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Acetaminophen (APAP) overdose is a leading cause of acute liver injury in the USA. The chitinase 3-like-1 (Chi3l1) protein contributes to APAP-induced liver injury (AILI) by promoting hepatic platelet recruitment. Here, we report the development of a Chi3l1-targeting antibody as a potential therapy for AILI. By immunizing a rabbit successively with the human and mouse Chi3l1 proteins, we isolated cross-reactive monoclonal antibodies (mAbs) from single memory B cells. One of the human and mouse Chi3l1 cross-reactive mAbs was humanized and characterized in both in vitro and in vivo biophysical and biological assays. X-ray crystallographic analysis of the lead antibody C59 in complex with the human Chi3l1 protein revealed that the kappa light contributes to majority of the antibody–antigen interaction; and that C59 binds to the 4α-5β loop and 4α-helix of Chi3l1, which is a functional epitope and hotspot for the development of Chi3l1 blocking antibodies. We humanized the C59 antibody by complementarity-determining region grafting and kappa chain framework region reverse mutations. The humanized C59 antibody exhibited similar efficacy as the parental rabbit antibody C59 in attenuating AILI in vivo. Our findings validate Chi3l1 as a potential drug target for AILI and provide proof of concept of developing Chi3l1 blocking antibody as a therapy for the treatment of AILI.

Beyond the classical description of eosinophil functions in parasite infections and allergic diseases, emerging evidence supports a critical role of eosinophils in resolving inflammation and promoting tissue remodeling. However, the role of eosinophils in liver injury and the underlying mechanism of their recruitment into the liver remain unclear.Hepatic eosinophils were detected and quantified using flow cytometry and immunohistochemical staining. Eosinophil-deficient (ΔdblGata1) mice were used to investigate the role of eosinophils in 3 models of acute liver injury. In vivo experiments using Il33Hepatic accumulation of eosinophils was observed in patients with acetaminophen (APAP)-induced liver failure, whereas few eosinophils were detectable in healthy liver tissues. In mice treated with APAP, carbon tetrachloride or concanavalin A, eosinophils were recruited into the liver and played a profound protective role. Mice deficient of macrophages or IL-33 exhibited impaired hepatic eosinophil recruitment during acute liver injury. CCL24, but not CCL11, was increased after treatment of each hepatotoxin in an IL-33 and macrophage-dependent manner. In vitro experiments demonstrated that IL-33, by stimulating IL-4 release from eosinophils, promoted the production of CCL24 by macrophages.This is the first study to demonstrate that hepatic recruitment of and protection by eosinophils occur commonly in various models of acute liver injury. Our findings support further exploration of eosinophils as a therapeutic target to treat APAP-induced acute liver injury.The current study unveils that eosinophils are recruited into the liver and play a protective function during acute liver injury caused by acetaminophen overdose. The data demonstrate that IL-33-activated eosinophils trigger macrophages to release high amounts of CCL24, which promotes hepatic eosinophil recruitment. Our findings suggest that eosinophils could be an effective cell-based therapy for the treatment of acetaminophen-induced acute liver injury.

OBJECTIVE: The aim of this study was to evaluate peak serum alanine aminotransferase (ALT) and postoperative clinical outcomes after hypothermic oxygenated machine perfusion (HOPE) versus static cold storage (SCS) in extended criteria donation (ECD) liver transplantation (LT) from donation after brain death (DBD).BACKGROUND: HOPE might improve outcomes in LT, particularly in high-risk settings such as ECD organs after DBD, but this hypothesis has not yet been tested in a randomized controlled clinical trial (RCT).METHODS: Between September 2017 and September 2020, 46 patients undergoing ECD-DBD LT from four centers were randomly assigned to HOPE (n = 23) or SCS (n = 23). Peak-ALT levels within 7 days following LT constituted the primary endpoint. Secondary endpoints included incidence of postoperative complications [Clavien-Dindo classification (CD), Comprehensive Complication Index (CCI)], length of intensive care- (ICU) and hospital-stay, and incidence of early allograft dysfunction (EAD).RESULTS: Demographics were equally distributed between both groups [donor age: 72 (IQR: 59-78) years, recipient age: 62 (IQR: 55-65) years, labMELD: 15 (IQR: 9-25), 38 male and 8 female recipients]. HOPE resulted in a 47% decrease in serum peak ALT [418 (IQR: 221-828) vs 796 (IQR: 477-1195) IU/L, P = 0.030], a significant reduction in 90-day complications [44% vs 74% CD grade ≥3, P = 0.036; 32 (IQR: 12-56) vs 52 (IQR: 35-98) CCI, P = 0.021], and shorter ICU- and hospital-stays [5 (IQR: 4-8) vs 8 (IQR: 5-18) days, P = 0.045; 20 (IQR: 16-27) vs 36 (IQR: 23-62) days, P = 0.002] compared to SCS. A trend toward reduced EAD was observed for HOPE (17% vs 35%; P = 0.314).CONCLUSION: This multicenter RCT demonstrates that HOPE, in comparison to SCS, significantly reduces early allograft injury and improves post-transplant outcomes in ECD-DBD liver transplantation.

BACKGROUND & AIMS: Sulfoconjugation of small molecules or protein peptides is a key mechanism to ensure biochemical and functional homeostasis in mammals. The PAPS synthase 2 (PAPSS2) is the primary enzyme to synthesize the universal sulfonate donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS). Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF), in which oxidative stress is a key pathogenic event, whereas sulfation of APAP contributes to its detoxification. The goal of this study is to determine whether and how PAPSS2 plays a role in APAP-induced ALF. METHODS: Gene expression was analyzed in APAP-induced ALF in patients and mice. Liver-specific Papss2 knockout mice using Alb-Cre (Papss2(ΔHC)) or AAV8-TBG-Cre (Papss2(iΔHC)) were created and subjected to APAP-induced ALF. Primary human and mouse hepatocytes were used for in vitro mechanistic analysis. RESULTS: The hepatic expression of PAPSS2 was decreased in APAP-induced ALF in patients and mice. Surprisingly, Papss2(ΔHC) mice were protected from APAP-induced hepatotoxicity despite having a decreased APAP sulfation, which was accompanied by increased hepatic antioxidative capacity through the activation of the p53-p2-Nrf2 axis. Treatment with a sulfation inhibitor also ameliorated APAP-induced hepatotoxicity. Gene knockdown experiments showed that the hepatoprotective effect of Papss2(ΔHC) was Nrf2-, p53- and p21-dependent. Mechanistically, we identified p53 as a novel substrate of sulfation. Papss2 ablation led to p53 protein accumulation by preventing p53 sulfation, which disrupts p53-MDM2 interaction and p53 ubiquitination, and increases p53 protein stability. CONCLUSIONS: We have uncovered a previously unrecognized and p53-mediated role of PAPSS2 in controlling oxidative response. Inhibition of p53 sulfation may be explored for the clinical management of APAP overdose.

Insufficient liver regeneration causes post-hepatectomy liver failure and small-for-size syndrome. Identifying therapeutic targets to enhance hepatic regenerative capacity remains urgent. Recently, increased IL-33 was observed in patients undergoing liver resection and in mice after partial hepatectomy (PHx). The present study aims to investigate the role of IL-33 in liver regeneration after PHx and to elucidate its underlying mechanisms.We performed PHx in IL-33Our study identified that IL-33 is pro-regenerative in a noninjurious model of liver resection. The underlying mechanism involved IL-33/ST2-induced increase of serotonin release from enterochromaffin cells to portal blood and subsequent HTR2A/p70S6K activation in hepatocytes by serotonin. The findings implicate the potential of targeting the IL-33/ST2/serotonin pathway to reduce the risk of post-hepatectomy liver failure and small-for-size syndrome.

Liver contains high frequency of group 1 innate lymphoid cells (ILC), which are composed of comparable number of type 1 ILC (ILC1) and natural killer (NK) cells in steady state. Little is known about whether and how the interaction between ILC1 and NK cells affects the development of alcoholic liver disease.A mouse model of chronic alcohol abuse plus single-binge (Gao-Binge model) was established. The levels of alanine aminotransferase/aspartate aminotransferase, hepatic lipid, and inflammatory cytokines or neutrophils were measured to evaluate the degree of liver injury, steatosis, and inflammation. Flow cytometric analysis, cell depletion, or adoptive transfer were used to interrogate the interaction between ILC1 and NK cells.Upon chronic alcohol consumption, NK cells, but not ILC1, underwent apoptosis, resulting in ILC1 dominance among group 1 ILC. Interleukin (IL) 17A expression was up-regulated, and increased IL17A was mainly derived from liver ILC1 after chronic alcohol feeding. Either depletion of ILC1 or neutralization of IL17A could significantly attenuate liver steatosis, inflammation, and injury in alcohol-fed mice. In contrast, normalization of the ILC1/NK cells ratio through NK cells transfer or expanding NK cells had a significant hepatoprotection against alcohol-induced steatohepatitis. Furthermore, NK cell-derived interferon gamma exerted a protective function via inhibiting IL17A production by liver ILC1 during alcoholic steatohepatitis.This is the first study showing that the interplay between liver ILC1 and NK cells occurs and drives the development of alcoholic steatohepatitis. Our findings support further exploration of liver ILC1 or NK cells as a therapeutic target for the treatment of alcohol-associated liver disease.

A better understanding of the underlying mechanism of acetaminophen (APAP)-induced liver injury (AILI) remains an important endeavor to develop therapeutic approaches. Eosinophils have been detected in liver biopsies of patients with APAP overdose. We recently demonstrated a profound protective role of eosinophils against AILI; however, the molecular mechanism had not been elucidated.In agreement with our previous data from experiments using genetic deletion of eosinophils, we found that depletion of eosinophils in wild-type (WT) mice by an anti-IL-15 antibody resulted in exacerbated AILI. Moreover, adoptive transfer of eosinophils significantly reduced liver injury and mortality rate in WT mice. Mechanistic studies using eosinophil-specific IL-4/IL-13 knockout mice demonstrated that these cytokines, through inhibiting interferon-γ, mediated the hepatoprotective function of eosinophils. Reverse phase protein array analyses and in vitro experiments using various inhibitors demonstrated that IL-33 stimulation of eosinophils activated p38 mitogen-activated protein kinase (MAPK), and in turn, cyclooxygenases (COX), which triggered NF-κB-mediated IL-4/IL-13 production. In vivo adoptive transfer experiments showed that in contrast to naive eosinophils, those pretreated with COX inhibitors failed to attenuate AILI.The current study revealed that eosinophil-derived IL-4/IL-13 accounted for the hepatoprotective effect of eosinophils during AILI. The data demonstrated that the p38 MAPK/COX/NF-κB signaling cascade played a critical role in inducing IL-4/IL-13 production by eosinophils in response to IL-33.

Macrophages, which are key cellular components of the liver, have emerged as essential players in the maintenance of hepatic homeostasis and in injury and repair processes in acute and chronic liver diseases. Upon liver injury, resident Kupffer cells (KCs) sense disturbances in homeostasis, interact with hepatic cell populations and release chemokines to recruit circulating leukocytes, including monocytes, which subsequently differentiate into monocyte-derived macrophages (MoMϕs) in the liver. Both KCs and MoMϕs contribute to both the progression and resolution of tissue inflammation and injury in various liver diseases. The diversity of hepatic macrophage subsets and their plasticity explain their different functional responses in distinct liver diseases. In this review, we highlight novel findings regarding the origins and functions of hepatic macrophages and discuss the potential of targeting macrophages as a therapeutic strategy for liver disease.

Primary sclerosing cholangitis (PSC) is a progressive fibrosing cholestatic liver disease that is strongly associated with inflammatory bowel disease (IBD). PSC-associated IBD (PSC-IBD) displays a unique phenotype characterized by right-side predominant colon inflammation and increased risk of colorectal cancer compared to non-PSC-IBD. The frequent association and unique phenotype of PSC-IBD suggest distinctive underlying disease mechanisms from other chronic liver diseases or IBD alone. Multidrug resistance protein 2 knockout (Mdr2−/−) mice develop spontaneous cholestatic liver injury and fibrosis mirroring human PSC. As a novel model of PSC-IBD, we treated Mdr2−/− mice with dextran sulfate sodium (DSS) to chemically induce colitis (Mdr2−/−/DSS). Mdr2−/− mice demonstrate alterations in fecal bile acid composition and enhanced colitis susceptibility with increased colonic adhesion molecule expression, particularly mucosal addressin cell adhesion molecule 1 (MAdCAM-1). In vitro, ursodeoxycholic acid (UDCA) co-treatment resulted in a dose dependent attenuation of TNF-α-induced endothelial MAdCAM-1 expression. In the combined Mdr2−/−/DSS model, UDCA supplementation attenuated colitis severity and down-regulated intestinal MAdCAM-1 expression. These findings suggest a potential mechanistic role for alterations in bile acid signaling in modulating MAdCAM-1 expression and colitis susceptibility in cholestasis-associated colitis. Together, our findings provide a novel model and new insight into the pathogenesis and potential treatment of PSC-IBD.

Background and aims 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. Approach and results 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. Conclusions 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.

Kupffer cells (KCs), which are liver-resident macrophages, originate from the fetal yolk sac and represent one of the largest macrophage populations in the body. However, the current data on the origin of the cells that restore macrophages during liver injury and regeneration remain controversial. Here, we address the question of whether liver macrophage restoration results from circulating monocyte infiltration or local KC proliferation in regenerating livers after partial hepatectomy (PHx) and uncover the underlying mechanisms. By using several strains of genetically modified mice and performing immunohistochemical analyses, we demonstrated that local KC proliferation mainly contributed to the restoration of liver macrophages after PHx. Peak KC proliferation was impaired in Il6-knockout (KO) mice and restored after the administration of IL-6 protein, whereas KC proliferation was not affected in Il4-KO or Csf2-KO mice. The source of IL-6 was identified using hepatocyte- and myeloid-specific Il6-KO mice and the results revealed that both hepatocytes and myeloid cells contribute to IL-6 production after PHx. Moreover, peak KC proliferation was also impaired in myeloid-specific Il6 receptor-KO mice after PHx, suggesting that IL-6 signaling directly promotes KC proliferation. Studies using several inhibitors to block the IL-6 signaling pathway revealed that sirtuin 1 (SIRT1) contributed to IL-6-mediated KC proliferation in vitro. Genetic deletion of the Sirt1 gene in myeloid cells, including KCs, impaired KC proliferation after PHx. In conclusion, our data suggest that KC repopulation after PHx is mainly driven by local KC proliferation, which is dependent on IL-6 and SIRT1 activation in KCs.

Background: Macrophage migration inhibitory factor (MIF) is an important stress-regulating mediator of acute ischemia/reperfusion (I/R) injury and ischemic conditioning. The present study aimed to investigate whether MIF is involved in the effects of remote ischemic conditioning (RIC) in a rat model of orthotopic liver transplantation (OLT). Methods: OLTs were performed in male Lewis rats (245 g-340 g). Recipients were allocated in a randomized fashion into three experimental groups: remote preconditioning-RIPC, remote post-conditioning-RIPOST, control. RIC was applied as 4x5-5 min I/R via clamping of the infrarenal aorta. Animals were followed for 1, 3, 24, 168 h post-reperfusion (n = 6 recipient/group/time point). Graft micro- and macrocirculation and hepatocellular damage were assessed. Messenger ribonucleic acid (mRNA) expression, serum, and tissue protein levels of MIF, as well as additional markers of I/R injury, were measured. Results: RIC resulted in a prominent downregulation of MIF mRNA, serum, and tissue protein. Compared with control, hepatocellular damage was significantly mitigated after RIPC or RIPOST (serum ALT; RIPC, RIPOST vs. Control, P = 0.008, P = 0.030, respectively). Graft circulation was better preserved in the RIC groups. Furthermore, there was a significant positive correlation between serum MIF and transaminase levels (r = 0.330; P = 0.02). RIC showed a significant effect on iNOS and STAT5 mRNA expressions. Supporting findings were obtained from the measurements of tissue CXCL12 mRNA expression and pAkt/Akt, pErk/Erk. Conclusion: In this sophisticated experimental model of OLT, RIC-induced hepatoprotective effects were associated with a downregulation of MIF at mRNA and protein levels, suggesting the role of MIF as a mediator in RIC-induced protection following OLT.

Non-small cell lung cancer remains a highly lethal malignancy. Using the tamoxifen inducible Hnf1b:CreERT2 (H) transgenic mouse crossed to the LsL-KrasG12D (K) transgenic mouse, we recently discovered that an Hnf1b positive cell type in the lung is sensitive to adenoma formation when expressing a mutant KrasG12D allele. In these mice, we observe adenoma formation over a time frame of three to six months. To study specificity of the inducible Hnf1b:CreERT2 in the lung, we employed lineage tracing using an mTmG (G) reporter allele. This technique revealed recombined, GFP+ cells were predominantly SPC+. We further employed this technique in HKG mice to determine Hnf1b+ cells give rise to adenomas that express SPC and TTF1. Review of murine lung tissue confirmed a diagnosis of adenoma and early adenocarcinoma, a pathologic subtype of non-small cell lung cancer. Our expanded mouse model revealed loss of Mst1/2 promotes aggressive lung adenocarcinoma and large-scale proteomic analysis revealed upregulation of PKM2 in the lungs of mice with genetic deletion of Mst1/2. PKM2 is a known metabolic regulator in proliferating cells and cancer. Using a human lung adenocarcinoma cell line, we show pharmacologic inhibition of Mst1/2 increases the abundance of PKM2, indicating genetic loss or pharmacologic inhibition of Mst1/2 directly modulates the abundance of PKM2. In conclusion, here we report a novel model of non-small cell lung cancer driven by a mutation in Kras and deletion of Mst1/2 kinases. Tumor development is restricted to a subset of alveolar type II cells expressing Hnf1b. Our data show loss of Mst1/2 regulates levels of a potent metabolic regulator, PKM2.

Alcoholic liver disease (ALD) is a significant health hazard and economic burden affecting approximately 10 million people in the United States. ALD stems from the production of toxic-reactive metabolites, oxidative stress and fat accumulation in hepatocytes which ultimately results in hepatocyte death promoting hepatitis and fibrosis deposition. Monocyte-derived infiltrating Ly6Chi and Ly6Clow macrophages are instrumental in perpetuating and resolving the hepatitis and fibrosis associated with ALD pathogenesis. In the present study we isolated liver infiltrating macrophages from mice on an ethanol diet and subjected them to metabolomic and proteomic analysis to provide a broad assessment of the cellular metabolite and protein differences between infiltrating macrophage phenotypes. We identified numerous differentially regulated metabolites and proteins between Ly6Chi and Ly6Clow macrophages. Bioinformatic analysis for pathway enrichment of the differentially regulated metabolites showed a significant number of metabolites involved in the processes of glycerophospholipid metabolism, arachidonic acid metabolism and phospholipid biosynthesis. From analysis of the infiltrating macrophage proteome, we observed a significant enrichment in the biological processes of antigen presentation, actin polymerization and organization, phagocytosis and apoptotic regulation. The data presented herein could yield exciting new research avenues for the analysis of signaling pathways regulating macrophage polarization in ALD.

Liver disease causing end organ failure is a growing cause of mortality. In most cases, the only therapy is liver transplantation. However, liver transplantation is a complex undertaking and its success is dependent on a number of factors. In particular, liver transplantation is subject to the risks of ischaemia-reperfusion injury (IRI). Liver IRI has significant effects on the function of a liver after transplantation. The cellular and molecular mechanisms governing IRI in liver transplantation are numerous. They involve multiple cells types such as liver sinusoidal endothelial cells, hepatocytes, Kupffer cells, neutrophils and platelets acting via an interconnected network of molecular pathways such as activation of toll-like receptor signalling, alterations in micro-RNA expression, production of ROS, regulation of autophagy and activation of hypoxia-inducible factors. Interestingly, the cellular and molecular events in liver IRI can be correlated with clinical risk factors for IRI in liver transplantation such as donor organ steatosis, ischaemic times, donor age, and donor and recipient coagulopathy. Thus, understanding the relationship of the clinical risk factors for liver IRI to the cellular and molecular mechanisms that govern it is critical to higher levels of success after liver transplantation. This in turn will help in the discovery of therapeutics for IRI in liver transplantation - a process that will lead to improved outcomes for patients suffering from end-stage liver disease.

Hepatic ischemia and reperfusion (IR) injury contributes to the morbidity and mortality associated with liver transplantation. microRNAs (miRNAs) constitute a family of noncoding RNAs that regulate gene expression at the posttranslational level through the repression of specific target genes. Here, we hypothesized that miRNAs could be targeted to enhance hepatic ischemia tolerance. A miRNA screen in a murine model of hepatic IR injury pointed us toward the liver-specific miRNA miR122. Subsequent studies in mice with hepatocyte-specific deletion of miR122 (miR122loxP/loxP Alb-Cre+ mice) during hepatic ischemia and reperfusion revealed exacerbated liver injury. Transcriptional studies implicated hypoxia-inducible factor-1α (HIF1α) in the induction of miR122 and identified the oxygen-sensing prolyl hydroxylase domain 1 (PHD1) as a miR122 target. Further studies indicated that HIF1α-dependent induction of miR122 participated in a feed-forward pathway for liver protection via the enhancement of hepatic HIF responses through PHD1 repression. Moreover, pharmacologic studies utilizing nanoparticle-mediated miR122 overexpression demonstrated attenuated liver injury. Finally, proof-of-principle studies in patients undergoing orthotopic liver transplantation showed elevated miR122 levels in conjunction with the repression of PHD1 in post-ischemic liver biopsies. Taken together, the present findings provide molecular insight into the functional role of miR122 in enhancing hepatic ischemia tolerance and suggest the potential utility of pharmacologic interventions targeting miR122 to dampen hepatic injury during liver transplantation. ispartof: JOURNAL OF CLINICAL INVESTIGATION vol:131 issue:7 ispartof: location:United States status: published

Eosinophils are a myeloid cell subpopulation that mediates type 2 T helper cell immune responses. Unexpectedly, we identified a rapid accumulation of eosinophils in 22 human liver grafts after hepatic transplantation. In contrast, no eosinophils were detectable in healthy liver tissues before transplantation. Studies with two genetic mouse models of eosinophil deficiency and a mouse model of antibody-mediated eosinophil depletion revealed exacerbated liver injury after hepatic ischemia and reperfusion. Adoptive transfer of bone marrow-derived eosinophils normalized liver injury of eosinophil-deficient mice and reduced hepatic ischemia and reperfusion injury in wild-type mice. Mechanistic studies combining genetic and adoptive transfer approaches identified a critical role of suppression of tumorigenicity (ST2)-dependent production of interleukin-13 by eosinophils in the hepatoprotection against ischemia-reperfusion-induced injury. Together, these data provide insight into a mechanism of eosinophil-mediated liver protection that could serve as a therapeutic target to improve outcomes of patients undergoing liver transplantation.

On November 18th, 2016 the 21st annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held at the Center for Translational Research and Education at Loyola University Chicago’s Health Sciences Campus in Maywood, IL. The 2016 meeting focused broadly on alcohol and inflammation, epigenetics, and the microbiome. The four plenary sessions of the meeting were: Alcohol, Inflammation, and Immunity; Alcohol and Epigenetics; Alcohol, Transcriptional Regulation, and Epigenetics; and Alcohol, Intestinal Mucosa, and the Gut Microbiome. Presentations in all sessions of the meeting explored putative underlying causes for chronic diseases and mortality associated with alcohol consumption, shedding light on future work and potential therapeutic targets to alleviate the negative effects of alcohol misuse.

Hepatic macrophages (MΦs) are important in the development and progression of alcoholic liver disease (ALD). This study investigates the role of gp91phox (nicotinamide adenine dinucleotide phosphate oxidase 2) in the severity of ALD and specifically in regulating hepatic MΦ efferocytic capability and the subsequent reprogramming associated with resolution of inflammation. After 4 weeks of ethanol feeding, more severe ALD developed in gp91phox-/- mice than in wild-type (WT) C57Bl/6J mice, evidenced by increased liver injury and inflammation. This phenomenon was not sex dependent, and thus the majority of experiments were performed with female mice. While total hepatic MΦ numbers did not differ between genotypes, hepatic infiltrating MΦs (IMs) were slightly more numerous in gp91phox-/- mice, and both IMs and resident Kupffer cells displayed enhanced proinflammatory and reduced tissue-restorative programming compared with these cells from WT mice. The ratio of proinflammatory IMs with higher expression of Ly6C (Ly6Chi) to anti-inflammatory IMs with lower expression of Ly6C (Ly6Clow) was significantly higher in gp91phox-/- mice compared to WT mice. Greater numbers of apoptotic cells accumulated in the liver of gp91phox-/- mice compared to WT mice, and receptors for binding and engulfing apoptotic cells were expressed at much lower levels on both Kupffer cells and IMs of gp91phox-/- mice. Interactions with apoptotic cells (binding and engulfment) in vitro were significantly fewer for gp91phox-/- MΦs than for WT MΦs, resulting in diminished expression of tissue restorative mediators by hepatic MΦs of gp91phox-/- mice. Conclusion: gp91phox plays a critical role in the differentiation of proinflammatory hepatic MΦs to a tissue-restorative phenotype, likely through programming for efferocytosis, and thereby lessens the severity of ALD. These findings enhance our understanding of the tissue environmental cues that regulate MΦ phenotypes. This knowledge could help in designing MΦ-targeting strategies to prevent and treat ALD. (Hepatology Communications 2017;1:765-779).

RNA virus invasion induces a cytosolic RIG-I-like receptor (RLR) signaling pathway by promoting assembly of the Mitochondrial antiviral-signaling protein (MAVS) signalosome and triggers the rapid production of type I interferons (IFNs) and proinflammatory cytokines. During this process, the pivotal kinase TANK binding kinase 1 (TBK1) is recruited to the MAVS signalosome to transduce a robust innate antiviral immune response by phosphorylating transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor (NF)-&kappa, B and promoting their nuclear translocation. However, the molecular mechanisms underlying the negative regulation of TBK1 are largely unknown. In the present study, we found that THO complex subunit 7 homolog (THOC7) negatively regulated the cellular antiviral response by promoting the proteasomal degradation of TBK1. THOC7 overexpression potently inhibited Sendai virus- or polyI:C-induced IRF3 dimerization and phosphorylation and IFN-&beta, production. In contrast, THOC7 knockdown had the opposite effects. Moreover, we simulated a node-activated pathway to show that THOC7 regulated the RIG-I-like receptors (RLR)-/MAVS-dependent signaling cascade at the TBK1 level. Furthermore, THOC7 was involved in the MAVS signalosome and promoted TBK1 degradation by increasing its K48 ubiquitin-associated polyubiquitination. Together, these findings suggest that THOC7 negatively regulates type I IFN production by promoting TBK1 proteasomal degradation, thus improving our understanding of innate antiviral immune responses.

Clinical studies have demonstrated a strong association between both acute toxic exposure and the repetitive, chronic exposure to acetaminophen (APAP) with pulmonary dysfunction. However, the mechanisms underlying this association are unknown. Preclinical reports have demonstrated that significant bronchiolar injury occurs with toxic APAP exposure, but very little information exists on how the distal lung is affected. However, cells in the alveolar space, including the pulmonary epithelium and resident macrophages, express the APAP-metabolizing enzyme CYP2E1 and are a potential source of toxic metabolites and subsequent distal lung injury. Thus, we hypothesized that distal lung injury would occur in a murine model of toxic APAP exposure. Following exposure of APAP (280 mg/kg, IP), adult male mice were found to have significant proximal lung histopathology as well as distal lung inflammation and emphysematous changes. Toxic APAP exposure was associated with increased CYP2E1 expression in the distal lung and accumulation of APAP-protein adducts. This injury was associated with distal lung activation of oxidant stress, endoplasmic reticulum stress, and inflammatory stress response pathways. Our findings confirm that following toxic APAP exposure, distal lung CYP2E1 expression is associated with APAP metabolism, tissue injury, and oxidant, inflammatory, and endoplasmic reticulum signaling. This previously unrecognized injury may help improve our understanding of the relationship between APAP and pulmonary-related morbidity.

Interferon regulatory factor 7 (IRF7), a crucial regulator of type I interferons (IFNs), plays a crucial role in resistance to viral infection. The abnormal production of type I IFNs is associated with many types of disease, such as cancer and inflammatory disorders. Thus, understanding the post-translational modifications of IRF7 is essential to promoting an appropriate immune response. We have recently showed that the TAR RNA binding protein 2 (TARBP2) suppresses IFN-β production and the innate antiviral response by targeting MAVS. Here, we further identified TARBP2 as a novel inhibitor of IRF7, which inhibits IRF7-mediated IFN-β production triggered by the Sendai virus in 293 T cells. Overexpression of TARBP2 inhibits the phosphorylation as well as the K63-linked ubiquitination of IRF7, whilst TARBP2 also impairs the stability of endogenous TRAF6. Furthermore, TARBP2 participates in the interaction between IRF7 and TRAF6, thereby suppressing TRAF6-mediated K63-linked ubiquitination of IRF7, which is a prerequisite of IRF7 phosphorylation. Our findings further reveal the mechanism by which TARBP2 regulates the antiviral signaling pathways of the innate immune system.

Currently there are very few pharmacological options available to treat acute liver injury. Because its natural exposure to noxious stimuli the liver has developed a strong endogenous hepatoprotective capacity. Indeed, experimental evidence exposed a variety of endogenous hepatic and systemic responses naturally activated to protect the hepatic parenchyma and to foster liver regeneration, therefore preserving individual’s survival. The fibroblast growth factor (FGF) family encompasses a range of polypeptides with important effects on cellular differentiation, growth survival and metabolic regulation in adult organisms. Among these FGFs, FGF19 and FGF21 are endocrine hormones that profoundly influence systemic metabolism but also exert important hepatoprotective activities. In this review, we revisit the biology of these factors and highlight their potential application for the clinical management of acute liver injury

After traumatic injury, some cells function as detectors to sense injury and to modulate the local immune response toward a restitution phase by affecting the local cytokine milieu. Using intravital microscopy, we observed that patrolling invariant natural killer T (iNKT) cells were initially excluded from a site of hepatic injury but subsequently were strategically arrested first via self-antigens and then by cytokines, circumscribing the injured site at exactly the location where monocytes co-localized and hepatocytes proliferated. Activation of iNKT cells by self-antigens resulted in the production of interleukin-4 (IL-4) but not interferon-γ (IFN-γ). This promoted increased hepatocyte proliferation, monocyte transition (from Ly6C

Hepatic macrophages (MΦs) are important in the development and progression of alcoholic liver disease (ALD). This study investigates the role of gp91phox (nicotinamide adenine dinucleotide phosphate oxidase 2) in the severity of ALD and specifically in regulating hepatic MΦ efferocytic capability and the subsequent reprogramming associated with resolution of inflammation. After 4 weeks of ethanol feeding, more severe ALD developed in gp91phox−/− mice than in wild‐type (WT) C57Bl/6J mice, evidenced by increased liver injury and inflammation. This phenomenon was not sex dependent, and thus the majority of experiments were performed with female mice. While total hepatic MΦ numbers did not differ between genotypes, hepatic infiltrating MΦs (IMs) were slightly more numerous in gp91phox−/− mice, and both IMs and resident Kupffer cells displayed enhanced proinflammatory and reduced tissue‐restorative programming compared with these cells from WT mice. The ratio of proinflammatory IMs with higher expression of Ly6C (Ly6Chi) to anti‐inflammatory IMs with lower expression of Ly6C (Ly6Clow) was significantly higher in gp91phox−/− mice compared to WT mice. Greater numbers of apoptotic cells accumulated in the liver of gp91phox−/− mice compared to WT mice, and receptors for binding and engulfing apoptotic cells were expressed at much lower levels on both Kupffer cells and IMs of gp91phox−/− mice. Interactions with apoptotic cells (binding and engulfment) in vitro were significantly fewer for gp91phox−/− MΦs than for WT MΦs, resulting in diminished expression of tissue restorative mediators by hepatic MΦs of gp91phox−/− mice. Conclusion: gp91phox plays a critical role in the differentiation of proinflammatory hepatic MΦs to a tissue‐restorative phenotype, likely through programming for efferocytosis, and thereby lessens the severity of ALD. These findings enhance our understanding of the tissue environmental cues that regulate MΦ phenotypes. This knowledge could help in designing MΦ‐targeting strategies to prevent and treat ALD. (Hepatology Communications 2017;1:765–779)

Macrophages are key effectors of innate immune response to pathogens and the initiation of inflammation, and they contribute to maintaining tissue homeostasis, tissue repair, and development (Wynn et al. 2013b). Macrophages also help shape the adaptive immune response through the presentation of foreign antigens and production of chemokines which facilitate the trafficking of adaptive immune cells to sights of inflammation and tissue damage (Newson et al. 2014). Macrophages are classified as either tissue resident (i.e., liver Kupffer cells, brain microglia, and alveolar macrophages in the lung) or recruited (monocyte−/bone marrow-derived macrophages). In mice, monocytes are subdivided based on their expression of Ly6C and CCR2. Ly6Chi CCR2+ monocytes rapidly infiltrate tissue upon injury (Karlmark et al. 2009). During acute or chronic liver injury, Ly6Chi monocyte-derived macrophages are the predominate macrophage subtype (Tacke and Randolph 2006; Karlmark et al. 2009). Ly6ClowCCR2− monocytes have been shown to exhibit a “patrolling” behavior and have been found crawling along the hepatic endothelium (Carlin et al. 2013). While monocyte-derived Ly6Chi initially exert tissue-destructive properties, they can differentiate into Ly6Clow monocyte-derived macrophages which promote tissue repair and resolution of inflammation (Dal-Secco et al. 2015). The human monocyte counterparts are classified as classical (CD14hiCD16−), intermediate (CD14+CD16+), and nonclassical (CD14dimCD16+) (Ingersoll et al. 2010). Mouse and human monocytes share much similarity but differ in several aspects that must be taken into consideration when translating mouse and human studies. For instance, based on receptor expression CD16− and Ly6Chi, monocytes highly express CCR2, whereas CD16hi and Ly6Clo monocytes express high levels of CX3CR1 (Geissmann et al. 2003; Tacke et al. 2007). However, in regard to FcγR1 expression, mRNA and protein expression is conserved between CD16− and Ly6Chi monocytes, whereas human CD16+ monocytes lose FcγR1 surface protein expression which is retained on Ly6Clow monocytes (Ingersoll et al. 2010). Furthermore, Ly6Clow monocytes are more instrumental in phagocytosis, whereas in humans, CD16− monocytes are more phagocytic (Wildgruber et al. 2009; Tacke et al. 2006). Macrophages display a remarkable ability to alter their phenotype based on tissue microenvironmental cues such as hypoxia, cytokines, lipids, and exposure to dead cells. In acute and chronic liver diseases, various subsets of hepatic macrophages have been found to be involved in disease pathogenesis. In this review, we will summarize the development, heterogeneity, and plasticity of macrophages in response to microenvironmental stimuli in physiological and pathophysiological conditions of the liver.

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.

Type 2 diabetes mellitus (T2DM) progresses from compensated insulin resistance to beta cell failure resulting in uncompensated hyperglycemia, a process replicated in the Zucker diabetic fatty (ZDF) rat. The Nlrp3 inflammasome has been implicated in obesity-induced insulin resistance and beta cell failure. Endocannabinoids contribute to insulin resistance through activation of peripheral CB1 receptors (CB₁Rs) and also promote beta cell failure. Here we show that beta cell failure in adult ZDF rats is not associated with CB₁R signaling in beta cells, but rather in M1 macrophages infiltrating into pancreatic islets, and that this leads to activation of the Nlrp3-ASC inflammasome in the macrophages. These effects are replicated in vitro by incubating wild-type human or rodent macrophages, but not macrophages from CB₁R-deficient (Cnr1(-/-)) or Nlrp3(-/-) mice, with the endocannabinoid anandamide. Peripheral CB₁R blockade, in vivo depletion of macrophages or macrophage-specific knockdown of CB₁R reverses or prevents these changes and restores normoglycemia and glucose-induced insulin secretion. These findings implicate endocannabinoids and inflammasome activation in beta cell failure and identify macrophage-expressed CB₁R as a therapeutic target in T2DM.

Evidence suggests that bio-activation of drugs to generate chemically reactive metabolites (RM) that act as haptens to form immunogenic protein conjugates may be an important cause of immune-mediated drug hypersensitivity reactions (IDHR). Although many drugs that form RMs raise concerns about producing IDHR, standard non-clinical testing methods are rarely able to identify compounds with the potential to produce IDHR in humans. The objective of this study was to develop a predictive assay for IDHR that involves: (1) the use of an in vitro drug-metabolizing system to generate the RM that is captured by GSH, (2) conjugating the RM-GSH conjugate to mouse serum albumin (MSA) by using a chemical cross-linker, (3) immunization of mice with RM-GSH-MSA adducts, and (4) ex vivo challenge with RM-GSH-MSA adduct and measurement of lymphocyte proliferation to determine if the RM is immunogenic. The predictivity of the assay was evaluated by using drugs that produce RM and have been strongly, weakly, or not associated with IDHRs in the clinic. While this method requires additional validation with more drugs, the results demonstrate the feasibility of identifying drugs strongly associated with IDHR and the utility of the assay for rank ordering drugs with respect to their potential to cause IDHR.

Alcohol consumption can lead to the increase in gut permeability and cause the translocation of bacteria-derived lipopolysaccharides from the gut to the liver, which subsequently activates immune responses. In this process, macrophages play a critical role and involve in the pathogenesis of alcoholic liver disease (ALD). To define the mechanism underpinning the function of macrophages, it is important to conduct extensive studies to further explicate the phenotypic diversity of macrophages in the context of ALD. In this review, the role of hepatic macrophages in the pathogenesis of ALD is discussed.

Recent studies have suggested that macrophages have a critical role in the development of alcohol-induced inflammation in the liver. To define the precise pathogenic function of these cells during alcoholic liver disease (ALD), it is extremely important to conduct extensive studies in clinical settings that further elucidate the phenotypic diversity of macrophages in the context of ALD. Research to date already has identified several characteristics of macrophages that underlie the cells’ actions, including macrophage polarization and their phenotypic diversity. Other analyses have focused on the contributions of resident versus infiltrating macrophages/monocytes, as well as on the roles of macrophage mediators, in the development of ALD. Findings point to the potential of macrophages as a therapeutic target in alcoholic liver injury. Future studies directed toward understanding how alcohol affects macrophage phenotypic switch in the liver and other tissues, whether the liver microenvironment determines macrophage function in ALD, and if targeting of macrophages alleviates alcoholic liver injury, will provide promising strategies to manage patients with alcoholic hepatitis.