BACKGROUND. The clearance of hepatitis B surface antigen (HBsAg) loss, defined as functional cure, is a clinical target in patients with chronic hepatitis B (CH). To understand the immune responses underlying functional cure, we evaluated cytokine and chemokine expression profiles from patients with resolving and nonresolving acute hepatitis B (AH). METHODS. We cross-sectionally evaluated 41 chemokines and cytokines at the peak of hepatitis in the sera from 41 self-limited AH patients who achieved HBsAg seroconversion, 8 AH patients who failed to clear HBsAg within 1 year after the diagnosis, 8 CH patients with hepatic flare, and 14 healthy volunteers. We longitudinally examined 41 chemokines and cytokines in the sera from 4 self-limited AH patients, 3 chimpanzees inoculated with hepatitis B virus (HBV), and 2 CH patients treated with nucleotide analogs and PEG–IFN-α, one resulting in functional cure. RESULTS. In AH patients and HBV-inoculated chimpanzees with HBsAg loss, CXCL9, CXCL10, CXCL11, CXCL13, and IL-21 were elevated at hepatitis with subsequent decline of HBsAg. Interestingly, IL-21 elevation was observed only in resolving AH patients but not in nonresolvers. CXCL13 and IL-21 elevation was not observed in CH patients who failed to attain HBsAg loss, even at hepatic flare. A concomitant increase of CXCL13 and IL-21 was significant in CH patients who attained HBsAg seroconversion with a sequential therapy. CONCLUSION. Elevation of serum CXCL9, CXCL10, CXCL11, CXCL13, and IL-21 might be a hallmark of functional cure of AH or CH patients.
Sachiyo Yoshio, Yohei Mano, Hiroyoshi Doi, Hirotaka Shoji, Tomonari Shimagaki, Yuzuru Sakamoto, Hironari Kawai, Michitaka Matsuda, Taizo Mori, Yosuke Osawa, Masaaki Korenaga, Masaya Sugiyama, Masashi Mizokami, Eiji Mita, Keiko Katayama, Junko Tanaka, Tatsuya Kanto
Macrophages polarize into heterogeneous proinflammatory M1 and antiinflammatory M2 subtypes. Heme oxygenase 1 (HO-1) protects against inflammatory processes such as ischemia-reperfusion injury (IRI), organ transplantation, and atherosclerosis. To test our hypothesis that HO-1 regulates macrophage polarization and protects against IRI, we generated myeloid-specific HO-1–knockout (mHO-1–KO) and –transgenic (mHO-1–Tg) mice, with deletion or overexpression of HO-1, in various macrophage populations. Bone marrow–derived macrophages (BMDMs) from mHO-1–KO mice, treated with M1-inducing LPS or M2-inducing IL-4, exhibited increased mRNA expression of M1 (CXCL10, IL-1β, MCP1) and decreased expression of M2 (Arg1 and CD163) markers as compared with controls, while BMDMs from mHO-1–Tg mice displayed the opposite. A similar pattern was observed in the hepatic M1/M2 expression profile in a mouse model of liver IRI. mHO-1–KO mice displayed increased hepatocellular damage, serum AST/ALT levels, Suzuki’s histological score of liver IRI, and neutrophil and macrophage infiltration, while mHO-1–Tg mice exhibited the opposite. In human liver transplant biopsies, subjects with higher HO-1 levels showed lower expression of M1 markers together with decreased hepatocellular damage and improved outcomes. In conclusion, myeloid HO-1 expression modulates macrophage polarization, and protects against liver IRI, at least in part by favoring an M2 phenotype.
Min Zhang, Kojiro Nakamura, Shoichi Kageyama, Akeem O. Lawal, Ke Wei Gong, May Bhetraratana, Takehiro Fujii, Dawoud Sulaiman, Hirofumi Hirao, Subhashini Bolisetty, Jerzy W. Kupiec-Weglinski, Jesus A. Araujo
The hepatic glucose fasting response is gaining traction as a therapeutic pathway to enhance hepatic and whole-host metabolism. However, the mechanisms underlying these metabolic effects remain unclear. Here, we demonstrate the epidermal-type lipoxygenase, eLOX3 (encoded by its gene, Aloxe3), is a potentially novel effector of the therapeutic fasting response. We show that Aloxe3 is activated during fasting, glucose withdrawal, or trehalose/trehalose analogue treatment. Hepatocyte-specific Aloxe3 expression reduced weight gain and hepatic steatosis in diet-induced and genetically obese (db/db) mouse models. Aloxe3 expression, moreover, enhanced basal thermogenesis and abrogated insulin resistance in db/db diabetic mice. Targeted metabolomics demonstrated accumulation of the PPARγ ligand 12-KETE in hepatocytes overexpressing Aloxe3. Strikingly, PPARγ inhibition reversed hepatic Aloxe3–mediated insulin sensitization, suppression of hepatocellular ATP production and oxygen consumption, and gene induction of PPARγ coactivator-1α (PGC1α) expression. Moreover, hepatocyte-specific PPARγ deletion reversed the therapeutic effect of hepatic Aloxe3 expression on diet-induced insulin intolerance. Aloxe3 is, therefore, a potentially novel effector of the hepatocellular fasting response that leverages both PPARγ-mediated and pleiotropic effects to augment hepatic and whole-host metabolism, and it is, thus, a promising target to ameliorate metabolic disease.
Cassandra B. Higgins, Yiming Zhang, Allyson L. Mayer, Hideji Fujiwara, Alicyn I. Stothard, Mark J. Graham, Benjamin M. Swarts, Brian J. DeBosch
Adeno-associated viral vector–mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.
Moumita Paul-Heng, Mario Leong, Eithne Cunningham, Daniel L. J. Bunker, Katherine Bremner, Zane Wang, Chuanmin Wang, Szun Szun Tay, Claire McGuffog, Grant J. Logan, Ian E. Alexander, Min Hu, Stephen I. Alexander, Tim D. Sparwasser, Patrick Bertolino, David G. Bowen, G. Alex Bishop, Alexandra Sharland
Chronic liver disease (CLD) is associated with tissue-destructive fibrosis. Considering that common mechanisms drive fibrosis across etiologies, and that steatosis is an important cofactor for pathology, we performed RNA sequencing on liver biopsies of patients with different fibrosis stages, resulting from infection with hepatitis C virus (HCV) (with or without steatosis) or fatty liver disease. In combination with enhanced liver fibrosis score correlation analysis, we reveal a common set of genes associated with advanced fibrosis, as exemplified by those encoding the transcription factor ETS-homologous factor (EHF) and the extracellular matrix protein versican (VCAN). We identified 17 fibrosis-associated genes as candidate EHF targets and demonstrated that EHF regulates multiple fibrosis-associated genes, including VCAN, in hepatic stellate cells. Serum VCAN levels were also elevated in advanced fibrosis patients. Comparing biopsies from patients with HCV with or without steatosis, we identified a steatosis-enriched gene set associated with advanced fibrosis, validating follistatin-like protein 1 (FSTL1) as an exemplar of this profile. In patients with advanced fibrosis, serum FSTL1 levels were elevated in those with steatosis (versus those without). Liver Fstl1 mRNA levels were also elevated in murine CLD models. We thus reveal a common gene signature for CLD-associated liver fibrosis and potential biomarkers and/or targets for steatosis-associated liver fibrosis.
Divya Ramnath, Katharine M. Irvine, Samuel W. Lukowski, Leigh U. Horsfall, Zhixuan Loh, Andrew D. Clouston, Preya J. Patel, Kevin J. Fagan, Abishek Iyer, Guy Lampe, Jennifer L. Stow, Kate Schroder, David P. Fairlie, Joseph E. Powell, Elizabeth E. Powell, Matthew J. Sweet
Liver damage is typically inferred from serum measurements of cytoplasmic liver enzymes. DNA molecules released from dying hepatocytes are an alternative biomarker, unexplored so far, potentially allowing for quantitative assessment of liver cell death. Here we describe a method for detecting acute hepatocyte death, based on quantification of circulating, cell-free DNA (cfDNA) fragments carrying hepatocyte-specific methylation patterns. We identified 3 genomic loci that are unmethylated specifically in hepatocytes, and used bisulfite conversion, PCR, and massively parallel sequencing to quantify the concentration of hepatocyte-derived DNA in mixed samples. Healthy donors had, on average, 30 hepatocyte genomes/ml plasma, reflective of basal cell turnover in the liver. We identified elevations of hepatocyte cfDNA in patients shortly after liver transplantation, during acute rejection of an established liver transplant, and also in healthy individuals after partial hepatectomy. Furthermore, patients with sepsis had high levels of hepatocyte cfDNA, which correlated with levels of liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Duchenne muscular dystrophy patients, in which elevated AST and ALT derive from damaged muscle rather than liver, did not have elevated hepatocyte cfDNA. We conclude that measurements of hepatocyte-derived cfDNA can provide specific and sensitive information on hepatocyte death, for monitoring human liver dynamics, disease, and toxicity.
Roni Lehmann-Werman, Judith Magenheim, Joshua Moss, Daniel Neiman, Ofri Abraham, Sheina Piyanzin, Hai Zemmour, Ilana Fox, Talya Dor, Markus Grompe, Giora Landesberg, Bao-Li Loza, Abraham Shaked, Kim Olthoff, Benjamin Glaser, Ruth Shemer, Yuval Dor
The gut-liver axis is of clinical importance as a potential therapeutic target in a wide range of liver diseases; however, the mechanisms underlying interactions between microbial products and immune responses in the liver remain unknown. In this study, we demonstrated that IL-10–producing macrophages contribute to immune tolerance in the inflamed liver under intestinal barrier disruption in a murine tandem model of dextran sulfate sodium (DSS) colitis and concanavalin A (Con A) hepatitis. Intestinal barrier disruption protected mice from subsequent liver injury, and the severity of colitis directly affected susceptibility to such injury. The protective effect of DSS–Con A was canceled in gut-sterilized mice, suggesting that gut microbiota play a substantial role in this process. Altered gut microbiota and their metabolites, along with a disrupted intestinal barrier, directly gave rise to immunological permissiveness in the inflamed liver. We identified 1-methylnicotinamide (1-MNA) as a candidate metabolite capable of suppressing liver injury with the potential to induce IL-10–producing macrophages. Consistently, expression of nicotinamide N-methyltransferase, which converts nicotinamide to 1-MNA, was upregulated in the liver of DSS–Con A mice, and this effect was abrogated by gut sterilization. Collectively, our results provide a mechanistic insight into the regulation of immunological balance in the liver via the gut-liver axis.
Nobuhito Taniki, Nobuhiro Nakamoto, Po-Sung Chu, Yohei Mikami, Takeru Amiya, Toshiaki Teratani, Takahiro Suzuki, Tomoya Tsukimi, Shinji Fukuda, Akihiro Yamaguchi, Shunsuke Shiba, Rei Miyake, Tadashi Katayama, Hirotoshi Ebinuma, Takanori Kanai
While several molecular targets are under consideration, mechanistic underpinnings of the transition from uncomplicated nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) remain unresolved. Here we apply multiscale chemical profiling technologies to mouse models of deranged hepatic ketogenesis to uncover potential NAFLD driver signatures. Use of stable-isotope tracers, quantitatively tracked by nuclear magnetic resonance (NMR) spectroscopy, supported previous observations that livers of wild-type mice maintained long term on a high-fat diet (HFD) exhibit a marked increase in hepatic energy charge. Fed-state ketogenesis rates increased nearly 3-fold in livers of HFD-fed mice, a greater proportionate increase than that observed for tricarboxylic acid (TCA) cycle flux, but both of these contributors to overall hepatic energy homeostasis fueled markedly increased hepatic glucose production (HGP). Thus, to selectively determine the role of the ketogenic conduit on HGP and oxidative hepatic fluxes, we studied a ketogenesis-insufficient mouse model generated by knockdown of the mitochondrial isoform of 3-hydroxymethylglutaryl-CoA synthase (HMGCS2). In response to ketogenic insufficiency, TCA cycle flux in the fed state doubled and HGP increased more than 60%, sourced by a 3-fold increase in glycogenolysis. Finally, high-resolution untargeted metabolomics and shotgun lipidomics performed using ketogenesis-insufficient livers in the fed state revealed accumulation of bis(monoacylglycero)phosphates, which also accumulated in livers of other models commonly used to study NAFLD. In summary, natural and interventional variations in ketogenesis in the fed state strongly influence hepatic energy homeostasis, glucose metabolism, and the lipidome. Importantly, HGP remains tightly linked to overall hepatic energy charge, which includes both terminal fat oxidation through the TCA cycle and partial oxidation via ketogenesis.
D. André d’Avignon, Patrycja Puchalska, Baris Ercal, YingJu Chang, Shannon E. Martin, Mark J. Graham, Gary J. Patti, Xianlin Han, Peter A. Crawford
Bile acids are signaling molecules that critically control hepatocellular function. Disrupted bile acid homeostasis may be implicated in the pathogenesis of chronic liver diseases. Glutathione is an important antioxidant that protects the liver against oxidative injury. Various forms of liver disease share the common characteristics of reduced cellular glutathione and elevated oxidative stress. This study reports a potentially novel physiological function of bile acids in regulating hepatic sulfur amino acid and glutathione metabolism. We found that bile acids strongly inhibited the cysteine dioxygenase type-1–mediated (CDO1-mediated) cysteine catabolic pathway via a farnesoid X receptor–dependent mechanism. Attenuating this bile acid repressive effect depleted the free cysteine pool and reduced the glutathione concentration in mouse liver. Upon acetaminophen challenge, cholestyramine-fed mice showed impaired hepatic glutathione regeneration capacity and markedly worsened liver injury, which was fully prevented by N-acetylcysteine administration. These effects were recapitulated in CDO1-overexpressing hepatocytes. Findings from this study support the importance of maintaining bile acid homeostasis under physiological and pathophysiological conditions, as altered hepatic bile acid signaling may negatively impact the antioxidant defense mechanism and sensitivity to oxidative injury. Furthermore, this finding provides a possible explanation for the reported mild hepatotoxicity associated with the clinical use of bile acid sequestrants in human patients.
Yifeng Wang, Jibiao Li, David Matye, Yuxia Zhang, Katie Dennis, Wen-Xing Ding, Tiangang Li
Excess lipid accumulation is an early signature of nonalcoholic fatty liver disease (NAFLD). Although liver receptor homolog 1 (LRH-1) (encoded by NR5A2) is suppressed in human NAFLD, evidence linking this phospholipid-bound nuclear receptor to hepatic lipid metabolism is lacking. Here, we report an essential role for LRH-1 in hepatic lipid storage and phospholipid composition based on an acute hepatic KO of LRH-1 in adult mice (LRH-1AAV8-Cre mice). Indeed, LRH-1–deficient hepatocytes exhibited large cytosolic lipid droplets and increased triglycerides (TGs). LRH-1–deficient mice fed high-fat diet displayed macrovesicular steatosis, liver injury, and glucose intolerance, all of which were reversed or improved by expressing wild-type human LRH-1. While hepatic lipid synthesis decreased and lipid export remained unchanged in mutants, elevated circulating free fatty acid helped explain the lipid imbalance in LRH-1AAV8-Cre mice. Lipidomic and genomic analyses revealed that loss of LRH-1 disrupts hepatic phospholipid composition, leading to lowered arachidonoyl (AA) phospholipids due to repression of Elovl5 and Fads2, two critical genes in AA biosynthesis. Our findings reveal a role for the phospholipid sensor LRH-1 in maintaining adequate pools of hepatic AA phospholipids, further supporting the idea that phospholipid diversity is an important contributor to healthy hepatic lipid storage.
Diego A. Miranda, William C. Krause, Amaury Cazenave-Gassiot, Miyuki Suzawa, Hazel Escusa, Juat Chin Foo, Diyala S. Shihadih, Andreas Stahl, Mark Fitch, Edna Nyangau, Marc Hellerstein, Markus R. Wenk, David L. Silver, Holly A. Ingraham
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