Primary Hepatocyte Cultures Research Articles

Environmental salinity differentiates responses to acute hypothermal stress in milkfish

Global warming has led to an increase in the frequency of cold extremes, causing significant economic losses in aquaculture, particularly in subtropical regions. Milkfish (Chanos chanos) holds significant importance in aquaculture within subtropical Asian regions. Despite milkfish's ability to tolerate varying salinity levels, frequent cold snaps associated with extreme weather events have caused substantial mortality. Understanding the molecular and cellular mechanisms underlying cold stress-induced cell death is crucial for developing effective strategies to mitigate such losses. Given the pivotal role of the liver in fish physiology, we established a primary milkfish hepatocyte culture demonstrating robust proliferation and expressing a unique marker leptin A. The molecular characterization of cold-treated hepatocytes at 18 °C showed that the mRNA levels of superoxide dismutase (sod1) and catalase (cat), responsible for neutralizing reactive oxygen species (ROS), were downregulated in freshwater (FW) conditions, while cat expression was upregulated in seawater (SW) conditions. This differential modulation of ROS signaling implies distinct responses in FW and SW, leading to higher ROS levels and increased cell death in FW condition compared to those in SW. Transcriptomic analysis of liver tissues from milkfish reared in FW or SW, with or without cold stress, revealed distinct gene expression patterns. Although cold stress affected a common set of genes in both FW and SW conditions, SW-specific cold responsive genes are associated with metabolic pathways while FW-specific genes were linked to cell proliferation pathways. Notably, gene set enrichment analysis highlighted the downregulation of cytochrome-related genes, a major source of ROS production, in response to cold stress in SW. In summary, our study unveils an insightful mechanism whereby the salinity of SW counteracts cold stress-induced ROS signaling, emphasizing the feasibility and practicality of preconditioning fish in SW as a preventive measure against cold stress-induced mortality.

Phenylephrine Enhances the Mitogenic Effect of S-Allyl-L-cysteine on Primary Cultured Hepatocytes through Protein Kinase C-Induced B-Raf Phosphorylation.

The co-mitogenic effects of the α1-adrenoceptor agonist phenylephrine on S-allyl-L-cysteine (SAC)-induced hepatocyte proliferation were examined in primary cultures of adult rat hepatocytes. The combination of phenylephrine (10-10-10-6 M) and SAC (10-6 M) exhibited a significant dose-dependent increase in the number of hepatocyte nuclei and viable cells compared to SAC alone. This combination also increased the progression of hepatocyte nuclei into the S-phase. The potentiating effect of phenylephrine on SAC-induced cell proliferation was counteracted by prazosin (an α1-adrenergic receptor antagonist) and GF109203X (selective protein kinase C (PKC) inhibitor). In addition, PMA (direct PKC activator) potentiated the proliferative effects of SAC similarly to phenylephrine. In essence, these findings suggest that PKC activity plays a crucial role in enhancing SAC-induced cell proliferation. Moreover, the effects of phenylephrine on SAC-induced Ras activity, Raf phosphorylation, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation were investigated. Phenylephrine (or PMA) in combination with SAC did not augment Ras activity, but further increased ERK2 phosphorylation and its upstream B-Raf phosphorylation. These results indicate that PKC activation, triggered by stimulating adrenergic α1 receptors, further amplifies SAC-induced cell proliferation through enhanced ERK2 phosphorylation via increased B-Raf-specific phosphorylation in primary cultured hepatocytes.

Liver mechanosignaling as a natural anti-hepatitis B virus mechanism

The mechanisms underlying the natural control of hepatitis B virus (HBV) infection have long been an intriguing question. Given the wide physiological range of liver stiffness and the growing attention to the role of mechanical microenvironment in homeostasis and diseases, we investigated how physical matrix cues impact HBV replication. High matrix stiffness significantly inhibited HBV replication and activated YAP in primary hepatocyte culture system, a key molecule in mechanosignaling. YAP activation notably suppressed HBV transcription and antigen expression. Several YAP-induced genes exhibited strong anti-HBV effects. Single-cell analysis of liver tissue from male individuals with active HBV replication revealed a strong significant negative correlation between YAP signature activation and HBV transcript levels. Intraperitoneal administration of YAP small molecule agonist potently controls HBV in male mouse models. These findings unveil a mechanism that involves the mechanical environment of hepatocytes and YAP to clear hepatotropic viral infection in the liver, providing new perspectives for HBV cure studies and antiviral development.

L-Glutamine mitigates bile acid-induced inhibition of growth factor activity in rat hepatocyte cultures

Bile acid-induced hepatotoxicity is inevitable in Cholestasis pathogenesis and L-Glutamine (L-Gln) has been reported to prevent total parenteral nutrition (TPN)-induced cholestasis in premature neonates. While mechanisms remain unknown, we hypothesize that bile acids impair growth factor (GF) function in hepatocytes which L-glutamine prevents through NAPDH oxidase (NOX) modulation. Glycochenodeoxycholic acid (GCDC, 0–100 µM) when added to primary hepatocyte cultures significantly (p < 0.01) decreased the FBS-induced BrdU incorporation, however inhibition of Fibroblast Growth factor (FGF)- or Hepatocyte growth factor (HGF)-induced DNA synthesis was more pronounced (p < 0.001). L-Gln markedly attenuated GCDC-mediated inhibition of DNA synthesis in both FBS and GF-treated cells. GCDC significantly increased the NADPH oxidase activity and NOX-1 protein expression that were markedly reduced by L-Gln and protein kinase c (PKC) inhibitor, LY-333531. Apocynin (APCN) and diphenyliodonium (DPI) significantly blocked the GCDC-mediated inhibition of GF-induced DNA synthesis. This study demonstrates that bile acid-induced hepatotoxicity involves dysfunction of certain growth factors via protein kinase c (PKC)- mediated NOX modulation which can be corrected, at least partly, by L-glutamine.

Genome Engineering of Primary and Pluripotent Stem Cell-Derived Hepatocytes for Modeling Liver Tumor Formation.

Genome editing has demonstrated its utility in generating isogenic cell-based disease models, enabling the precise introduction of genetic alterations into wild-type cells to mimic disease phenotypes and explore underlying mechanisms. However, its application in liver-related diseases has been limited by challenges in genetic modification of mature hepatocytes in a dish. Here, we conducted a systematic comparison of various methods for primary hepatocyte culture and gene delivery to achieve robust genome editing of hepatocytes ex vivo. Our efforts yielded editing efficiencies of up to 80% in primary murine hepatocytes cultured in monolayer and 20% in organoids. To model human hepatic tumorigenesis, we utilized hepatocytes differentiated from human pluripotent stem cells (hPSCs) as an alternative human hepatocyte source. We developed a series of cellular models by introducing various single or combined oncogenic alterations into hPSC-derived hepatocytes. Our findings demonstrated that distinct mutational patterns led to phenotypic variances, affecting both overgrowth and transcriptional profiles. Notably, we discovered that the PI3KCA E542K mutant, whether alone or in combination with exogenous c-MYC, significantly impaired hepatocyte functions and facilitated cancer metabolic reprogramming, highlighting the critical roles of these frequently mutated genes in driving liver neoplasia. In conclusion, our study demonstrates genome-engineered hepatocytes as valuable cellular models of hepatocarcinoma, providing insights into early tumorigenesis mechanisms.

Investigation of the therapeutic effect of melatonin on deltamethrin applied mouse primary hepatocyte culture

Objective In recent years, it has been known that the melatonin hormone, secreted from the pineal gland, possesses significant antioxidant activity. This study explores the therapeutic effect of melatonin on the deleterious effects of deltamethrin, a pyrethroid pesticide extensively used worldwide, including in Türkiye, on mouse liver cells. Methods Hepatocytes from Balb/C mice were isolated using a two-stage perfusion method, resulting in over 85% live hepatocytes. The isolated cells were cultured with different doses of deltamethrin (1 and 10 µM) and melatonin (100 µM) for 24 and 48 hours. At the conclusion of the culture period, hepatocytes were extracted at the 24th and 48th hours, and Malondialdehyde (MDA), Total Antioxidant Capacity (TAC), Total Oxidation Status (TOS), and DNA damages (8-hydroxy-2’-deoxyguanosine (8-OHdG)) were examined. Results While an increase in MDA, TOS, and DNA damage was observed in the deltamethrin-administered groups of hepatocytes, a decrease in TAC level was noted. It was determined that the applied deltamethrin had no effect on cell viability throughout the application period. Conclusion Furthermore, it was observed that melatonin, when administered concurrently with deltamethrin, reduced the toxic effect of deltamethrin. This study suggests that melatonin has a protective effect against deltamethrin-induced damage in mouse hepatocyte cells.

Quercetin ameliorates lipid deposition in primary hepatocytes of the chicken embryo

ABSTRACT 1. The accumulation of excessive fat plays a role in the development of non-alcoholic fatty liver disease (NAFLD) and phytogenic feed additives have the potential to ameliorate this. This study involved the isolation and culture of primary hepatocytes from chicken embryos to establish a model of hepatic steatosis induced by oleic acid/dexamethasone (OA/DEX). Lipid accumulation and cell viability were assessed using Nile Red staining, Oil Red O staining and cell count Kit −8 (CCK8) following treatment with varying concentrations of quercetin (Que). The potential mechanism by which Que exerts its effects was preliminarily investigated. 2. The results indicated that OA effectively treated lipid accumulation in hepatocytes. There was no notable variance in cell proliferation between the normal and OA/DEX groups when subjected to Que treatment at concentrations of 1000 ng/ml and 10 000 ng/ml. Triglycerides and cholesterol (low and high density) decreased with Que treatment, with the most substantial reduction observed at 10 000 ng/ml. 3. Gene expression levels decreased to levels similar to those in the control groups. Western blot data demonstrated that sterol regulatory element-binding protein 1 (SREBP-1) protein expression correlated with its mRNA expression level. Que mitigated lipid accumulation through the alpha serine/threonine protein kinase (AKT) and extracellular signal-regulated kinase (ERK) pathways. Expression levels of lipid-related genes (APOB, PPARα, CYP3A5 and SREBP-1) decreased to levels similar to the control groups. Western blot data demonstrated that the SREBP-1 protein expression correlated with its mRNA expression level. 4. Supplementation with Que ameliorated lipid accumulation through AKT and ERK signalling pathway in OA/DEX-induced high-fat hepatocytes.

A Novel Role for the Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Delta Isoform in Hepatocellular Proliferation

The phosphatidylinositol-4,5-bisphosphate 3-kinase delta isoform (Pik3cd), usually considered immune-specific, was unexpectedly identified as a gene potentially related to either regeneration and/or differentiation in animals lacking hepatocellular Integrin Linked Kinase (ILK). Since a specific inhibitor (Idelalisib, or CAL101) for the catalytic subunit encoded by Pik3cd (p110δ) has reported hepatotoxicity when used for treating Chronic Lymphocytic Leukemia and other lymphomas, we aimed to elucidate if there is a role for p110δ in normal liver function. To determine the effect on normal liver regeneration, partial hepatectomy (PHx) was performed using mice in which p110δ was first inhibited using CAL101. Inhibition led to over a 50% decrease in proliferating hepatocytes in the first two days after PHx. This difference correlated with phosphorylation changes in the HGF and EGF receptors (MET and EGFR, respectively) and NF-κB signaling. Ingenuity Pathway Analyses implicated C/EBPβ, HGF and the EGFR heterodimeric partner, ERBB2, as three of the top 20 regulators downstream of p110δ signaling as their pathways were suppressed in the presence of CAL101 at one day post-PHx. A regulatory role for p110δ signaling in mouse and rat hepatocytes through MET and EGFR was further verified using hepatocyte primary cultures, in the presence or absence of CAL101. Combined, this data supports a role for p110δ as a downstream regulator of normal hepatocytes when stimulated to proliferate.

Apolipoprotein B Secretion Assay from Primary Hepatocytes.

Apolipoprotein B (APOB) is the primary structural protein of atherogenic lipoproteins, which drive atherogenesis and thereby lead to deadly cardiovascular diseases (CVDs). Plasma levels of APOB-containing lipoproteins are tightly modulated by LDL receptor-mediated endocytic trafficking and cargo receptor-initiated exocytic route; the latter is much less well understood. This protocol aims to present an uncomplicated yet effective method for detecting APOB/lipoprotein secretion. We perform primary mouse hepatocyte isolation and culture coupled with well-established techniques such as immunoblotting for highly sensitive, specific, and semi-quantitative analysis of the lipoprotein secretion process. Its inherent simplicity facilitates ease of operation, rendering it a valuable tool widely utilized to explore the intricate landscape of cellular lipid metabolism and unravel the mechanistic complexities underlying lipoprotein-related diseases. Key features • A pipeline for the isolation and subsequent culture of mouse primary hepatocytes. • A procedure for tracking the secretion of APOB-containing lipoproteins. • A rapid and sensitive assay for detecting the APOB level based on immunoblotting.

Human Hepatic Spheroid Coculture Model for the Assessment of Drug-Induced Liver Injury.

Accurate evaluation of potential drug risks such as drug-induced liver injury (DILI) continues to be a challenge faced by pharmaceutical industry and regulatory agencies. Preclinical testing has served as a foundation for the evaluation of the potential risks and effectiveness of investigational new drug (IND) products in humans. However, current two-dimensional (2D) in vitro human primary hepatocyte (HPH) culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, while animal studies present inherited species-specific differences and low throughput scales. Thus, there is a continued demand to establish new approaches that can better characterize DILI during drug discovery and development. Among others, the three-dimensional (3D) hepatic spheroid model comprising self-aggregated primary human hepatocytes cocultured with non-parenchymal cells (NPCs) appears to be a more accurate representation of the natural hepatic microenvironment with intercellular interactions between hepatocytes, stellate cells, Kupffer cells, liver sinusoidal endothelial cells (LSECs), and other cell types. This model holds the potential to improve the ability for long-term functional and toxicological studies. Here, we provide methodological details for this human hepatic spheroid coculture model system.

AML Inhibits Lipid Uptake By the Liver through an HGF/c-MET Dependent Mechanism Which Results in an Increased Supply of Free Fatty Acid to the Tumor

Acute myeloid leukemia (AML) requires a high metabolic turnover to allow for constant expansion. We and others have previously shown that primary human AML blasts rely on fatty acid (FA)-oxidation for survival and proliferation. (Shafat MS, et al. 2017, Jones CL, et al. 2018). The liver plays a major role in carbohydrate, protein, amino acid, and lipid metabolism (Hodson L, and Gunn PJ. 2019). Given that AML is a FA dependent tumor, we have studied the interactions between AML and the liver, and specifically how these affect lipid metabolism, the availability of pro-tumoral FFA and ultimately the mechanism by which this supports AML growth. To understand the role of lipid metabolism we used two syngeneic models of AML (HOXA9/Meis1 or MN1). When engrafted with AML cells, mice body weight during the latter stages of disease. After sacrifice, the size of epididymal and inguinal fat pads were significantly reduced in mice with AML compared to controls. Mice with AML also had elevated free fatty acid (FFA) and reduced glucose in the plasma. We and others have shown that AML induces lipolysis of white adipose tissue for uptake by blasts and resulting in tumor expansion (Shafat MS, et al. 2017, Ye H, et al. 2016). Since the liver is the master regulator of available FA in the plasma, we isolated livers from AML engrafted mice. Transcriptomics revealed down-regulation of genes involved in FA uptake and metabolism. RT-qPCR of RNA isolated from liver samples confirmed these results showing down-regulation of FA transport proteins, (FABP1, SLC27A2, SLC27A5, and CD36) and FA metabolism genes (CPT1, ACADM, and HMGCS2). Furthermore, primary hepatocytes isolated and cultured with AML conditioned media showed a down-regulation of FA uptake and metabolism genes. These data support the hypothesis that AML secretes a factor that alters lipid metabolism in the liver. To discover the factor that AML secretes, and which regulates FA uptake and metabolism in the liver, a proteome profile of the serum from AML engrafted mice was compared to conditioned media from AML cultured cells. Pathway analysis revealed hepatocyte growth factor (HGF), IL-1B and Granulocyte colony stimulating factor (G-CSF) as possible key factors involved in liver FA metabolism (Jing Y, et al. 2019, Kaibori M, et al. 2002). In vitro gene expressions analysis of primary hepatocytes revealed that addition of HGF to the medium, but not Il-1b or G-CSF, inhibited expression of FA uptake genes. In addition, using Seahorse mitochondrial stress kit we confirmed that primary hepatocytes decreased FA oxidation when cultured with AML conditioned media or HGF. Next, we confirmed that AML derived HGF was responsible for inhibition of hepatocyte FA genes by using a neutralizing antibody to HGF which blocked the transcriptional changes previously observed in hepatocytes cultured in AML conditioned media. This showed that AML derived HGF downregulates hepatocyte FA uptake and metabolism. Finally, to determine the role of AML in the uptake of FA by hepatocytes, cultures of primary murine hepatocytes were pretreated with AML conditioned media or HGF with and without neutralizing antibody. A BODIPY conjugated long chain FA was then added to the culture. Images analysis showed hepatocytes had reduced lipid uptake in the presence of AML condition media or HGF, but this was restored with the addition of HGF neutralizing antibody. Here we report that AML inhibition of lipid uptake in the liver, through an HGF dependent mechanism, increases the availability of plasma FA and forms a fundamental part of the AML disease. Ongoing work to develop our understanding of how AML increases access to FA to aid proliferation, may aid in the development of new therapeutics to target liver metabolism in combination with other AML therapies.

"Small Hepatocytes" in the Liver.

Mature hepatocytes (MHs) in an adult rodent liver are categorized into the following three subpopulations based on their proliferative capability: type I cells (MH-I), which are committed progenitor cells that possess a high growth capability and basal hepatocytic functions; type II cells (MH-II), which possess a limited proliferative capability; and type III cells (MH-III), which lose the ability to divide (replicative senescence) and reach the final differentiated state. These subpopulations may explain the liver's development and growth after birth. Generally, small-sized hepatocytes emerge in mammal livers. The cells are characterized by being morphologically identical to hepatocytes except for their size, which is substantially smaller than that of ordinary MHs. We initially discovered small hepatocytes (SHs) in the primary culture of rat hepatocytes. We believe that SHs are derived from MH-I and play a role as hepatocytic progenitors to supply MHs. The population of MH-I (SHs) is distributed in the whole lobules, a part of which possesses a self-renewal capability, and decreases with age. Conversely, injured livers of experimental models and clinical cases showed the emergence of SHs. Studies demonstrate the involvement of SHs in liver regeneration. SHs that appeared in the injured livers are not a pure population but a mixture of two distinct origins, MH-derived and hepatic-stem-cell-derived cells. The predominant cell-derived SHs depend on the proliferative capability of the remaining MHs after the injury. This review will focus on the SHs that appeared in the liver and discuss the significance of SHs in liver regeneration.

SIRT1 regulates hepatocyte programmed cell death via GSDME - IL18 axis in human and mouse liver transplantation

Sirtuin 1 (SIRT1) is a histone/protein deacetylase in the cellular response to inflammatory, metabolic, and oxidative stressors. We previously reported that myeloid SIRT1 regulates the inflamed liver’s canonical pyroptosis cell death pathway. However, whether/how hepatocyte SIRT1 is engaged in programmed cell death in the cold-stressed liver remains uncertain. Here, we undertook translational studies in human and mouse orthotopic liver transplantation (OLT) to interrogate the significance of hepatocyte-specific SIRT1 in cold-stored donor livers and liver grafts after reperfusion. In the clinical arm of sixty human OLT patients, hepatic SIRT1 levels in cold-preserved donor livers correlated with the anti-apoptotic Bcl-2 expression. After reperfusion, improved OLT function was accompanied by hepatic SIRT1 levels negatively associated with cleaved caspase-3 expression. In the experimental arm, we compared FLOX-control with hepatocyte-specific SIRT1-KO livers after orthotopic transplantation into WT mouse recipients, parallel with primary murine hepatocyte cultures subjected to cold activation with/without knockdown of SIRT1, GSDME, and IL18Rβ. Indeed, hepatocyte SIRT1 deficiency upregulated apoptosis and GSDME-mediated programmed cell death, deteriorating hepatocellular function and shortening OLT survival. Augmented GSDME processing, accompanied by increased secretion of IL18 by stressed hepatocytes, was prominent in SIRT1-deficient, cold-stored livers. Hepatocyte SIRT1 expression regulated anti-apoptotic Bcl-2/XIAP proteins, suppressed cold stress-triggered apoptosis, and mitigated GSDME licensing to release IL18. Notably, consistent with the ability of IL18 to depress hepatocyte SIRT1 and Bcl-2/XIAP in vitro, IL18 neutralization in vivo prevented hepatocellular damage and restored the anti-apoptotic phenotype in otherwise injury-prone SIRT1-deficient OLTs. In conclusion, this translational study identifies a novel hepatocyte SIRT1-IL18 molecular circuit as a therapeutic target in the mechanism underpinning hepatocyte death pathways in human and mouse liver transplantation.

Low concentrations of ethylene bisdithiocarbamate pesticides maneb and mancozeb impair manganese and zinc homeostasis to induce oxidative stress and caspase-dependent apoptosis in human hepatocytes

The worldwide and intensive use of phytosanitary compounds results in environmental and food contamination by chemical residues. Human exposure to multiple pesticide residues is a major health issue. Considering that the liver is not only the main organ for metabolizing pesticides but also a major target of toxicities induced by xenobiotics, we studied the effects of a mixture of 7 pesticides (chlorpyrifos-ethyl, dimethoate, diazinon, iprodione, imazalil, maneb, mancozeb) often detected in food samples. Effects of the mixture was investigated using metabolically competent HepaRG cells and human hepatocytes in primary culture. We report the strong cytotoxicity of the pesticide mixture towards hepatocytes-like HepaRG cells and human hepatocytes upon acute and chronic exposures at low concentrations extrapolated from the Acceptable Daily Intake (ADI) of each compound. Unexpectedly, we demonstrated that the manganese (Mn)-containing dithiocarbamates (DTCs) maneb and mancozeb were solely responsible for the cytotoxicity induced by the mixture. The mechanism of cell death involved the induction of oxidative stress, which led to cell death by intrinsic apoptosis involving caspases 3 and 9. Importantly, this cytotoxic effect was found only in cells metabolizing these pesticides. Herein, we unveil a novel mechanism of toxicity of the Mn-containing DTCs maneb and mancozeb through their metabolization in hepatocytes generating the main metabolite ethylene thiourea (ETU) and the release of Mn leading to intracellular Mn overload and depletion in zinc (Zn). Alteration of the Mn and Zn homeostasis provokes the oxidative stress and the induction of apoptosis, which can be prevented by Zn supplementation. Our data demonstrate the hepatotoxicity of Mn-containing fungicides at very low doses and unveil their adverse effect in disrupting Mn and Zn homeostasis and triggering oxidative stress in human hepatocytes.

The Coumarin-Derivative Esculetin Protects against Lipotoxicity in Primary Rat Hepatocytes via Attenuating JNK-Mediated Oxidative Stress and Attenuates Free Fatty Acid-Induced Lipid Accumulation.

Coumarin derivates have been proposed as a potential treatment for metabolic-dysfunction-associated fatty liver disease (MAFLD). However, the mechanisms underlying their beneficial effects remain unclear. In the present study, we explored the potential of the coumarin derivate esculetin in MAFLD, focusing on hepatocyte lipotoxicity and lipid accumulation. Primary cultures of rat hepatocytes were exposed to palmitic acid (PA) and palmitic acid plus oleic acid (OA/PA) as models of lipotoxicity and lipid accumulation, respectively. Esculetin significantly reduced oxidative stress in PA-treated hepatocytes, as shown by decreased total reactive oxygen species (ROS) and mitochondrial superoxide production and elevated expression of antioxidant genes, including Nrf2 and Gpx1. In addition, esculetin protects against PA-induced necrosis. Esculetin also improved lipid metabolism in primary hepatocytes exposed to nonlipotoxic OA/PA by decreasing the expression of the lipogenesis-related gene Srebp1c and increasing the expression of the fatty acid β-oxidation-related gene Ppar-α. Moreover, esculetin attenuated lipid accumulation in OA/PA-treated hepatocytes. The protective effects of esculetin against lipotoxicity and lipid accumulation were shown to be dependent on the inhibition of JNK and the activation of AMPK, respectively. We conclude that esculetin is a promising compound to target lipotoxicity and lipid accumulation in the treatment of MAFLD.

Identification of CCZ1 as an essential lysosomal trafficking regulator in Marburg and Ebola virus infections

Marburg and Ebola filoviruses are two of the deadliest infectious agents and several outbreaks have occurred in the last decades. Although several receptors and co-receptors have been reported for Ebola virus, key host factors remain to be elucidated. In this study, using a haploid cell screening platform, we identify the guanine nucleotide exchange factor CCZ1 as a key host factor in the early stage of filovirus replication. The critical role of CCZ1 for filovirus infections is validated in 3D primary human hepatocyte cultures and human blood-vessel organoids, both critical target sites for Ebola and Marburg virus tropism. Mechanistically, CCZ1 controls early to late endosomal trafficking of these viruses. In addition, we report that CCZ1 has a role in the endosomal trafficking of endocytosis-dependent SARS-CoV-2 infections, but not in infections by Lassa virus, which enters endo-lysosomal trafficking at the late endosome stage. Thus, we have identified an essential host pathway for filovirus infections in cell lines and engineered human target tissues. Inhibition of CCZ1 nearly completely abolishes Marburg and Ebola infections. Thus, targeting CCZ1 could potentially serve as a promising drug target for controlling infections caused by various viruses, such as SARS-CoV-2, Marburg, and Ebola.

Wnt/β-catenin and NFκB signaling synergize to trigger growth factor-free regeneration of adult primary human hepatocytes.

The liver has a remarkable capacity to regenerate, which is sustained by the ability of hepatocytes to act as facultative stem cells that, while normally quiescent, re-enter the cell cycle after injury. Growth factor signaling is indispensable in rodents, whereas Wnt/β-catenin is not required for effective tissue repair. However, the molecular networks that control human liver regeneration remain unclear. Organotypic 3D spheroid cultures of primary human or murine hepatocytes were used to identify the signaling network underlying cell cycle re-entry. Furthermore, we performed chemogenomic screening of a library enriched for epigenetic regulators and modulators of immune function to determine the importance of epigenomic control for human hepatocyte regeneration. Our results showed that, unlike in rodents, activation of Wnt/β-catenin signaling is the major mitogenic cue for adult primary human hepatocytes. Furthermore, we identified TGFβ inhibition and inflammatory signaling through NF-κB as essential steps for the quiescent-to-regenerative switch that allows Wnt/β-catenin-induced proliferation of human cells. In contrast, growth factors, but not Wnt/β-catenin signaling, triggered hyperplasia in murine hepatocytes. High-throughput screening in a human model confirmed the relevance of NFκB and revealed the critical roles of polycomb repressive complex 2, as well as of the bromodomain families I, II, and IV. This study revealed a network of NFκB, TGFβ, and Wnt/β-catenin that controls human hepatocyte regeneration in the absence of exogenous growth factors, identified novel regulators of hepatocyte proliferation, and highlighted the potential of organotypic culture systems for chemogenomic interrogation of complex physiological processes.

Liver Extracellular Matrix-Based Nanofiber Scaffolds for the Culture of Primary Hepatocytes and Drug Screening.

Immortalized liver cell lines and primary hepatocytes are currently used as in vitro models for hepatotoxic drug screening. However, a decline in the viability and functionality of hepatocytes with time is an important limitation of these culture models. Advancements in tissue engineering techniques have allowed us to overcome this challenge by designing suitable scaffolds for maintaining viable and functional primary hepatocytes for a longer period of time in culture. In the current study, we fabricated liver-specific nanofiber scaffolds with polylactic acid (PLA) along with a decellularized liver extracellular matrix (LEM) by the electrospinning technique. The fabricated hybrid PLA-LEM scaffolds were more hydrophilic and had better swelling properties than the PLA scaffolds. The hybrid scaffolds had a pore size of 38 ± 8 μm and supported primary rat hepatocyte cultures for 10 days. Increased viability (2-fold increase in the number of live cells) and functionality (5-fold increase in albumin secretion) were observed in primary hepatocytes cultured on the PLA-LEM scaffolds as compared to those on conventional collagen-coated plates on day 10 of culture. A significant increase in CYP1A2 enzyme activity was observed in hepatocytes cultured on PLA-LEM hybrid scaffolds in comparison to those on collagen upon induction with phenobarbital. Drugs like acetaminophen and rifampicin showed the highest toxicity in hepatocytes cultured on hybrid scaffolds. Also, the lethal dose of these drugs in rodents was accurately predicted as 1.6 g/kg and 594 mg/kg, respectively, from the corresponding IC50 values obtained from drug-treated hepatocytes on hybrid scaffolds. Thus, the fabricated liver-specific electrospun scaffolds maintained primary hepatocyte viability and functionality for an extended period in culture and served as an effective ex vivo drug screening platform to predict an accurate in vivo drug-induced hepatotoxicity.

Hepatic SPARC Expression Is Associated with Inflammasome Activation during the Progression of Non-Alcoholic Fatty Liver Disease in Both Mice and Morbidly Obese Patients.

The severity of non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to steatohepatitis, and it is not yet clearly understood which patients will progress to liver fibrosis or cirrhosis. SPARC (Secreted Protein Acidic and Rich in Cysteine) has been involved in NAFLD pathogenesis in mice and humans. The aim of this study was to investigate the role of SPARC in inflammasome activation, and to evaluate the relationship between the hepatic expression of inflammasome genes and the biochemical and histological characteristics of NAFLD in obese patients. In vitro studies were conducted in a macrophage cell line and primary hepatocyte cultures to assess the effect of SPARC on inflammasome. A NAFLD model was established in SPARC knockout (SPARC-/-) and SPARC+/+ mice to explore inflammasome activation. A hepatic RNAseq database from NAFLD patients was analyzed to identify genes associated with SPARC expression. The results were validated in a prospective cohort of 59 morbidly obese patients with NAFLD undergoing bariatric surgery. Our results reveal that SPARC alone or in combination with saturated fatty acids promoted IL-1β expression in cell cultures. SPARC-/- mice had reduced hepatic inflammasome activation during the progression of NAFLD. NAFLD patients showed increased expression of SPARC, NLRP3, CASP1, and IL-1β. Gene ontology analysis revealed that genes positively correlated with SPARC are linked to inflammasome-related pathways during the progression of the disease, enabling the differentiation of patients between steatosis and steatohepatitis. In conclusion, SPARC may play a role in hepatic inflammasome activation in NAFLD.

Elafibranor upregulates the EMT-inducer S100A4 via PPARβ/δ

Elafibranor is a dual peroxisome proliferator-activated receptor (PPAR)α and β/δ agonist that has reached a phase III clinical trial for the treatment of metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we examined the effects of elafibranor in mice fed a choline-deficient high-fat diet (CD-HFD), a model of metabolic dysfunction-associated steatohepatitis (MASH) that presents obesity and insulin resistance. Our findings revealed that elafibranor treatment ameliorated steatosis, inflammation, and fibrogenesis in the livers of CD-HFD-fed mice. Unexpectedly, elafibranor also increased the levels of the epithelial-mesenchymal transition (EMT)-promoting protein S100A4 via PPARβ/δ activation. The increase in S100A4 protein levels caused by elafibranor was accompanied by changes in the levels of markers associated with the EMT program. The S100A4 induction caused by elafibranor was confirmed in the BRL-3A rat liver cells and a mouse primary hepatocyte culture. Furthermore, elafibranor reduced the levels of ASB2, a protein that promotes S100A4 degradation, while ASB2 overexpression prevented the stimulating effect of elafibranor on S100A4. Collectively, these findings reveal an unexpected hepatic effect of elafibranor on increasing S100A4 and promoting the EMT program.