treated hepatocytes. Collectively, Mincle can regulate the inflammatory response of KCs, which is necessary for the complete progression of hepatotoxicity induced by APAP. SIGNIFICANCE STATEMENT: Acetaminophen (APAP) overdose is becoming a main cause of drug-induced acute liver damage in the developed world. This study showed that macrophage-inducible C-type lectin (Mincle) deletion or inhibition of Mincle downstream signaling attenuates APAP hepatotoxicity. Furthermore, Mincle as a modulator of Kupffer cell activation contributes to the full process of hepatotoxicity induced by APAP. This mechanism will offer valuable insights to overcome the limitation of APAP hepatotoxicity treatment.

Zhao J ，Kim JW ，Zhou Z ，Qi J ，Tian W ，Lim CW ，Han KM ，Kim B ... -  《-》