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 ... -  《-》

Hepatic hypoxia-inducible factors inhibit PPARα expression to exacerbate acetaminophen induced oxidative stress and hepatotoxicity.

Oxidative stress has a critical role in the pathogenesis of acetaminophen (APAP) induced hepatocellular necrosis, and the identification of novel approaches to attenuate oxidative stress is essential to prevent/revert the disease. This study investigated the role of both HIF-1 and HIF-2 in the pathogenesis of APAP-induced oxidative stress, as well as the underlying mechanisms. In the present study, we initially found that knockout of HIF-1α or HIF-2α reduced APAP toxicity, and double knockout afforded the best protection. APAP treatment led to stabilization of both HIF-1α and HIF-2α in mouse livers. Moreover, the protective effects of HIF deficiency were related to the attenuated oxidative stress. Further experiments proved that PPARα, a master regulator in cellular metabolism accounted for the HIF deficiency-caused protective impact on APAP toxicity. Inactivation of HIFs promoted the expression of peroxisome proliferator-activated receptor α (PPARα) in the liver, which in turn activated nuclear factor erythroid 2-related factor 2 (Nrf2). Knockdown of PPARα or Nrf2 negated the hepatoprotection afforded by HIF deficiency. At last，examination of the PPARα promoter identified a HIF-binding site and HIF-dependent repression of PPARα in hepatocytes by luciferase reporter and EMSA study. Taken together， Our results demonstrate that HIFs are key suppressors of PPARα in the liver, thereby compromising the adaptive defense mechanisms against oxidative stress when confronted with APAP. These findings are important to the etiology and therapeutics of APAP hepatotoxicity. The functional link between HIFs and PPARα may have more implications in liver physiology and other pathologic conditions than APAP injury.

Li D ，Du Y ，Yuan X ，Han X ，Dong Z ，Chen X ，Wu H ，Zhang J ，Xu L ，Han C ，Zhang M ，Xia Q ... -  《-》

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 ... -  《-》

Interleukin 33 mediates hepatocyte autophagy and innate immune response in the early phase of acetaminophen-induced acute liver injury.

Despite interleukin 33 (IL-33) functions as an "alarmin" released from hepatic dead cells in response to tissue damages, the interrelationship between IL-33-mediated hepatocyte autophagy and innate immune response in the acetaminophen (APAP)-induced liver injury (AILI) process remains obscure. This study aimed to explore the regulation of IL-33 on hepatocyte autophagy and macrophage polarization after APAP challenge in vivo and vitro. We found IL-33 released from hepatic necrosis was elevated in the AILI mouse model. Blockage of IL-33 exacerbated liver injury by consuming liver-resident macrophages cells (Kupffer cells, KCs) and promoting hepatic inflammatory factors secretion, such as TNF-α, IL-6 and IL-1β in the early phase of liver injury. Interestingly, IL-33 deficiency further activated hepatocyte autophagy and disrupted M2 macrophage polarization post-APAP challenge in vivo and vitro, which can be reversed by recombinant IL-33 treatment. Mechanistically, administration of IL-33 can directly enhance M2 polarization via PI3K/Akt signaling pathway and activate protective hepatocyte autophagy via AMPKα/mTOR signaling pathway in the AILI process. In conclusion, our data firstly demonstrates that IL-33 exerts protective effects on hepatocytes through the activation of autophagy and functions as an innate immunity regulator mediating macrophage polarization in the early phase of AILI.

Wang Z ，Wu L ，Pan B ，Chen Y ，Zhang T ，Tang N ... -  《-》

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 ... -  《-》