Response Of Human Hepatocytes Research Articles

Dexamethasone mitigates remdesivir-induced liver toxicity in human primary hepatocytes and COVID-19 patients.

Coronavirus disease 2019 (COVID-19) is a global pandemic that has caused more than 600 million cases and over six million deaths worldwide. Despite the availability of vaccination, COVID-19 cases continue to grow making pharmacological interventions essential. Remdesivir (RDV) is an FDA-approved antiviral drug for treatment of both hospitalized and non-hospitalized COVID-19 patients, albeit with potential for hepatotoxicity. This study characterizes the hepatotoxicity of RDV and its interaction with dexamethasone (DEX), a corticosteroid often co-administered with RDV for inpatient treatment of COVID-19. Human primary hepatocytes and HepG2 cells were used as in vitro models for toxicity and drug-drug interaction studies. Real-world data from hospitalized COVID-19 patients were analyzed for drug-induced elevation of serum ALT and AST. In cultured hepatocytes, RDV markedly reduced the hepatocyte viability and albumin synthesis, while it increased the cleavage of caspase-8 and caspase-3, phosphorylation of histone H2AX, and release of ALT and AST in a concentration-dependent manner. Importantly, co-treatment with DEX partially reversed RDV-induced cytotoxic responses in human hepatocytes. Moreover, data from COVID-19 patients treated with RDV with and without DEX co-treatment suggested that among 1037 patients matched by propensity score, receiving the drug combination was less likely to result in elevation of serum AST and ALT levels (≥ 3 × ULN) compared to the RDV alone treated patients (OR = 0.44, 95% CI = 0.22-0.92, p = 0.03). Our findings obtained from in vitro cell-based experiments and patient data analysis provide evidence suggesting combination of DEX and RDV holds the potential to reduce the likelihood of RDV-induced liver injury in hospitalized COVID-19 patients.

Platelet activating factor receptor acts to limit colitis-induced liver inflammation.

Liver inflammation is a common extraintestinal manifestation in inflammatory bowel disease (IBD), yet, the mechanisms driving gut-liver axis inflammation remain poorly understood. IBD leads to a breakdown in the integrity of the intestinal barrier causing an increase in portal and systemic gut-derived antigens, which challenge the liver. Here, we examined the role of platelet activating factor receptor (PAFR) in colitis-associated liver damage using dextran sulfate sodium (DSS) and anti-CD40-induced colitis models. Both DSS and anti-CD40 models exhibited liver inflammation associated with colitis. Colitis reduced global PAFR protein expression in mouse livers causing an exclusive re-localization of PAFR to the portal triad. The global decrease in liver PAFR was associated with increased sirtuin 1 while relocalized PAFR expression was limited to Kupffer cells (KCs) and co-localized with toll-like receptor 4. DSS activated the NLRP3-inflammasome and increased interleukin (IL)-1β in the liver. Antagonism of PAFR amplified the inflammasome response by increasing NLRP3, caspase-1, and IL-1β protein levels in the liver. LPS also increased NLRP3 response in human hepatocytes, however, overexpression of PAFR restored the levels of NLPR3 and caspase-1 proteins. Interestingly, KCs depletion also increased IL-1β protein in mouse liver after DSS challenge. These data suggest a protective role for PAFR-expressing KCs during colitis and that regulation of PAFR is important for gut-liver axis homeostasis.

Robust hepatitis E virus infection and transcriptional response in human hepatocytes

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 105 and 106 FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral-host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.

Investigation of the Synergistic Effect of Brown Sugar, Longan, Ginger, and Jujube (Brown Sugar Longan Ginger Tea) on Antioxidation and Anti-Inflammation in In Vitro Models.

This research unveils the synergistic effect of brown sugar, longan, ginger, and jujube on the beneficial effects of antioxidation and anti-inflammation. Longan, ginger, and jujube are ubiquitous herbs in traditional Chinese medicine (TCM) and are frequently used in folk remedies. Longan and ginger have been reported to be beneficial for antioxidation, anti-inflammation, ant-obesity, and nonalcoholic fatty liver disease (NAFLD) improvements. However, the potential scientific and medical benefits of their combination Brown Sugar Longan Ginger Tea (BSLGT), a popular drink in Chinese cultures, are elusive. Through the in vitro methodologies, we discovered that BSLGT could significantly improve the mitochondrial activity, antioxidant capacity, lipid content, and inflammatory response in human hepatocytes. In addition, BSLGT also exerted positive effects on the downregulation of atherosclerosis-associated, vasoconstrictor, and thrombosis-related gene expression in human umbilical vein endothelial cells. In short, our experimental results successfully revealed that the antioxidative and anti-inflammatory effects of BSLGT may have the potential to improve liver metabolism and cardiovascular inflammation although solid evidence requires further investigation.

Orexin A ameliorates HBV X protein-induced cytotoxicity and inflammatory response in human hepatocytes

Hepatitis B virus is one of the main causes of hepatitis and hepatocellular carcinoma (HCC). Hepatitis B virus-encoded X protein (HBx) has been shown to be involved in many aspects of the pathogenicity of liver diseases. Orexin A is a small peptide produced in the hippocampus. Orexin A and its receptor have become important therapeutic targets for certain metabolic disorders. In this study, we show that orexin A has a protective role against HBx-induced cytotoxicity and inflammation in hepatocytes. The ectopic expression of HBx in hepatocytes reduces orexin A receptor 1 (OX1R) expression. When orexin A is added to the cells, it mitigates HBx-induced oxidative stress indicator 4-hydroxynonenal (4-HNE) and reactive oxygen species (ROS) as well the NADPH subunit NADPH oxidase 4 (NOX-4). Orexin A also ameliorates HBx-mediated mitochondrial membrane potential and adenosine triphosphate (ATP) reduction. Moreover, orexin A significantly inhibits HBx-induced production of pro-inflammatory cytokines including interleukin 8 (IL-8), tumour necrosis factor α (TNF-α) and chemokine ligand 2 (CXCL2). The presence of orexin A ameliorates HBx-induced lactate dehydrogenase (LDH) release, indicating that it could protect hepatocytes from cytotoxicity. Mechanistically, we found that orexin A suppresses c-Jun N-terminal kinase (JNK) phosphorylation, accumulation of nuclear factor-κB (NF-κB) protein p65 in nuclei, and NF-κB promoter activity, suggesting that orexin A suppresses JNK and NF-κB pathway activation. In conclusion, our study demonstrates that orexin A peptide possesses a protective role against HBx-mediated cytotoxicity and inflammation in hepatocytes.

In vitro Drug Metabolism Investigation of 7-Ethoxycoumarin in Human, Monkey, Dog and Rat Hepatocytes by High Resolution LC-MS/MS.

Background: Recently, it has been an increasing concern on the bioactivation and adverse re-actions associated with consumption of herbal and nature products. 7-Ethoxycoumarin is one of coumarin family compounds, but little information is available regarding its potential reactive metabolites.Method: 7-ethoxylcoumarin was incubated individually with human, monkey, dog and rat hepatocytes for 2 hr, metabolites were detected, identified and characterized using high resolution liquid chromagraphy – tandem mass spectrometry.Results: Twenty-eight metabolites (M1 - M28) were detected and identified. O-deethylation, glucuronida-tion, sulfation, oxygenation, oxidative ring-opening, hydrogenation, glutathionation, dehydrogenation, cysteination, glucosidation, methylation, and hydrolysis were observed. At least sixteen metabolites not reported previously, were newly identified. M1 (O-deethylation, mono-oxygenation and glucuronidation), M3 (O-deethylation and glucuronidation), M5 (hydrolysis and mono-oxygenation), M14 (O-deethylation), M16 (hydrolysis), M22 (oxidative ring-opening and oxygenation) and M27 (mono-oxygenation) exhibited high mass spectrometric responses in human hepatocytes. M3, M5, M8, M13 (mono-oxygenation), M14, M16, M18 (O-deethylation and sulfation), M22 and M27 exhibited high mass spectrometric responses in monkey hepatocytes. M14, M16, M18, M20 (glutathionation and dehy-drogenation) and M27 exhibited high mass spectrometric responses in dog hepatocytes. M1 (O-deethylation, mono-oxygenation and glucuronidation), M3, M5, M13, M14, M16, M17 (cysteination), M18, M20, and M22 exhibited high mass spectrometric responses in rat hepatocytes.Conclusion: Most of new metabolites via oxidative ring-opening and glutathionation were identified. Species differences in metabolism of 7-ethoxylcoumarin in hepatocytes were observed. The analysis of metabolites suggests that 7-ethoxylcoumarin may undergo 3,4-epoxidation responsible for formation of glutathione and its derived cysteine conjugates, carboxylic acid and its glucuronides, glucosides and sul-fate.

ULBP1 is induced by hepatitis C virus infection and is the target of the NK cell-mediated innate immune response in human hepatocytes.

Natural killer (NK) cells through their NK group 2 member D (NKG2D) receptors recognize NKG2D ligands such as UL16‐binding proteins (ULBPs) on virus‐infected cells and subsequently trigger the host innate immune response. In the present study, we demonstrated that hepatitis C virus (HCV) induced the cell surface expression of ULBP1 in human immortalized hepatocyte PH5CH8 cells and human hepatoma HuH‐7 cell‐derived RSc cells. Interestingly, NK cell line NK‐92 induced cytotoxicity and interferon‐γ mRNA expression and subsequently reduced the levels of HCV RNA replication during co‐culture with HCV‐infected RSc cells. From these results, we conclude that ULBP1 is a target of the NK cell‐mediated innate immune response in HCV‐infected human hepatocytes.

Combined incubation of cadmium, docosahexaenoic and eicosapentaenoic acid affecting the oxidative stress and antioxidant response in human hepatocytes in vitro.

Human hepatocellular cells Hep G2 were used to investigate the effects of the intake of contaminated fish on oxidative stress. Uptake of heavy metal contaminated fish was mimicked by incubating the cells with a combination of cadmium chloride (Cd(2+)) as possible contaminant and a combination of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as important fatty acids (FA) specific for fish. The main aim of this study was to determine the effects of these co-incubations (FA, Cd(2+)) on lipid and protein oxidation. In addition we also evaluated the antioxidant response of the cells using two different methods (SOD and TAC). Pre-incubation with the chosen FA significantly reduced the oxidative stress caused by incubation with Cd(2+). We measured an increased level of carbonyl proteins (CP) in the cells pre-incubated with bovine serum albumin (BSA) and post-incubated with Cd(2+).

Interferon-lambda (IFN-λ) induces signal transduction and gene expression in human hepatocytes, but not in lymphocytes or monocytes

This study compared the ability of IFN-α and IFN-λ to induce signal transduction and gene expression in primary human hepatocytes, PBLs, and monocytes. IFN-α drug products are widely used to treat chronic HCV infection; however, IFN-α therapy often induces hematologic toxicities as a result of the broad expression of IFNARs on many cell types, including most leukocytes. rIFN-λ1 is currently being tested as a potential alternative to IFN-α for treating chronic HCV. Although IFN-λ has been shown to be active on hepatoma cell lines, such as HepG2 and Huh-7, its ability to induce responses in primary human hepatocytes or leukocytes has not been examined. We found that IFN-λ induces activation of Jak/STAT signaling in mouse and human hepatocytes, and the ability of IFN-λ to induce STAT activation correlates with induction of numerous ISGs. Although the magnitude of ISG expression induced by IFN-λ in hepatocytes was generally lower than that induced by IFN-α, the repertoire of regulated genes was quite similar. Our findings demonstrate that although IFN-α and IFN-λ signal through distinct receptors, they induce expression of a common set of ISGs in hepatocytes. However, unlike IFN-α, IFN-λ did not induce STAT activation or ISG expression by purified lymphocytes or monocytes. This important functional difference may provide a clinical advantage for IFN-λ as a treatment for chronic HCV infection, as it is less likely to induce the leukopenias that are often associated with IFN-α therapy.

I18 Microrna’s regulating gene expression as novel markers and therapeutic markers

The epigenetic regulatory mechanisms involving all biological processes in health and disease include acetylation of histones, methylation of certain CPG islands in promoters, phosphorylation, sumoylation/ubiquination and miRs or microRNAs. All these processes are intimately intertwined. In this regard, microRNAs can be methylated and regulated by specific histone marks. The best example was shown recently in a most comprehensive study performed by Stefan Kuchen et al characterizing the distinct sets of micro RNAs and their controlling histone marks regulating the immune response in mice (1). Our laboratory studies the various epigenetic regulations in rheumatic diseases (2),With respect to microRNAs, which are short RNA sequences comprised of 20-22 nucleotides, bind to complementary mRNA sequences along with argonaute proteins resulting in translational repression and thereby silencing of gene expression. More than 1000 microRNAs are involved in the modulation of gene expression. For example, we could demonstrate that microRNA 155 is regulated by TNFa, IL-6 and TLR signaling (3). Moreover, based on the fact that microRNA 155 is involved into B and T cell development, we could show that microRNA ko mice do not develop arthritis (4). On the other hand, we could report that microRNA 203 is regulating the expression of IL-6 and that this microRNA is induced by hypomethylation of its promoter (5).On the other hand, IL-6 regulates the microRNA cluster 17/92 in the down regulation of bone morphogenic protein receptor 2 (BMPR2) in vascular cells during the development of pulmonary hypertension (6). In related studies Matthias Brock in our lab could show that microRNA-18a enhances the interleukin-6-mediated production of the acute-phase proteins fibrinogen and haptoglobin in human hepatocytes (7). These data reveal, for the first time, a microRNA-mediated positive feedback loop of IL-6 signal transduction leading to an enhanced acute-phase response in human hepatocytes. MicroRNAs will be novel diagnostic biological markers and new therapeutic targets

MicroRNA-18a Enhances the Interleukin-6-mediated Production of the Acute-phase Proteins Fibrinogen and Haptoglobin in Human Hepatocytes

The acute-phase response is an inflammatory process triggered mainly by the cytokine IL-6. Signaling of IL-6 is transduced by activation of STAT3 (signal transducer and activator of transcription 3), which rapidly induces the production of acute-phase proteins such as haptoglobin and fibrinogen. Another target of the IL-6/STAT3 signal transduction pathway is the microRNA cluster miR-17/92. Here, we investigated the interplay of miR-17/92 and STAT3 signaling and its impact on the acute-phase response in primary human hepatocytes and hepatoma (HepG2) cells. Employing a reporter gene system consisting of STAT3-sensitive promoter sequences, we show that the miR-17/92 cluster member miR-18a enhanced the transcriptional activity of STAT3. IL-6 stimulation experiments in miR-18a-overexpressing hepatocytes and HepG2 cells revealed an augmented acute-phase response indicated by increased expression and secretion of haptoglobin and fibrinogen. This effect was due, at least in part, to repression of PIAS3 (protein inhibitor of activated STAT, 3), a repressor of STAT3 activity, which we identified as a novel direct target of miR-18a. Finally, we demonstrate that the expression of miR-17/92 in primary hepatocytes and HepG2 cells is modulated by IL-6. Our data reveal, for the first time, a microRNA-mediated positive feedback loop of IL-6 signal transduction leading to an enhanced acute-phase response in human hepatocytes.

Catecholamines induce an inflammatory response in human hepatocytes

The liver is an early target organ in sepsis, severe sepsis, and septic shock, contributing to multiple organ failure, and both lipopolysaccharide and gut-derived catecholamines are implicated in the occurrence of hepatocellular dysfunction. Treatment of septic shock involves administration of vasoactive agents such as exogenous catecholamines or vasopressin in order to reestablish blood pressure. As a prelude to clinical application, we tested the hypothesis that catecholamines could modulate the lipopolysaccharide-induced inflammatory response and function in human liver. An in vitro human cell culture study. Research laboratory of an academic institution. Primary human hepatocytes and human hepatoma HepaRG cells. Primary human hepatocytes and human hepatoma HepaRG cells were exposed to lipopolysaccharide to evaluate effects of epinephrine and several other compounds (norepinephrine, dobutamine, dopamine, dopexamine, phenylephrine, clonidine, salbutamol, and vasopressin). Markers of inflammation (interleukin-6, C-reactive protein) and drug metabolism (cytochrome P450 [CYP] 3A4, CYP2B6, CYP1A2, CYP2E1, constitutive androstane receptor, pregnane X receptor) were analyzed. Transcripts of C-reactive protein and CYP3A4 were strongly increased and depressed respectively after a 24-hr treatment with 10 ng/mL lipopolysaccharide. Co-treatment with either of the catecholamines failed to reverse lipopolysaccharide effects, whereas when added alone, epinephrine, and to a lesser extent norepinephrine, salbutamol, and dobutamine, mimicked lipopolysaccharide effects. Suppression of CYP3A4 implicated beta-adrenergic receptors and was mediated through overproduction of interleukin-6. By contrast, vasopressin did not elicit an inflammatory response or modify CYP3A4 expression. Some catecholamines can induce an inflammatory response and exacerbate the hepatic dysfunction observed during sepsis, favoring the idea that catecholamines could alter the biotransformation of drugs metabolized by CYP3A4 and that alternative vasoactive agents, such as vasopressin, merit further investigation in septic shock patients.

The cachectic mediator proteolysis inducing factor activates NF-κB and STAT3 in human Kupffer cells and monocytes

A novel proteoglycan, proteolysis inducing factor (PIF), is capable of inducing muscle proteolysis during the process of cancer cachexia, and of inducing an acute phase response in human hepatocytes. We investigated whether PIF is able to activate pro-inflammatory pathways in human Kupffer cells, the resident macrophages of the liver, and in monocytes, resulting in the production of pro-inflammatory cytokines. Normal liver tissue was obtained from patients undergoing partial hepatectomy and Kupffer cells were isolated. Monocytes were isolated from peripheral blood. Following exposure to native PIF, pro-inflammatory cytokine production from Kupffer cells and monocytes was measured and the NF-kappaB and STAT3 transcriptional pathways were investigated using electrophoretic mobility shift assays. We demonstrate that PIF is able to activate the transcription factor NF-kappaB and NF-kappaB-inducible genes in human Kupffer cells, and in monocytes, resulting in the production of pro-inflammatory cytokines such as TNF-alpha, IL-8 and IL-6. PIF enhances the expression of the cell surface molecules LFA-1 and CD14 on macrophages. PIF also activates the transcription factor STAT3 in Kupffer cells. The pro-inflammatory effects of PIF, mediated via NF-kappaB and STAT3, are important in macrophage behaviour and may contribute to the inflammatory pro-cachectic process in the liver.

Acute-phase response of human hepatocytes after infection with Chlamydia pneumoniae and cytomegalovirus.

There is increasing evidence that chronic inflammation plays a pivotal role in the development of atherosclerosis. Whether inflammation is the cause or consequence of vascular damage is unclear. Also, the source of inflammation is unknown, but may well be infection by Cytomegalovirus (CMV) or Chlamydia pneumoniae (C. pneumoniae). Infection of the liver by CMV or C. pneumoniae may induce a general inflammatory reaction contributing to accelerated atherogenesis. In this study we investigated the production of interleukin-6 (IL-6), fibrinogen and plasminogen activator inhibitor-1 (PAI-1) by hepatocytes after infection with CMV or C. pneumoniae. HepG2 cell monolayers were grown to confluence in 48-well tissue culture plates. Hepatocytes were infected with 50 microL or 100 microL of suspension of CMV (10(2.70) TCID50 mL(-1)) and C. pneumoniae (10(4.75) TCID50 mL(-1)). The medium of the inoculated cells was collected every 24 h, from day 1 to day 4, for determination of IL-6, PAI-1 and fibrinogen concentrations. Fibrinogen production was increased significantly in a dose-dependent manner after infection with CMV (50 microL: P=0.022 and 100 microL: P<0.001) and C. pneumoniae (P=0.012). Cytomegalovirus infection resulted in an increase of IL-6 production compared with uninfected cells (P=0.048). Cytomegalovirus and C. pneumoniae infection did not result in a significantly increase of PAI-1 production by hepatocytes. We conclude that in addition to direct vascular wall infection by C. pneumoniae and CMV, virus-related development of atherosclerosis might also be initiated by chronic liver infection and subsequent production of inflammatory and procoagulant mediators released in the circulation. This may be another pathophysiological link for the observed relation between infections and the development of atherosclerosis.

Low proliferative response of human hepatocytes to growth factors due to lack of cyclin D

Low proliferative response of human hepatocytes to growth factors due to lack of cyclin D

Low proliferative response of human hepatocytes to growth factors due to lack of cyclin D

Low proliferative response of human hepatocytes to growth factors due to lack of cyclin D

Comparison in human and rat hepatocytes of the DNA-damaging activity of five chemicals probably carcinogenic to humans

Five chemicals—acrylonitrile, adriamycin, bischloroethyl nitrosourea, phenacetin and procarbazine—classified by the International Agency for Research on Cancer as probably carcinogenic to humans were assayed for DNA-damaging activity in primary cultures of human and rat hepatocytes in order to assess possible interspecies differences that might cast doubt on the extrapolation to humans of results obtained in rodents. DNA damage was measured by the alkaline elution technique. In the range of subtoxic concentrations indicated, dose-related increases in the frequencies of DNA single-strand breaks were induced in cells of both species by acrylonitrile (1.0–5.6 m m) and procarbazine (5.6–18 m m), whereas phenacetin was inactive up to the maximal soluble dose (3.2 m m). Adriamycin (1.8–5.6 μ m) and bischloroethyl nitrosourea (18–56 μ m) produced in cells of both species dose-dependent increases in the frequencies of both DNA breaks and cross-links. The responses of human hepatocytes were qualitatively similar to those of rat hepatocytes, but modest statistically significant differences between the two species in the average frequencies of DNA lesions were observed with the four active agents: the amount of DNA damage was greater in rat than in human hepatocytes with acrylonitrile (1.7-fold), adriamycin (1.4-fold), and BCNU (1.3-fold), whereas procarbazine was more genotoxic (1.4-fold) for human hepatocytes. However, as the interindividual variability of the response was greater than that occurring between the two species, the results should be interpreted as indicating that rat hepatocytes are good predictors of metabolic activation/detoxification and DNA-damaging activity in humans for the five chemicals studied.