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Potential conflict of interest: Nothing to report.
Supported by the Christian-Doppler Research Society and Austrian Science Fund grant ZFP215300 (to A. K.).
Pre–B cell colony–enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase or visfatin, plays an important role in metabolic, inflammatory, and malignant diseases. Recent evidence suggests that blocking its enzymatic activity using a specific small-molecule inhibitor (FK866) might be beneficial in acute experimental inflammation. We investigated the role of PBEF in human liver disease and experimental hepatitis. PBEF serum levels and hepatic expression were determined in patients with chronic liver diseases. These studies were followed by in vivo experiments using concanavalin A (ConA) and D-galactosamine/lipopolysaccharide (LPS) models of experimental hepatitis. PBEF was either overexpressed by hydrodynamic perfusion or inhibited by FK866. In vivo findings were corroborated studying inflammatory responses of lentivirally PBEF-silenced or control FL83B mouse hepatocytes. Here, we demonstrate that PBEF serum levels were increased in patients with chronic liver diseases irrespective of disease stage and etiology. In particular, we observed enhanced PBEF expression in hepatocytes. Liver-targeted overexpression of PBEF rendered mice more susceptible to ConA- and D-galactosamine/LPS–induced hepatitis compared with control animals. In contrast, inhibition of PBEF using FK866 protected mice from ConA-induced liver damage and apoptosis. Administration of FK866 resulted in depletion of liver nicotinamide adenine dinucleotide+ levels and reduced proinflammatory cytokine expression. Additionally, FK866 protected mice in the D-galactosamine/LPS model of acute hepatitis. In vitro, PBEF-silenced mouse hepatocytes showed decreased responses after stimulation with LPS, lipoteichoic acid, and tumor necrosis factor α. In primary murine Kupffer cells, FK866 suppressed LPS-induced interleukin (IL)-6 production, whereas incubation with recombinant PBEF resulted in increased IL-6 release. Conclusion: Our data suggest that PBEF is of key importance in experimental hepatitis. Its specific inhibition might be considered a novel treatment option for inflammatory liver diseases. (HEPATOLOGY 2011;)
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C hronic inflammation of the liver caused by viral infections such as hepatitis B or C viruses, chronic alcohol abuse, autoimmune tissue damage, metabolic disorders, and hereditary disorders often leads to tissue damage with progressive fibrosis and cancer in later life.1 Liver inflammation is often characterized by T cell activation, inflammatory infiltration, and necrotic and apoptotic tissue damage accompanied by liver regeneration. Numerous proinflammatory cytokines such as tumor necrosis factor α (TNFα) or interferon-γ (IFNγ) promote tissue damage, whereas others such as interleukin (IL)-10 and IL-22 protect the liver from these harmful effects.2, 3 So far, only limited therapeutic options are available to ameliorate the long-term outcome of hepatic inflammatory disorders.
Pre–B cell colony–enhancing factor (PBEF) was first identified by Samal et al.4 in a search for novel cytokine-like molecules. The PBEF transcript was strongly up-regulated in lymphocytes by pokeweed mitogen and cycloheximide and functionally synergized with IL-7 and stem cell factor in pre–B cell colony formation. We and others reported that PBEF preferentially activates mononuclear cells, in particular monocytes, thereby combining all features of a proinflammatory cytokine.5, 6 Beyond that, PBEF turned out to be the postulated enzyme catalyzing the rate-limiting step in nicotinamide adenine dinucleotide (NAD) synthesis.7, 8 NAD is a classic coenzyme with well-established roles in cellular redox reactions.9 In mammals, NAD+ biosynthesis comprises two pathways: the de novo pathway produces nicotinic acid (NA) mononucleotide by way of tryptophan and quinolinic acid. NA mononucleotide is transformed into NAD through Nam/NA mononucleotide adenylyltransferase 1/2 and NAD+ synthetase.10 The salvage pathway reuses nicotinamide (Nam), the end-product of NAD-consuming enzymes such as poly (adenosine diphosphate-ribose) polymerases (PARPs) or sirtuins (SIRTs) .11 Nam is further converted to nicotinamide mononucleotide through nicotinamide phosphoribosyltransferase (Nampt), which in turn is converted to NAD by Nam/NA mononucleotide adenylyltransferase 1/2.12 Nampt represents the rate-limiting enzyme in this cascade.8 Most recently, PBEF's enzymatic activity has been suggested to modulate immune functions by regulating NAD+ replenishment. FK866, a specific noncompetitive Nampt inhibitor, causes intracellular NAD+ shortage, specifically in activated immune cells. This leads to functional inactivity of NAD+-dependent enzymes such as PARP-1 and SIRT-6 that promote cellular activation.13, 14
Numerous studies have described an association between elevated PBEF expression with acute and chronic inflammatory conditions in humans and in mice. PBEF expression is elevated in neutrophils of septic patients preventing neutrophil apoptosis.15 PBEF has been found in diseased tissues of critically ill patients with acute lung injury.16 Its transcription is also highly elevated in a variety of chronic inflammatory conditions such as rheumatoid arthritis,17, 18 severe generalized psoriasis,19 and inflammatory bowel disease.5 These data suggest that PBEF displays many features of a proinflammatory mediator.
We hypothesized that PBEF might play a role in acute and chronic liver damage. We show that PBEF is strongly up-regulated in human chronic liver diseases and acute experimental hepatitis. Mice overexpressing hepatic PBEF at baseline are more susceptible to liver damage during ConA- or D-galactosamine/lipopolysaccharide (LPS)–mediated hepatitis, whereas FK866 protected mice from acute hepatic injury induced by either ConA or D-galactosamine/LPS.
Serum samples were obtained from 83 randomly selected, consecutive patients with clinically, biochemically, radiologically, and histologically confirmed diagnosis of chronic liver disease. Chronic liver disease was staged according to Child-Turcotte-Pugh (CTP) criteria.20 Thirty-nine age- and sex-matched healthy subjects served as a control group. Baseline characteristics of chronic liver disease patients are reported in Table 1. Informed consent was obtained, and the study was approved by the local ethics committee of the Innsbruck Medical University. Nine milliliters of blood were collected in Sarstedt Monovette tubes. Blood was centrifuged at 1,200g for 15 minutes and 1-mL aliquots were stored at −80°C until assayed. Each sample was assigned an encoding number, and all assays were performed in duplicate in a blinded manner.
Table 1. Patient Characteristics
Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus.
Age, years, mean (SD) [range]
56.5 (8.3) [41-72]
51.5 (11.4) [23-72]
Etiology of disease
Viral (HBV and/or HCV)
Six- to eight-week old female C57BL/6 mice were obtained from Charles River (Sulzfeld, Germany). Mice were housed in accordance with institutional animal care with open access to standard chow and water. Animal experiments were approved by the Austrian Federal Ministry of Science and Research (license number: 66011/34-II/10b/2009).
Unless stated otherwise, mice were injected intravenously through the lateral tail vein with 15 mg/kg ConA from Canavalia ensiformis (Sigma-Aldrich, St. Louis, MO) in endotoxin-free phosphate-buffered saline.
Mice received an intraperitoneal injection of 700 mg/kg D-galactosamine (Carl Roth, Karlsruhe, Germany) and 1 μg/kg lipopolysaccharide (InvivoGen, San Diego, CA). Mice were euthanized at the indicated time after injection.
FK866 was purchased from Axon Medchem (Groningen, Netherlands) and dissolved in dimethyl sulfoxide; 25-mg/mL aliquots were stored at −80°C until further use. Mice received three intraperitoneal injections of 10 mg/kg FK866 further diluted in phosphate-buffered saline 24, 12, and 0.5 hours prior to treatment with ConA or D-galactosamine/LPS.
Hydrodynamic perfusion was performed essentially as described.21, 22 Details are reported in the Supporting Methods.
Sampling of Material
For the detection of aminotransferases, blood was drawn by way of cardiac puncture 15 hours after ConA injection. Livers were removed in toto. The left lower lobe was split into two equal parts. One half was encapsulated together with the left upper lobe for histology. The remaining half was divided into three parts—one part in RNAlater (Qiagen, Hilden, Germany), and two parts were snap-frozen together with the right upper lobe in liquid nitrogen. The right middle lobe was embedded in Tissue-TEK OCT compound (Sakura, Alphen aan den Rijn, the Netherlands) and stored at −80°C until further analysis.
Liver damage was quantified by measurement of plasma enzyme activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) using an automated procedure.
One half of the left lower lobe and the left upper lobe were encapsulated and fixed in 10% buffered formalin overnight at room temperature. Tissue was then embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sections were photographed using a 5× objective lens, and necrosis fields were marked and quantified using ImageJ processing and analysis software (National Institutes of Health, Bethesda, MD).
Cell Culture, Reagents, and PBEF/Nampt Silencing.
All experiments were performed using FL83B murine hepatocytes (American Type Culture Collection No. CRL-2390). For PBEF gene silencing, FL83B cells were either stably transfected using short hairpin RNA (shRNA) -producing lentiviral particles or transfected with PBEF1-specific small interfering RNAs. Primary Kupffer cells were prepared as described.23 Details including apoptosis experiments are described in the Supporting Methods.
RNA Isolation and Real-Time Reverse-Transcription Polymerase Chain Reaction.
Total RNA was extracted from cells and tissues using TRIZOL reagent according to the manufacturer's instructions (Invitrogen). Further polymerase chain reaction details are outlined in the Supporting Methods.
Protein Extraction and Western Blot Analysis.
Protein from cells and tissue was extracted using M-PER mammalian protein extraction reagent supplemented with Halt protease inhibitor cocktail according to the manufacturer's instructions (Pierce, Rockford, IL). Protein concentrations were determined using Bradford reagent (Bio-Rad Laboratories, Hercules, CA). Western Blot analyses are detailed in the Supporting Methods.
Detection of Cytokines.
Concentrations of CXCL1/KC in cell culture supernatants were determined using commercially available antibody pairs and protein standards from R&D Systems (McKinley Place, Minneapolis, MN). Circulating human PBEF was assayed using a human visfatin (C-terminal) enzyme immunometric assay (Phoenix Pharmaceuticals, Burlingame, CA). Mouse serum PBEF was determined using a mouse visfatin/PBEF enzyme-linked immunosorbent assay (ELISA) kit (MBL, Woburn, MA). Absorption was determined with a PowerWave Microplate Spectrophotometer (BioTek, Winooski, VT). Kupffer cell supernatants were assayed for cytokines using a Bio-Plex 2200 Multiplex Array System with Bio-Plex reagents according to the manufacturer's instructions (Bio-Rad Laboratories).
Measurement of NAD/NADH Tissue Expression.
NAD+ tissue concentrations were determined using an enzymatic cycling assay essentially as described (Supporting Methods).24
Immunohistochemistry, Immunofluorescence, and Terminal Deoxynucleotidyl Transferase–Mediated Deoxyuridine Triphosphate Nick-End Labeling.
The protocol for PBEF staining has been reported elsewhere.25 Details of immunofluorescence double staining and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) tests are described in the Supporting Methods.
Quantitative data are presented as the mean ± SE. Analyses are described in the Supporting Methods.
Serum Levels and Liver Expression of PBEF Are Increased in Liver Cirrhosis.
We assessed PBEF serum levels in 83 patients with cirrhosis and 39 age- and sex- matched healthy controls. As depicted in Fig. 1A, PBEF serum levels in patients with cirrhosis were significantly elevated irrespective of disease etiology compared with control subjects. Serum concentrations of PBEF were not different between patients with alcoholic (3,094 ± 483 ng/mL), viral (3,129 ± 322 ng/mL), or cryptogenic (3,416 ± 744 ng/mL) cirrhosis (Fig. 1A). Regarding the stage of liver disease, no significant differences of PBEF serum concentrations were found between patients with CTP class A (3,299 ± 465 ng/mL), CTP class B (2,973 ± 345 ng/mL), or CTP class C liver cirrhosis (3,944 ± 1,356 ng/mL). Again, PBEF levels of all CTP subclasses were significantly higher compared with the control population (996 ± 133 ng/mL) (Fig. 1B). In patients with liver cirrhosis, we observed significant positive correlations of PBEF serum levels with γ-glutamyltransferase (rs = 0.413, P < 0.001) and patients' age (rs = 0.327, P < 0.001; data not shown). No significant associations were evident between PBEF and sex, body mass index, and creatinine clearance (estimated glomerular filtration rate).
Paraffin-embedded tissue sections from patients with alcoholic or viral liver disease patients were specifically stained for PBEF. Immunoperoxidase staining showed strong expression of PBEF in hepatocytes regardless of the underlying disease (Fig. 1C,D). In general, staining intensity was moderate to strong in degree. Kupffer cells within the sinusoidal lumen also stained positively for PBEF. In line with its reported cellular distribution, nuclei stained positive for PBEF. A variable staining displayed some cells of the interlobular septa and portal fields as well as the bile duct epithelium. As demonstrated by immunofluorescence double-staining, liver sinusoidal endothelial cells stained positive for PBEF (Fig. 1E). An antibody specifically detecting smooth muscle actin was used to identify activated stellate cells.26 No colocalization was noticed between PBEF and activated stellate cells (Fig. 1F).
Induction of Acute Liver Injury by Injection of Con-A Up-regulates Hepatic PBEF Expression.
To test whether PBEF expression was induced in ConA-mediated experimental hepatitis, specific pathogen-free C57BL/6 mice were injected with 15 mg/kg ConA, and livers were harvested after 2, 4, 6, and 8 hours. Induction of hepatitis was associated with a time-dependent up-regulation of hepatic PBEF messenger RNA (mRNA) expression. As shown in Fig. 2A, PBEF expression was induced 4.4-fold after 2 hours, 15.5-fold after 4 hours, and peaked after 6 hours showing a 46.3-fold expression. mRNA data were confirmed by way of western blot analysis (Fig. 2B). In accordance with the mRNA data, protein expression peaked after 6 hours. Cellular origins were determined by way of immmunofluorescent microscopy. Apart from hepatocytes, PBEF colocalized with F4/80-positive Kupffer cells (Fig. 2C) and CD31-positive liver sinusoidal endothelial cells (Fig. 2D).
PBEF Gene Delivery Aggravates ConA- and D-Galactosamine/LPS–Induced Liver Injury.
To study the role of mouse PBEF in mediating ConA-induced hepatitis, we first introduced the PBEF gene into mouse livers using hydrodynamic delivery. Hydrodynamic perfusion represents an effective method for in vivo gene transfer into mouse livers.27 The mouse PBEF gene was cloned into a pCI-neo mammalian expression vector constitutively expressing murine PBEF gene under control of the cytomegalovirus immediate-early enhancer/promoter region (pCI-Pbef1). The same vector containing a nonsense sequence was used as a control (pCI-Ctrl). Quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that PBEF mRNA was efficiently overexpressed in mouse livers 24 hours after injection (Supporting Fig. 1A). RT-PCR results were further confirmed by way of western blot analysis (Supporting Fig. 1B). Of note, circulating PBEF concentrations in pCI-Pbef1–injected animals were increased eight-fold compared with pCI-control injected mice (Supporting Fig. 1C).
Experiments included four groups: (1) mice receiving the PBEF1-overexpressing plasmid (pCI-Pbef1) and 12.5 mg/kg ConA (Fig. 3A), (2) mice receiving the control plasmid (pCI-Ctrl) and 12.5 mg/kg ConA (Fig. 3B), (3) mice injected with the pCI-Pbef1 and saline (Fig. 3C), and (4) mice injected with the pCI-Ctrl and saline (Fig. 3D). With respect to liver pathology, we found more severe lesions in group 1 (pCI-Pbef1 + ConA) compared with group 2 (pCI-Ctrl + ConA) (Fig. 3A,B). No necrotic areas were present in saline-injected control mice (group 3 and group 4) (Fig. 3C,D). Correspondingly, liver damage as measured by the release of liver enzymes (AST and ALT) was significantly higher in group 1 (pCI-Pbef1 + ConA) compared with group 2 (pCI-Ctrl + ConA) (Fig. 3E). AST and ALT tended to be higher in group 3 (pCI-Pbef1 + saline) versus group 4 (pCI-Ctrl + saline), with P values of 0.108 and 0.124, respectively (Fig. 3E). Potential differences in hepatic inflammatory cell infiltration were determined by way of immunohistochemistry. A vivid, comparable T cell infiltration was found in group 1 (pCI-Pbef1 + ConA) and group 2 (pCI-Ctrl + ConA), but not in ConA-naïve groups 3 and 4 (Supporting Fig. 1D). The abundance of Mac-2–positive mononuclear cells was significantly increased in group 1 (pCI-Pbef1 + ConA) compared with group 2 (pCI-Ctrl + ConA) mice (Supporting Fig. 1E). Compared with pCI-Ctrl–treated animals, pCI-Pbef1–treated animals displayed significantly elevated levels of hepatic mRNA expression of CXCL-1, IL-6, and IL-1β after ConA challenge. No difference was observed in liver TNFα, IFNγ, and IL-10 inductions (Fig. 3F). The experiments performed in the ConA model were repeated in D-galactosamine/LPS–induced experimental hepatitis. Disease outcome was compared between pCI-Pbef1– and pCI-Ctrl–injected animals. Again, overexpression of PBEF by hydrodynamic perfusion deteriorated liver damage in D-galactosamine/LPS-induced hepatitis as demonstrated by significantly elevated liver enzymes (Supporting Fig. 2A) and increased hepatic mRNA expression of CXCL-1 and IL-1β (Supporting Fig. 2B) when compared with pCI-Ctrl–injected mice.
Pharmacological Inhibition of Nampt by FK866 Prevents Liver Damage in Animal Models of Acute Liver Failure.
The above studies indicated that Nampt is strongly up-regulated during experimental hepatitis as well as in human chronic liver disease. Therefore, FK866—a highly specific, noncompetitive inhibitor of Nampt—was used to block Nampt in vivo. Importantly, whereas vehicle treatment did not affect the course of ConA hepatitis, the preadministration of FK866 resulted in reduced ConA-induced liver toxicity. By the time of liver explantation, control livers appeared macroscopically more severely affected with abundant subcapsular necrotic areas (data not shown). Upon examination of hematoxylin and eosin–stained liver sections, vehicle-treated control mice showed more extensive and more numerous necrotic lesions (Fig. 4B) compared with FK866-treated mice (Fig. 4A). Quantification of liver necrosis revealed a 12.2-fold reduction in necrotic areas (Fig. 4C). FK866-treated animals displayed a marked reduction of hepatocyte apoptosis as detected and quantified by TUNEL staining (Supporting Fig. 3A) compared with their vector-treated littermates (Supporting Fig. 3B). In support of these data, FK866-treated mice displayed a 5.1-fold decrease in AST plasma levels and a 4.2-fold decrease in ALT plasma levels (Fig. 4D). Examination of liver tissue NAD+ concentrations revealed that FK866 effectively suppressed Nampt-mediated NAD+ production. Liver NAD+ concentrations were 6.1-fold lower in FK866 compared with vehicle-treated mice (Fig. 4E). Determination of liver cytokine expression in FK866-treated mice showed a significant reduction in the relative expression of CXCL1, IL-1β, TNFα, IFNγ, and IL-10 compared with control-treated animals (Fig. 4F). Once more, we tested FK866 in another model of acute liver failure, namely the D-galactosamine/LPS model. Again, treatment with the Nampt inhibitor protected mice from macrophage-driven D-galactosamine/LPS hepatitis, as shown by significant decreases of plasma AST and ALT activities (Supporting Fig. 3C). Again, treatment with FK866 was associated with a significant decrease in hepatic NAD concentration (Supporting Fig. 3D).
Knockdown of PBEF in Murine Hepatocytes Results in Reduced Susceptibility to Proinflammatory Stimuli and Increased Cell Survival upon Stimulation with D-Galactosamine/LPS.
We next asked whether PBEF deficiency might affect innate immune responses in murine hepatocytes. Therefore, FL83B cells were stably transfected with a lentiviral vector containing a PBEF-specific shRNA (FL83B-iPbef1). Control cells were stably transfected with a lentivirus producing a nonbinding shRNA (FL83B-Ctrl). CXCL-1 expression was significantly impaired in FL83B-iPbef1 cells after stimulation with TNFα, LPS, or lipoteichoic acid (LTA) compared with FL83B-Ctrl cells (Fig. 5A). mRNA data were confirmed by measurement of CXCL-1/KC release in cell culture supernatants using a specific ELISA (Fig. 5B). In another set of experiments, FL83B cells were activated with LPS with or without FK866 in the indicated concentrations (Fig. 5C). Again, CXCL-1 release was significantly suppressed in the presence of 10 nM and 100 nM FK866 compared with vector-treated cells (Fig. 5C). Moreover, we investigated the effect of PBEF on hepatocyte survival upon stimulation with D-galactosamine/LPS. As demonstrated in Supporting Fig. 5, PBEF-silenced cells showed significantly increased survival after stimulation with D-galactosamine/LPS. No effect was observed in PBEF-overexpressing cells or by addition of extracellular recombinant PBEF. As determined by way of quantitative PCR, silencing efficiency was between 80% and 90% for stably transfected cells (Supporting Fig. 4A) and transient transfected cells (data not shown). As confirmed on western blot analysis, PBEF was efficiently silenced in unstimulated as well as LPS-challenged FL83B-iPbef1–transfected cells compared with FL83B-Ctrl–transfected cells (Supporting Fig. 3B).
Intracellular and Extracellular PBEF Is Involved in Kupffer Cell Function.
In order to link PBEF with liver cell function, we stimulated murine Kupffer cells with LPS with or without FK866 in increasing dosing. As shown in Fig. 5D, FK866 dose-dependently suppressed IL-6 production in LPS-stimulated primary Kupffer cells. The same effect was found for other macrophage cytokines, including RANTES (Supporting Fig. 4C), MIP-1β, and MCP-1 (data not shown). Kupffer cells were also incubated with recombinant murine PBEF. Stimulation with recombinant PBEF was associated with a significant increase in Kupffer cell IL-6 release (Fig. 5E). Moreover, stimulation with PBEF resulted in a significant increase in TNFα and inducible nitric oxide synthase mRNA expression (data not shown).
PBEF exhibits dual functions in that it acts extracellularly as a proinflammatory cytokine and intracellularly as an enzyme catalyzing the rate-limiting step of the NAD salvage pathway from nicotinamide.28 Here we demonstrate that PBEF liver expression and serum levels are increased in human chronic liver diseases. Similarly, PBEF is strongly up-regulated in ConA-induced experimental hepatitis, and produced by hepatocytes, Kupffer cells, and liver sinusoidal endothelial cells. In the ConA model, PBEF gene delivery aggravates liver disease, resulting in enhanced hepatic inflammation and liver cell death. Similar effects are observed in D-galactosamine/LPS–induced hepatitis. Importantly, blocking PBEF using FK866, a specific, noncompetitive small-molecule inhibitor of PBEF/Nampt,27 is associated with a remarkable protection of mice in Con-A– and D-galactosamine/LPS–induced hepatitis. PBEF modifies immune functions in hepatocytes, because PBEF-silenced hepatocytes have a reduced capacity to produce CXCL-1 after stimulation with TNFα and TLR-ligands and show increased cell survival after stimulation with D-galactosamine/LPS in vitro. Whereas FK866 suppresses Kupffer cell functions, these cells can by activated by extracellular recombinant PBEF. Our findings suggest that both extracellular and intracellular PBEF might therefore play a role in inflammatory liver diseases.
We have reported that obesity as a chronic inflammatory condition is associated with enhanced PBEF levels, and both hepatic as well as systemic concentrations decline after successful weight loss.25 In the present study, we report that PBEF serum concentrations in patients with cirrhosis are significantly higher compared with a healthy control population irrespective of disease etiology or disease stage. Immunohistochemical and immunofluorecence analyses proved the relative abundance and tissue distribution of PBEF in human liver disease. It should be noted that our data are different from those presented by de Boer et al.,29 who found decreased PBEF serum levels in 19 patients with cirrhosis compared with healthy controls. However, other studies have also demonstrated that PBEF levels are increased either in patients with chronic hepatitis C30 or in the ascites fluid of liver cirrhosis patients irrespective of etiology,31 supporting that PBEF serum/ascites concentrations are rather increased in chronic liver diseases.
Garten et al.32 reported that human hepatocytes represent a potential source for circulating PBEF. This complies with our data studying primary mouse liver cell cultures. PBEF was readily detected in supernatants from primary hepatocytes (data not shown). In vivo, we showed that liver PBEF expression is strongly induced during ConA hepatitis and apart from hepatocytes, Kupffer cells and liver sinusoidal endothelial cells proved to be PBEF sources. PBEF deficiency in FL83B cells dampened their proinflammatory capacity after stimulation with LPS, LTA, and TNFα. PBEF-silenced hepatocytes showed an increased cellular survival after stimulation with D-galactosamine/LPS in vitro, suggesting that intracellular PBEF might be involved in apoptosis and cell death regulation, especially in inflammatory conditions.
Injection of the plant-derived lectin ConA is a well-described model of acute liver injury that induces fulminant hepatitis within 8 hours after application.33 In this model, liver inflammation is driven by Kupffer cell–derived TNFα34, 35 and T cell–derived IFNγ.36, 37 In addition to proinflammatory mediators, anti-inflammatory cytokines such as IL-10 and IL-22 counterbalance these destructive effects by suppressing the aggressive activities of immune cells.3, 38 Liver expression of PBEF was strongly up-regulated during ConA hepatitis. Overexpression of PBEF by hydrodynamic perfusion aggravated ConA- and D-galactosamine–induced liver damage. The cytokine profile observed in these mice revealed increased levels of CXCL1, IL-1β, and IL-6, suggesting that PBEF promotes innate immune responses. We demonstrated that extracellular PBEF activates Kupffer cells. Given the high serum concentrations in PBEF-injected mice, Kupffer cell activation by circulating PBEF may contribute to the observed effects. Blocking PBEF with FK866 protected mice from ConA-induced liver damage. These effects were paralleled by a significant reduction of the key proinflammatory cytokines TNFα, IFNγ, IL-1β, and CXCL-1. Administration of FK866 was associated with a significant decrease in liver tissue NAD+/NADH concentrations in this model. Of note, FK866-treated mice also exhibited a reduction of anti-inflammatory IL-10 as well as mitigation in the up-regulation of PBEF itself in the course of hepatitis (data not shown). Altogether, these data suggest that blocking PBEF might interfere at an early step in the disease process, reducing the overall proinflammatory tonus in the liver. Notably, such an effect is also supported by the fact that a similar protective effect for FK866 was observed in the D-galactosamine/LPS model of hepatitis.
Two recently published studies investigated the effect of the specific Nampt inhibitor FK866 in animal models of inflammation. Busso et al.39 demonstrated that administration of FK866 significantly protected mice from the deleterious effects of collagen-induced arthritis. Mechanistically, the authors found that FK866 suppressed the activity of mononuclear cells. Specifically, FK866 dose-dependently depleted intracellular NAD+ concentrations in thioglycollate-elicited mouse macrophages and human monocytes, rendering them less responsive to stimulation with LPS.39 Bruzzone et al.13 investigated the effect of FK866 on T lymphocyte function and demonstrated that activated T lymphocytes specifically undergo a massive NAD+ depletion when treated with FK866. NAD+ depletion inhibits critical T cell functions such as proliferation and IFNγ/TNFα production, eventually leading to cell death. In vitro, these authors were able to reverse the effects by adding nicotinic acid to the cell culture, thereby preventing NAD+ shortage. A mechanistic link between intracellular NAD levels and inflammation has been reported by Van Gool et al.,14 who demonstrated that intracellular NAD promotes TNF synthesis, probably in a Sirt6-dependent manner.14 Thus, there is emerging evidence that specifically blocking PBEF's enzymatic activity may have promise as a potential therapy for acute and chronic inflammatory diseases. Moreover, our data are supportive of a concept in which FK866 suppresses immune activation of different cell types leading to NAD shortage and thereby protecting the liver from the deleterious effects of an overwhelming immune activation.
In conclusion, our data demonstrate that enhanced PBEF liver expression and increased PBEF serum levels are a feature of human and experimental liver diseases. Hepatic overexpression of PBEF promotes and pharmacological inhibition of PBEF suppresses inflammation in both a T cell–mediated and macrophage-mediated hepatitis model. Our data indicate that both intracellular and extracellular PBEF might be involved and modulate hepatic inflammation. The potent suppression of experimental liver inflammation by the specific Nampt inhibitor FK866 suggests that targeting this mediator could be a useful strategy in the treatment of hepatic inflammation.
We thank Sabine Geiger, Alexandra Bichler, and Barbara Enrich for excellent technical assistance. We thank Patrizia Moser and Ines Brosch for supporting us in histological work-up at the Institute of Pathology. We are also indebted to Gottfried Baier and Natascha Kleiter for technical advice.