Oleic acid availability impacts thymocyte preprogramming and subsequent peripheral Treg cell differentiation.

The nature of activation signals is essential in determining T cell subset differentiation; however, the features that determine T cell subset preference acquired during intrathymic development remain elusive. Here we show that naive CD4+ T cells generated in the mouse thymic microenvironment lacking Scd1, encoding the enzyme catalyzing oleic acid (OA) production, exhibit enhanced regulatory T (Treg) cell differentiation and attenuated development of experimental autoimmune encephalomyelitis. Scd1 deletion in K14+ thymic epithelia recapitulated the enhanced Treg cell differentiation phenotype of Scd1-deficient mice. The dearth of OA permitted DOT1L to increase H3K79me2 levels at the Atp2a2 locus of thymocytes at the DN2-DN3 transition stage. Such epigenetic modification persisted in naive CD4+ T cells and facilitated Atp2a2 expression. Upon T cell receptor activation, ATP2A2 enhanced the activity of the calcium-NFAT1-Foxp3 axis to promote naive CD4+ T cells to differentiate into Treg cells. Therefore, OA availability is critical for preprogramming thymocytes with Treg cell differentiation propensities in the periphery.

Necrotic liver lesion resolution: another mode of liver regeneration.

The liver has the great ability to regenerate after partial resection or injury, and the mechanisms underlying liver regeneration have been extensively investigated. Interestingly, acute liver injuries triggered by various etiologies are associated with the formation of necrotic lesions, and such necrotic lesions are also rapidly resolved. However, how necrotic liver lesions are repaired has not been carefully investigated until recently. In this review, we briefly summarize the spatiotemporal process of necrotic liver lesion resolution in several liver injury models including immune-mediated liver injury and drug-induced liver injury. The roles of liver non-parenchymal cells and infiltrating immune cells in controlling necrotic liver lesion resolution are discussed, which may help identify potential therapies for acute liver injury and failure.

Macrophages in necrotic liver lesion repair: opportunities for therapeutical applications.

Healthy livers contain 80% of body resident macrophages known as Kupffer cells. In diseased livers, the number of Kupffer cells usually drops but is compensated by infiltration of monocyte-derived macrophages, some of which can differentiate into Kupffer-like cells. Early studies suggest that Kupffer cells play important roles in both promoting liver injury and liver regeneration. Yet, the distinction between the functionalities of resident and infiltrating macrophages is not always made. By using more specific macrophage markers and targeted cell depletion and single-cell RNA sequencing, recent studies revealed several subsets of monocyte-derived macrophages that play important functions in inducing liver damage and inflammation as well as in liver repair and regeneration. In this review, we discuss the different roles that hepatic macrophages play in promoting necrotic liver lesion resolution and dead cell clearance, as well as the targeting of these cells as potential tools for the development of novel therapies for acute liver failure and acute-on-chronic liver failure.

Targeting hepatic serine-arginine protein kinase 2 ameliorates alcohol-associated liver disease by alternative splicing control of lipogenesis.

BACKGROUND AND AIMS: Lipid accumulation induced by alcohol consumption is not only an early pathophysiological response but also a prerequisite for the progression of alcohol-associated liver disease (ALD). Alternative splicing regulates gene expression and protein diversity; dysregulation of this process is implicated in human liver diseases. However, how the alternative splicing regulation of lipid metabolism contributes to the pathogenesis of ALD remains undefined. APPROACH AND RESULTS: Serine-arginine-rich protein kinase 2 (SRPK2), a key kinase controlling alternative splicing, is activated in hepatocytes in response to alcohol, in mice with chronic-plus-binge alcohol feeding, and in patients with ALD. Such induction activates sterol regulatory element-binding protein 1 and promotes lipogenesis in ALD. Overexpression of FGF21 in transgenic mice abolishes alcohol-mediated induction of SRPK2 and its associated steatosis, lipotoxicity, and inflammation; these alcohol-induced pathologies are exacerbated in FGF21 knockout mice. Mechanistically, SRPK2 is required for alcohol-mediated impairment of serine-arginine splicing factor 10, which generates exon 7 inclusion in lipin 1 and triggers concurrent induction of lipogenic regulators-lipin 1ß and sterol regulatory element-binding protein 1. FGF21 suppresses alcohol-induced SRPK2 accumulation through mammalian target of rapamycin complex 1 inhibition-dependent degradation of SRPK2. Silencing SRPK2 rescues alcohol-induced splicing dysregulation and liver injury in FGF21 knockout mice. CONCLUSIONS: These studies reveal that (1) the regulation of alternative splicing by SRPK2 is implicated in lipogenesis in humans with ALD; (2) FGF21 is a key hepatokine that ameliorates ALD pathologies largely by inhibiting SRPK2; and (3) targeting SRPK2 signaling by FGF21 may offer potential therapeutic approaches to combat ALD.

MicroRNA-223 attenuates hepatocarcinogenesis by blocking hypoxia-driven angiogenesis and immunosuppression.

OBJECTIVE: The current treatment for hepatocellular carcinoma (HCC) to block angiogenesis and immunosuppression provides some benefits only for a subset of patients with HCC, thus optimised therapeutic regimens are unmet needs, which require a thorough understanding of the underlying mechanisms by which tumour cells orchestrate an inflamed tumour microenvironment with significant myeloid cell infiltration. MicroRNA-223 (miR-223) is highly expressed in myeloid cells but its role in regulating tumour microenvironment remains unknown. DESIGN: Wild-type and miR-223 knockout mice were subjected to two mouse models of inflammation-associated HCC induced by injection of diethylnitrosamine (DEN) or orthotopic HCC cell implantation in chronic carbon tetrachloride (CCl4)-treated mice. RESULTS: Genetic deletion of miR-223 markedly exacerbated tumourigenesis in inflammation-associated HCC. Compared with wild-type mice, miR-223 knockout mice had more infiltrated programmed cell death 1 (PD-1+) T cells and programmed cell death ligand 1 (PD-L1+) macrophages after DEN+CCl4 administration. Bioinformatic analyses of RNA sequencing data revealed a strong correlation between miR-223 levels and tumour hypoxia, a condition that is well-documented to regulate PD-1/PD-L1. In vivo and in vitro mechanistic studies demonstrated that miR-223 did not directly target PD-1 and PD-L1 in immune cells rather than indirectly downregulated them by modulating tumour microenvironment via the suppression of hypoxia-inducible factor 1α-driven CD39/CD73-adenosine pathway in HCC. Moreover, gene delivery of miR-223 via adenovirus inhibited angiogenesis and hypoxia-mediated PD-1/PD-L1 activation in both HCC models, thereby hindering HCC progression. CONCLUSION: The miR-223 plays a critical role in modulating hypoxia-induced tumour immunosuppression and angiogenesis, which may serve as a novel therapeutic target for HCC.

Inhibition of p53 Sulfoconjugation Prevents Oxidative Hepatotoxicity and Acute Liver Failure.

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 was 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 (Papss2ΔHC) or AAV8-TBG-Cre (Papss2iΔHC) 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, Papss2ΔHC 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 Papss2ΔHC 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.

Aging exaggerates acute-on-chronic alcohol-induced liver injury in mice and humans by inhibiting neutrophilic sirtuin 1-C/EBPα-miRNA-223 axis.

BACKGROUND AND AIMS: Aging exacerbates liver neutrophil infiltration and alcohol-associated liver disease (ALD) in mice and humans, but the underlying mechanisms remain obscure. This study aimed to examine the effect of aging and alcohol consumption on neutrophilic Sirtuin 1 (SIRT1) and microRNA-223 (miR-223), and their contribution to ALD pathogeneses. APPROACH AND RESULTS: Young and aged myeloid-specific Sirt1 knockout mice were subjected to chronic-plus-binge ethanol feeding. Blood samples from healthy controls and patients with chronic alcohol drinking who presented with acute intoxication were analyzed. Neutrophilic Sirt1 and miR-223 expression were down-regulated in aged mice compared with young mice. Deletion of the Sirt1 gene in myeloid cells including neutrophils exacerbated chronic-plus-binge ethanol-induced liver injury and inflammation and down-regulated neutrophilic miR-223 expression. Immunoprecipitation experiments revealed that SIRT1 promoted C/EBPα deacetylation by directly interacting with C/EBPα, a key transcription factor that controls miR-223 biogenesis, and subsequently elevated miR-223 expression in neutrophils. Importantly, down-regulation of SIRT1 and miR-223 expression was also observed in circulating neutrophils from middle-aged and elderly subjects compared with those from young individuals. Chronic alcohol users with acute intoxication had a reduction in neutrophilic SIRT1 expression in young and middle-aged patients, with a greater reduction in the latter group. The neutrophilic SIRT1 expression correlated with neutrophilic miR-223 and serum alanine transaminase levels in those patients. CONCLUSIONS: Aging increases the susceptibility of alcohol-induced liver injury in mice and humans through the down-regulation of the neutrophilic SIRT1-C/EBPα-miR-223 axis, which could be a therapeutic target for the prevention and/or treatment of ALD.

17-Beta Hydroxysteroid Dehydrogenase 13 Deficiency Does Not Protect Mice From Obesogenic Diet Injury.

BACKGROUND AND AIMS: 17-Beta hydroxysteroid dehydrogenase 13 (HSD17B13) is genetically associated with human nonalcoholic fatty liver disease (NAFLD). Inactivating mutations in HSD17B13 protect humans from NAFLD-associated and alcohol-associated liver injury, fibrosis, cirrhosis, and hepatocellular carcinoma, leading to clinical trials of anti-HSD17B13 therapeutic agents in humans. We aimed to study the in vivo function of HSD17B13 using a mouse model. APPROACH AND RESULTS: Single-cell RNA-sequencing and quantitative RT-PCR data revealed that hepatocytes are the main HSD17B13-expressing cells in mice and humans. We compared Hsd17b13 whole-body knockout (KO) mice and wild-type (WT) littermate controls fed regular chow (RC), a high-fat diet (HFD), a Western diet (WD), or the National Institute on Alcohol Abuse and Alcoholism model of alcohol exposure. HFD and WD induced significant weight gain, hepatic steatosis, and inflammation. However, there was no difference between genotypes with regard to body weight, liver weight, hepatic triglycerides (TG), histological inflammatory scores, expression of inflammation-related and fibrosis-related genes, and hepatic retinoid levels. Compared to WT, KO mice on the HFD had hepatic enrichment of most cholesterol esters, monoglycerides, and certain sphingolipid species. Extended feeding with the WD for 10 months led to extensive liver injury, fibrosis, and hepatocellular carcinoma, with no difference between genotypes. Under alcohol exposure, KO and WT mice showed similar hepatic TG and liver enzyme levels. Interestingly, chow-fed KO mice showed significantly higher body and liver weights compared to WT mice, while KO mice on obesogenic diets had a shift toward larger lipid droplets. CONCLUSIONS: Extensive evaluation of Hsd17b13 deficiency in mice under several fatty liver-inducing dietary conditions did not reproduce the protective role of HSD17B13 loss-of-function mutants in human NAFLD. Moreover, mouse Hsd17b13 deficiency induces weight gain under RC. It is crucial to understand interspecies differences prior to leveraging HSD17B13 therapies.

RUNX1 and CBFß-SMMHC transactivate target genes together in abnormal myeloid progenitors for leukemia development.

Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia, which generates a CBFB-MYH11 fusion gene. It is generally considered that CBFß-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1. However, recent findings challenge the RUNX1-repression model for CBFß-SMMHC-mediated leukemogenesis. To definitively address the role of Runx1 in CBFB-MYH11-induced leukemia, we crossed conditional Runx1 knockout mice (Runx1f/f) with conditional Cbfb-MYH11 knockin mice (Cbfb+/56M). On Mx1-Cre activation in hematopoietic cells induced by poly (I:C) injection, all Mx1-CreCbfb+/56M mice developed leukemia in 5 months, whereas no leukemia developed in Runx1f/fMx1-CreCbfb+/56M mice, and this effect was cell autonomous. Importantly, the abnormal myeloid progenitors (AMPs), a leukemia-initiating cell population induced by Cbfb-MYH11 in the bone marrow, decreased and disappeared in Runx1f/fMx1-CreCbfb+/56M mice. RNA-seq analysis of AMP cells showed that genes associated with proliferation, differentiation blockage, and leukemia initiation were differentially expressed between Mx1-CreCbfb+/56M and Runx1f/fMx1-CreCbfb+/56M mice. In addition, with the chromatin immunocleavage sequencing assay, we observed a significant enrichment of RUNX1/CBFß-SMMHC target genes in Runx1f/fMx1-CreCbfb+/56M cells, especially among downregulated genes, suggesting that RUNX1 and CBFß-SMMHC mainly function together as activators of gene expression through direct target gene binding. These data indicate that Runx1 is indispensable for Cbfb-MYH11-induced leukemogenesis by working together with CBFß-SMMHC to regulate critical genes associated with the generation of a functional AMP population.

Hepatic CYP2B10 is highly induced by binge ethanol and contributes to acute-on-chronic alcohol-induced liver injury.

BACKGROUND: The chronic-plus-binge model of ethanol consumption, where chronically (8-week) ethanol-fed mice are gavaged a single dose of ethanol (E8G1), is known to induce steatohepatitis in mice. However, how chronically ethanol-fed mice respond to multiple binges of ethanol remains unknown. METHODS: We extended the E8G1 model to three gavages of ethanol (E8G3) spaced 24 h apart, sacrificed each group 9 h after the final gavage, analyzed liver injury, and examined gene expression changes using microarray analyses in each group to identify mechanisms contributing to liver responses to binge ethanol. RESULTS: Surprisingly, E8G3 treatment induced lower levels of liver injury, steatosis, inflammation, and fibrosis as compared to mice after E8G1 treatment. Microarray analyses identified several pathways that may contribute to the reduced liver injury after E8G3 treatment compared to E8G1 treatment. The gene encoding cytochrome P450 2B10 (Cyp2b10) was one of the top upregulated genes in the E8G1 group and was further upregulated in the E8G3 group, but only moderately induced after chronic ethanol consumption, as confirmed by RT-qPCR and western blot analyses. Genetic disruption of Cyp2b10 worsened liver injury in E8G1 and E8G3 mice with higher blood ethanol levels compared to wild-type control mice, while in vitro experiments revealed that CYP2b10 did not directly promote ethanol metabolism. Metabolomic analyses revealed significant differences in hepatic metabolites from E8G1-treated Cyp2b10 knockout and WT mice, and these metabolic alterations may contribute to the reduced liver injury in Cyp2b10 knockout mice. CONCLUSION: Hepatic Cyp2b10 expression is highly induced after ethanol binge, and such upregulation reduces acute-on-chronic ethanol-induced liver injury via the indirect modification of ethanol metabolism.

Interleukin-20 exacerbates acute hepatitis and bacterial infection by downregulating IκBζ target genes in hepatocytes.

BACKGROUND & AIMS: Interleukin (IL)-20 and IL-22 belong to the IL-10 family. IL-10 is a well-documented anti-inflammatory cytokine while IL-22 is well known for epithelial protection and its antibacterial function, showing great therapeutic potential for organ damage; however, the function of IL-20 remains largely unknown. METHODS: Il20 knockout (Il20-/-) mice and wild-type littermates were generated and injected with Concanavalin A (ConA) and Klebsiella pneumoniae (K.P.) to induce acute hepatitis and bacterial infection, respectively. RESULTS: Il20-/- mice were resistant to acute hepatitis and exhibited selectively elevated levels of the hepatoprotective cytokine IL-6. Such selective inhibition of IL-6 by IL-20 was due to IL-20 targeting hepatocytes that produce high levels of IL-6 but a limited number of other cytokines. Mechanistically, IL-20 upregulated NAD(P)H: quinone oxidoreductase 1 (NQO1) expression and subsequently promoted the protein degradation of transcription factor IκBζ, resulting in selective downregulation of the IκBζ-dependent gene Il6 as well several other IκBζ-dependent genes including lipocalin-2 (Lcn2). Given the important role of IL-6 and LCN2 in limiting bacterial infection, we examined the effect of IL-20 on bacterial infection and found Il20-/- mice were resistant to K.P. infection and exhibited elevated levels of hepatic IκBζ-dependent antibacterial genes. Moreover, IL-20 upregulated hepatic NQO1 by binding to IL-22R1/IL-20R2 and activating ERK/p38MAPK/NRF2 signaling pathways. Finally, the levels of hepatic IL1B, IL20, and IκBζ target genes were elevated, and correlated with each other, in patients with severe alcoholic hepatitis. CONCLUSIONS: IL-20 selectively inhibits hepatic IL-6 production rather than exerting IL-10-like broad anti-inflammatory properties. Unlike IL-22, IL-20 aggravates acute hepatitis and bacterial infection. Thus, anti-IL-20 therapy could be a promising option to control acute hepatitis and bacterial infection. LAY SUMMARY: Several interleukin (IL)-20 family cytokines have been shown to play important roles in controllimg inflammatory responses, infection and tissue damage, but the role of IL-20 remains unclear. Herein, we elucidated the role of IL-20 in liver disease and bacterial infection. We show that IL-20 can aggravate hepatitis and bacterial infection; thus, targeting IL-20 holds promise for the treatment of patients with liver disease.

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

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.

Protective and Detrimental Roles of p38α Mitogen-Activated Protein Kinase in Different Stages of Nonalcoholic Fatty Liver Disease.

BACKGROUND AND AIMS: Neutrophil infiltration is a hallmark of nonalcoholic steatohepatitis (NASH), but how this occurs during the progression from steatosis to NASH remains obscure. Human NASH features hepatic neutrophil infiltration and up-regulation of major neutrophil-recruiting chemokines (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1] and interleukin [IL]-8). However, mice fed a high-fat diet (HFD) only develop fatty liver without significant neutrophil infiltration or elevation of chemokines. The aim of this study was to determine why mice are resistant to NASH development and the involvement of p38 mitogen-activated protein kinase (p38) activated by neutrophil-derived oxidative stress in the pathogenesis of NASH. APPROACH AND RESULTS: Inflamed human hepatocytes attracted neutrophils more effectively than inflamed mouse hepatocytes because of the greater induction of CXCL1 and IL-8 in human hepatocytes. Hepatic overexpression of Cxcl1 and/or IL-8 promoted steatosis-to-NASH progression in HFD-fed mice by inducing liver inflammation, injury, and p38 activation. Pharmacological inhibition of p38α/ß or hepatocyte-specific deletion of p38a (a predominant form in the liver) attenuated liver injury and fibrosis in the HFD+Cxcl1 -induced NASH model that is associated with strong hepatic p38α activation. In contrast, hepatocyte-specific deletion of p38a in HFD-induced fatty liver where p38α activation is relatively weak exacerbated steatosis and liver injury. Mechanistically, weak p38α activation in fatty liver up-regulated the genes involved in fatty acid ß-oxidation through peroxisome proliferator-activated receptor alpha phosphorylation, thereby reducing steatosis. Conversely, strong p38α activation in NASH promoted caspase-3 cleavage, CCAAT-enhancer-binding proteins homologous protein expression, and B cell lymphoma 2 phosphorylation, thereby exacerbating hepatocyte death. CONCLUSIONS: Genetic ablation of hepatic p38a increases simple steatosis but ameliorates oxidative stress-driven NASH, indicating that p38α plays distinct roles depending on the disease stages, which may set the stage for investigating p38α as a therapeutic target for the treatment of NASH.

Interleukin-22 Ameliorates Neutrophil-Driven Nonalcoholic Steatohepatitis Through Multiple Targets.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease encompasses a spectrum of diseases ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. At present, how simple steatosis progresses to NASH remains obscure and effective pharmacological therapies are lacking. Hepatic expression of C-X-C motif chemokine ligand 1 (CXCL1), a key chemokine for neutrophil infiltration (a hallmark of NASH), is highly elevated in NASH patients but not in fatty livers in obese individuals or in high-fat diet (HFD)-fed mice. The aim of this study was to test whether overexpression of CXCL1 itself in the liver can induce NASH in HFD-fed mice and to test the therapeutic potential of IL-22 in this new NASH model. APPROACH AND RESULTS: Overexpression of Cxcl1 in the liver alone promotes steatosis-to-NASH progression in HFD-fed mice by inducing neutrophil infiltration, oxidative stress, and stress kinase (such as apoptosis signal-regulating kinase 1 and p38 mitogen-activated protein kinase) activation. Myeloid cell-specific deletion of the neutrophil cytosolic factor 1 (Ncf1)/p47phox gene, which encodes a component of the NADPH oxidase 2 complex that mediates neutrophil oxidative burst, markedly reduced CXCL1-induced NASH and stress kinase activation in HFD-fed mice. Treatment with interleukin (IL)-22, a cytokine with multiple targets, ameliorated CXCL1/HFD-induced NASH or methionine-choline deficient diet-induced NASH in mice. Mechanistically, IL-22 blocked hepatic oxidative stress and its associated stress kinases via the induction of metallothionein, one of the most potent antioxidant proteins. Moreover, although it does not target immune cells, IL-22 treatment attenuated the inflammatory functions of hepatocyte-derived, mitochondrial DNA-enriched extracellular vesicles, thereby suppressing liver inflammation in NASH. CONCLUSIONS: Hepatic overexpression of CXCL1 is sufficient to drive steatosis-to-NASH progression in HFD-fed mice through neutrophil-derived reactive oxygen species and activation of stress kinases, which can be reversed by IL-22 treatment via the induction of metallothionein.

An Open-Label, Dose-Escalation Study to Assess the Safety and Efficacy of IL-22 Agonist F-652 in Patients With Alcohol-associated Hepatitis.

BACKGROUND AND AIMS: Interleukin-22 has beneficial effects on inflammation and impaired hepatic regeneration that characterize alcohol-associated hepatitis (AH). F-652 is a recombinant fusion protein of human interleukin-22 and immunoglobulin G2 fragment crystallizable. This study aims to assess the safety and efficacy signals of F-652 in patients with moderate and severe AH. APPROACH AND RESULTS: A phase-2 dose-escalating study was carried out. F-652 (10 µg/kg, 30 µg/kg, or 45 µg/kg) administered on days 1 and 7 was tested in 3 patients each with moderate (Model for End-Stage Liver Disease [MELD] scores: 11-20) and severe AH (MELD scores: 21-28). Safety was defined by absence of serious adverse events and efficacy was assessed by Lille score, changes in MELD score, and serum bilirubin and aminotransferases at days 28 and 42. Three independent propensity-matched comparator patient cohorts were used. Plasma extracellular vesicles and multiplex serum cytokines were measured to assess inflammation and hepatic regeneration. Eighteen patients (9 moderate and 9 severe AH) were enrolled, 66% were male, and the mean age was 48 years. The half-life of F-652 following the first dose was 61-85 hours. There were no serious adverse events leading to discontinuation. The MELD score and serum aminotransferases decreased significantly at days 28 and 42 from baseline (P < 0.05). Day-7 Lille score was 0.45 or less in 83% patients as compared with 6%, 12%, and 56% among the comparator cohorts. Extracellular vesicle counts decreased significantly at day 28 (P < 0.013). Cytokine inflammatory markers were down-regulated, and regeneration markers were up-regulated at days 28 and 42. CONCLUSIONS: F-652 is safe in doses up to 45 µg/kg and associated with a high rate of improvement as determined by Lille and MELD scores, reductions in markers of inflammation and increases in markers of hepatic regeneration. This study supports the need for randomized placebo-controlled trials to test the efficacy of F-652 in AH.

Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in mice.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is a clinical syndrome defined by liver failure on pre-existing chronic liver disease. It is often associated with bacterial infection and high short-term mortality. Experimental models that fully reproduce ACLF are lacking, so too are effective pharmacological therapies for this condition. METHODS: To mimic ACLF conditions, we developed a severe liver injury model by combining chronic injury (chronic carbon tetrachloride [CCl4] injection), acute hepatic insult (injection of a double dose of CCl4), and bacterial infection (intraperitoneal injection of bacteria). Serum and liver samples from patients with ACLF or acute drug-induced liver injury (DILI) were used. Liver injury and regeneration were assessed to ascertain the potential benefits of interleukin-22 (IL-22Fc) administration. RESULTS: This severe liver injury model recapitulated some of the key features of clinical ACLF, including acute-on-chronic liver injury, bacterial infection, multi-organ injury, and high mortality. Liver regeneration in this model was severely impaired because of a shift from the activation of the pro-regenerative IL-6/STAT3 pathway to the anti-regenerative IFN-γ/STAT1 pathway. The impaired IL-6/STAT3 activation was due to the inability of Kupffer cells to produce IL-6; whereas the enhanced STAT1 activation was due to a strong innate immune response and subsequent production of IFN-γ. Compared to patients with DILI, patients with ACLF had higher levels of IFN-γ but lower liver regeneration. IL-22Fc treatment improved survival in ACLF mice by reversing the STAT1/STAT3 pathway imbalance and enhancing expression of many antibacterial genes in a manner involving the anti-apoptotic protein BCL2. CONCLUSIONS: Acute-on-chronic liver injury or bacterial infection is associated with impaired liver regeneration due to a shift from a pro-regenerative to an anti-regenerative pathway. IL-22Fc therapy reverses this shift and attenuates bacterial infection, thus IL-22Fc may have therapeutic potential for ACLF treatment. LAY SUMMARY: A mouse model combining chronic liver injury, acute hepatic insult, and bacterial infection recapitulates some of the key features of acute-on-chronic liver failure (ACLF) in patients. Both fibrosis and bacterial infection contribute to the impaired regenerative capacity of the liver in patients with ACLF. Herein, we show that IL-22Fc therapy improves ACLF by reprogramming impaired regenerative pathways and attenuating bacterial infection. Thus, it may have therapeutic potential for patients with ACLF.

MicroRNA-223 Ameliorates Nonalcoholic Steatohepatitis and Cancer by Targeting Multiple Inflammatory and Oncogenic Genes in Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of diseases ranging from simple steatosis to more severe forms of liver injury including nonalcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC). In humans, only 20%-40% of patients with fatty liver progress to NASH, and mice fed a high-fat diet (HFD) develop fatty liver but are resistant to NASH development. To understand how simple steatosis progresses to NASH, we examined hepatic expression of anti-inflammatory microRNA-223 (miR-223) and found that this miRNA was highly elevated in hepatocytes in HFD-fed mice and in human NASH samples. Genetic deletion of miR-223 induced a full spectrum of NAFLD in long-term HFD-fed mice including steatosis, inflammation, fibrosis, and HCC. Furthermore, microarray analyses revealed that, compared to wild-type mice, HFD-fed miR-223 knockout (miR-223KO) mice had greater hepatic expression of many inflammatory genes and cancer-related genes, including (C-X-C motif) chemokine 10 (Cxcl10) and transcriptional coactivator with PDZ-binding motif (Taz), two well-known factors that promote NASH development. In vitro experiments demonstrated that Cxcl10 and Taz are two downstream targets of miR-223 and that overexpression of miR-223 reduced their expression in cultured hepatocytes. Hepatic levels of miR-223, CXCL10, and TAZ mRNA were elevated in human NASH samples, which positively correlated with hepatic levels of several miR-223 targeted genes as well as several proinflammatory, cancer-related, and fibrogenic genes. Conclusion: HFD-fed miR-223KO mice develop a full spectrum of NAFLD, representing a clinically relevant mouse NAFLD model; miR-223 plays a key role in controlling steatosis-to-NASH progression by inhibiting hepatic Cxcl10 and Taz expression and may be a therapeutic target for the treatment of NASH.

Endoplasmic Reticulum Stress Causes Liver Cancer Cells to Release Exosomal miR-23a-3p and Up-regulate Programmed Death Ligand 1 Expression in Macrophages.

Endoplasmic reticulum (ER) stress promotes tumor cell escape from immunosurveillance. However, the underlying mechanisms remain unknown. We hypothesized that ER stress induces hepatocellular carcinoma (HCC) cells to release exosomes, which attenuate antitumor immunity by modulating the expression of programmed death ligand 1 (PD-L1) in macrophages. In this study, we demonstrated that expression of several ER stress markers (glucose-regulated protein 78, activating transcription factor 6, protein kinase R-like ER kinase, and inositol-requiring enzyme 1α) was up-regulated in HCC tissues and negatively correlated with the overall survival and clinicopathological scores in patients with HCC. Expression of ER stress-related proteins positively correlated with CD68+ macrophage recruitment and PD-L1 expression in HCC tissues. High-throughput sequencing analysis identified miR-23a-3p as one of the most abundant microRNAs in exosomes derived from tunicamycin (TM)-treated HCC cells (Exo-TMs). miR-23a-3p levels in HCC tissues negatively correlated with overall survival. Treatment with Exo-TMs up-regulated the expression of PD-L1 in macrophages in vitro and in vivo. Bioinformatics analysis suggests that miR-23a-3p regulates PD-L1 expression through the phosphatase and tensin homolog (PTEN)-phosphatidylinositol 3-kinase-protein kinase B (AKT) pathway. This notion was confirmed by in vitro transfection and coculture experiments, which revealed that miR-23a-3p inhibited PTEN expression and subsequently elevated phosphorylated AKT and PD-L1 expression in macrophages. Finally, coculture of T cells with Exo-TM-stimulated macrophages decreased CD8+ T-cell ratio and interleukin-2 production but increased T-cell apoptosis in vitro. Conclusion: ER-stressed HCC cells release exosomes to up-regulate PD-L1 expression in macrophages, which subsequently inhibits T-cell function through an exosome miR-23a-PTEN-AKT pathway. Our findings provide insight into the mechanism how tumor cells escape from antitumor immunity.

Pregnane X Receptor Regulates Liver Size and Liver Cell Fate by Yes-Associated Protein Activation in Mice.

Activation of pregnane X receptor (PXR), a nuclear receptor that controls xenobiotic and endobiotic metabolism, is known to induce liver enlargement, but the molecular signals and cell types responding to PXR-induced hepatomegaly remain unknown. In this study, the effect of PXR activation on liver enlargement and cell change was evaluated in several strains of genetically modified mice and animal models. Lineage labeling using AAV-Tbg-Cre-treated Rosa26EYFP mice or Sox9-CreERT , Rosa26EYFP mice was performed and Pxr-null mice or AAV Yap short hairpin RNA (shRNA)-treated mice were used to confirm the role of PXR or yes-associated protein (YAP). Treatment with selective PXR activators induced liver enlargement and accelerated regeneration in wild-type (WT) and PXR-humanized mice, but not in Pxr-null mice, by increase of cell size, induction of a regenerative hybrid hepatocyte (HybHP) reprogramming, and promotion of hepatocyte and HybHP proliferation. Mechanistically, PXR interacted with YAP and PXR activation induced nuclear translocation of YAP. Blockade of YAP abolished PXR-induced liver enlargement in mice. Conclusion: These findings revealed a function of PXR in enlarging liver size and changing liver cell fate by activation of the YAP signaling pathway. These results have implications for understanding the physiological functions of PXR and suggest the potential for manipulation of liver size and liver cell fate.

Adipocyte Death Preferentially Induces Liver Injury and Inflammation Through the Activation of Chemokine (C-C Motif) Receptor 2-Positive Macrophages and Lipolysis.

Adipocyte death occurs under various physiopathological conditions, including obesity and alcohol drinking, and can trigger organ damage particularly in the liver, but the underlying mechanisms remain obscure. To explore these mechanisms, we developed a mouse model of inducible adipocyte death by overexpressing the human CD59 (hCD59) on adipocytes (adipocyte-specific hCD59 transgenic mice). Injection of these mice with intermedilysin (ILY), which rapidly lyses hCD59 expressing cells exclusively by binding to the hCD59 but not mouse CD59, resulted in the acute selective death of adipocytes, adipose macrophage infiltration, and elevation of serum free fatty acid (FFA) levels. ILY injection also resulted in the secondary damage to multiple organs with the strongest injury observed in the liver, with inflammation and hepatic macrophage activation. Mechanistically, acute adipocyte death elevated epinephrine and norepinephrine levels and activated lipolysis pathways in adipose tissue in a chemokine (C-C motif) receptor 2-positive (CCR2+ ) macrophage-dependent manner, which was followed by FFA release and lipotoxicity in the liver. Additionally, acute adipocyte death caused hepatic CCR2+ macrophage activation and infiltration, further exacerbating liver injury. Conclusion: Adipocyte death predominantly induces liver injury and inflammation, which is probably due to the superior sensitivity of hepatocytes to lipotoxicity and the abundance of macrophages in the liver.