Generation of Multicellular 3D Liver Organoids From Induced Pluripotent Stem Cells as a Tool for Modelling Liver Diseases.

The liver is an essential organ that is involved in the metabolism, synthesis, and secretion of serum proteins and detoxification of xenobiotic compounds and alcohol. Studies on liver diseases have largely relied on cancer-derived cell lines that have proven to be inferior due to the lack of drug-metabolising enzymes. Primary human hepatocytes are considered the gold-standard for evaluating drug metabolism. However, several factors such as lack of donors, high cost of cells, and loss of polarity of the cells have limited their widescale adoption and utility. Stem cells have emerged as an alternative source for liver cells that could be utilised for studying liver diseases, developmental biology, toxicology testing, and regenerative medicine. In this article, we describe in detail an optimised protocol for the generation of multicellular 3D liver organoids composed of hepatocytes, stellate cells, and Kupffer cells as a tractable robust model of the liver. Key features • Optimising a protocol for generating multicellular 3D liver organoids from induced pluripotent stem cells. Graphical overview.

The pivotal role of dysregulated autophagy in the progression of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a clinicopathologic syndrome characterized by excessive fat deposition in hepatocytes and a major cause of end-stage liver disease. Autophagy is a metabolic pathway responsible for degrading cytoplasmic products and damaged organelles, playing a pivotal role in maintaining the homeostasis and functionality of hepatocytes. Recent studies have shown that pharmacological intervention to activate or restore autophagy provides benefits for liver function recovery by promoting the clearance of lipid droplets (LDs) in hepatocytes, decreasing the production of pro-inflammatory factors, and inhibiting activated hepatic stellate cells (HSCs), thus improving liver fibrosis and slowing down the progression of NAFLD. This article summarizes the physiological process of autophagy, elucidates the close relationship between NAFLD and autophagy, and discusses the effects of drugs on autophagy and signaling pathways from the perspectives of hepatocytes, kupffer cells (KCs), and HSCs to provide assistance in the clinical management of NAFLD.

Mid1 aggravates hepatic ischemia-reperfusion injury by inducing immune cell infiltration.

Hepatic ischemia-reperfusion injury (HIRI) represents a major risk factor in liver transplantation and resection surgeries. Kupffer cells (KCs) produce proinflammatory cytokines and lead to hepatic neutrophil infiltration in the liver, which is one of the leading causes of HIRI. Mid1 is involved in immune infiltration, but the role of Mid1 remains poorly understood. Herin, our study aimed to investigate the effect of Mid1 on HIRI progression. Male C57BL/6 mice aged 6 weeks were used for the HIRI model established. The function of Mid1 on liver injury and hepatic inflammation was evaluated. In vitro, KCs were used to investigate the function and mechanism of Mid1 in modulating KC inflammation upon lipopolysaccharide (LPS) stimulation. We found that Mid1 expression was up-regulated upon HIRI. Mid1 inhibition alleviated liver damage, as evidenced by neutrophil infiltration, intrahepatic inflammation, and hepatocyte apoptosis. In vitro experiments further revealed that Mid1 knockdown reduced the secretion of proinflammatory cytokines and chemokines in KCs. Moreover, silenced-Mid1 suppressed proinflammatory responses by the inhibition of NF-κB, JNK, and p38 signaling pathways. Taken together, Mid1 contributes to HIRI via regulating the proinflammatory response of KCs and inducing neutrophil infiltration. Targeting Mid1 may be a promising strategy to protect against HIRI.

Spinal cord injury-induced metabolic impairment and steatohepatitis develops in non-obese rats and is exacerbated by premorbid obesity.

Impaired sensorimotor functions are prominent complications of spinal cord injury (SCI). A clinically important but less obvious consequence is development of metabolic syndrome (MetS), including increased adiposity, hyperglycemia/insulin resistance, and hyperlipidemia. MetS predisposes SCI individuals to earlier and more severe diabetes and cardiovascular disease compared to the general population, which trigger life-threatening complications (e.g., stroke, myocardial infarcts). Although each comorbidity is known to be a risk factor for diabetes and other health problems in obese individuals, their relative contribution or perceived importance in propagating systemic pathology after SCI has received less attention. This could be explained by an incomplete understanding of MetS promoted by SCI compared with that from the canonical trigger diet-induced obesity (DIO). Thus, here we compared metabolic-related outcomes after SCI in lean rats to those of uninjured rats with DIO. Surprisingly, SCI-induced MetS features were equal to or greater than those in obese uninjured rats, including insulin resistance, endotoxemia, hyperlipidemia, liver inflammation and steatosis. Considering the endemic nature of obesity, we also evaluated the effect of premorbid obesity in rats receiving SCI; the combination of DIO + SCI exacerbated MetS and liver pathology compared to either alone, suggesting that obese individuals that sustain a SCI are especially vulnerable to metabolic dysfunction. Notably, premorbid obesity also exacerbated intraspinal lesion pathology and worsened locomotor recovery after SCI. Overall, these results highlight that normal metabolic function requires intact spinal circuitry and that SCI is not just a sensory-motor disorder, but also has significant metabolic consequences.

Metabolic reprogramming in liver fibrosis.

Chronic liver diseases, primarily metabolic dysfunction-associated steatotic liver disease (MASLD), harmful use of alcohol, or viral hepatitis, may result in liver fibrosis, cirrhosis, and cancer. Hepatic fibrogenesis is a complex process with interactions between different resident and non-resident heterogeneous liver cell populations, ultimately leading to deposition of extracellular matrix and organ failure. Shifts in cell phenotypes and functions involve pronounced transcriptional and protein synthesis changes that require metabolic adaptations in cellular substrate metabolism, including glucose and lipid metabolism, resembling changes associated with the Warburg effect in cancer cells. Cell activation and metabolic changes are regulated by metabolic stress responses, including the unfolded protein response, endoplasmic reticulum stress, autophagy, ferroptosis, and nuclear receptor signaling. These metabolic adaptations are crucial for inflammatory and fibrogenic activation of macrophages, lymphoid cells, and hepatic stellate cells. Modulation of these pathways, therefore, offers opportunities for novel therapeutic approaches to halt or even reverse liver fibrosis progression.

Carbon Dot-Loaded Apoptotic Vesicles Improve the Liver Kupffer Cell-Mediated Antibacterial Effect to Synergistically Alleviate Sepsis.

Sepsis is a lethal systemic inflammatory disease against infection that lacks effective therapeutic approaches. Liver resident macrophage Kupffer cell (KC)-initiated bacterial clearance is crucial for the host to defend against infection. However, it remains unclear whether this process also governs the antibacterial therapy of sepsis that would be used to improve therapeutic outcomes. Here, we found that copper-doped carbon dots (Cu-CDs) exhibited superior antibacterial capabilities in vitro but displayed limited therapeutic effects in septic mice due to their limited ability to target the liver and restore KC antimicrobial capacity. Thus, we developed a composite nanodrug of copper-doped carbon dot-loaded apoVs (CC-apoVs) that combined the antibacterial ability of Cu-CDs and liver KC targeting features of apoV. Moreover, intravenous injection of CC-apoVs markedly alleviated the systemic infection and decreased the mortality of septic mice compared to Cu-CD and apoV infusion alone. Mechanistically, CC-apoV injection rescued impaired liver KCs during sepsis and enhanced their ability to capture and kill bloodborne bacteria. In addition, apoV-promoted macrophage killing of bacteria could be blocked by the inhibition of small GTPase Rab5. This study reveals a liver KC-targeted therapeutic strategy for sepsis and provides a nanodrug CC-apoV to improve the host antibacterial defense and amplify the therapeutic effect of the nanodrug.

The Coming Age of Antisense Oligos for the Treatment of Hepatic Ischemia/Reperfusion (IRI) and Other Liver Disorders: Role of Oxidative Stress and Potential Antioxidant Effect.

Imbalances in the redox state of the liver arise during metabolic processes, inflammatory injuries, and proliferative liver disorders. Acute exposure to intracellular reactive oxygen species (ROS) results from high levels of oxidative stress (OxS) that occur in response to hepatic ischemia/reperfusion injury (IRI) and metabolic diseases of the liver. Antisense oligonucleotides (ASOs) are an emerging class of gene expression modulators that target RNA molecules by Watson-Crick binding specificity, leading to RNA degradation, splicing modulation, and/or translation interference. Here, we review ASO inhibitor/activator strategies to modulate transcription and translation that control the expression of enzymes, transcription factors, and intracellular sensors of DNA damage. Several small-interfering RNA (siRNA) drugs with N-acetyl galactosamine moieties for the liver have recently been approved. Preclinical studies using short-activating RNAs (saRNAs), phosphorodiamidate morpholino oligomers (PMOs), and locked nucleic acids (LNAs) are at the forefront of proof-in-concept therapeutics. Future research targeting intracellular OxS-related pathways in the liver may help realize the promise of precision medicine, revolutionizing the customary approach to caring for and treating individuals afflicted with liver-specific conditions.

Liver Cell Mitophagy in Metabolic Dysfunction-Associated Steatotic Liver Disease and Liver Fibrosis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects approximately one-third of the global population. MASLD and its advanced-stage liver fibrosis and cirrhosis are the leading causes of liver failure and liver-related death worldwide. Mitochondria are crucial organelles in liver cells for energy generation and the oxidative metabolism of fatty acids and carbohydrates. Recently, mitochondrial dysfunction in liver cells has been shown to play a vital role in the pathogenesis of MASLD and liver fibrosis. Mitophagy, a selective form of autophagy, removes and recycles impaired mitochondria. Although significant advances have been made in understanding mitophagy in liver diseases, adequate summaries concerning the contribution of liver cell mitophagy to MASLD and liver fibrosis are lacking. This review will clarify the mechanism of liver cell mitophagy in the development of MASLD and liver fibrosis, including in hepatocytes, macrophages, hepatic stellate cells, and liver sinusoidal endothelial cells. In addition, therapeutic strategies or compounds related to hepatic mitophagy are also summarized. In conclusion, mitophagy-related therapeutic strategies or compounds might be translational for the clinical treatment of MASLD and liver fibrosis.

Reactive oxygen species regulation by NCF1 governs ferroptosis susceptibility of Kupffer cells to MASH.

Impaired self-renewal of Kupffer cells (KCs) leads to inflammation in metabolic dysfunction-associated steatohepatitis (MASH). Here, we identify neutrophil cytosolic factor 1 (NCF1) as a critical regulator of iron homeostasis in KCs. NCF1 is upregulated in liver macrophages and dendritic cells in humans with metabolic dysfunction-associated steatotic liver disease and in MASH mice. Macrophage NCF1, but not dendritic cell NCF1, triggers KC iron overload, ferroptosis, and monocyte-derived macrophage infiltration, thus aggravating MASH progression. Mechanistically, elevated oxidized phospholipids induced by macrophage NCF1 promote Toll-like receptor (TLR4)-dependent hepatocyte hepcidin production, leading to increased KC iron deposition and subsequent KC ferroptosis. Importantly, the human low-functional polymorphic variant NCF190H alleviates KC ferroptosis and MASH in mice. In conclusion, macrophage NCF1 impairs iron homeostasis in KCs by oxidizing phospholipids, triggering hepatocyte hepcidin release and KC ferroptosis in MASH, highlighting NCF1 as a therapeutic target for improving KC fate and limiting MASH progression.

Angiocrine signaling in sinusoidal homeostasis and liver diseases.

The hepatic sinusoids are composed of liver sinusoidal endothelial cells (LSECs), which are surrounded by hepatic stellate cells (HSCs) and contain liver-resident macrophages called Kupffer cells, and other patrolling immune cells. All these cells communicate with each other and with hepatocytes to maintain sinusoidal homeostasis and a spectrum of hepatic functions under healthy conditions. Sinusoidal homeostasis is disrupted by metabolites, toxins, viruses, and other pathological factors, leading to liver injury, chronic liver diseases, and cirrhosis. Alterations in hepatic sinusoids are linked to fibrosis progression and portal hypertension. LSECs are crucial regulators of cellular crosstalk within their microenvironment via angiocrine signaling. This review discusses the mechanisms by which angiocrine signaling orchestrates sinusoidal homeostasis, as well as the development of liver diseases. Here, we summarise the crosstalk between LSECs, HSCs, hepatocytes, cholangiocytes, and immune cells in health and disease and comment on potential novel therapeutic methods for treating liver diseases.

Mimicking the cellular environment does not cause monocyte-derived macrophages to become phenotypically similar to Kupffer cells.

Resident macrophages of various mammalian organs are characterized by several distinctive features in their gene expression profile and phenotype, including involvement in the regulation of organ functions, as well as reduced sensitivity to proinflammatory activation factors. The reasons for the formation of such a specific phenotype remain the subject of intensive research. Some papers emphasize the role of the origin of organ macrophages. Other studies indicate that monocytes that develop in the red bone marrow are also able to form resident macrophages with a phenotype characteristic of a particular organ, but this requires appropriate microenvironmental conditions. In this article, we studied the possibility of differentiation of monocyte-derived macrophages into cells with a Kupffer-like phenotype under the influence of the main stromal components of Kupffer cells macrophage niche: Ito cells, liver sinusoid endotheliocytes and hepatocyte growth factor (HGF). It was found that Kupffer cells are characterized by several features, including increased expression of transcription factors Spic and Id3, as well as MUP family genes, Clusterin and Ngp genes. In addition, Kupffer cells were characterized by a higher proliferative activity. The expression of marker genes of Kupffer cells (i.e. Id3, Spic, Marco and Timd4) increased in monocyte-derived macrophages during coculture with Ito cells, liver sinusoid endothelial cells, macrophage colony-stimulating factor and HGF cells only by 3 days. However, the expression level of these genes was always higher in Kupffer cells. In addition, a complete coincidence of the expressed gene profile in monocyte-derived macrophages and Kupffer cells did not occur even after 3 days of culturing.

Integrated Single Cell Analysis Reveals An Atlas of Tumor Associated Macrophages in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), one of the most prevalent cancers globally, is closely associated with tumor-associated macrophages (TAMs), including monocyte-derived macrophages and liver-resident Kupffer cells. Understanding TAM heterogeneity at the cellular level is crucial for developing effective HCC prevention and treatment strategies. In this study, we conducted an integrated single-cell analysis of four cohorts (GSE140228, GSE125449, GSE149614 and GSE156625) to elucidate the TAM landscape in HCC. We identified 284 gene markers, termed Panmyeloid markers, that characterize myeloid cells within this context. Our analysis distinguished six clusters of monocyte-derived macrophages (Macro1-Macro6) and four clusters of Kupffer cells (Kupffer1-Kupffer4). Notably, CXCL10 + macrophages and MT1G + Kupffer cells, predominantly located within tumor tissues, exhibited distinct functional characteristics relevant to HCC. We also explored cellular communication between TAMs and T cells, uncovering potential signaling pathways such as the CXCL10/CXCL11-CXCR3 and CXCL12-CXCR4 networks. These findings enhance our understanding of TAMs in HCC and open new avenues for targeted therapeutic interventions.

Succinic Acid Ameliorates Concanavalin A-Induced Hepatitis by Altering the Inflammatory Microenvironment and Expression of BCL-2 Family Proteins.

Autoimmune hepatitis (AIH) is a severe immune-mediated inflammatory liver disease that currently lacks feasible drug treatment methods. Our study aimed to evaluate the protective effect of succinic acid against AIH and provide a reliable method for the clinical treatment of AIH. We performed an in vivo study of the effects of succinic acid on concanavalin A (ConA)-induced liver injury in mice. We examined liver transaminase levels, performed hematoxylin and eosin (HE) staining, and observed apoptotic phenotypes in mice. We performed flow cytometry to detect changes in the number of neutrophils and monocytes, and used liposomes to eliminate the liver Kupffer cells and evaluate their role. We performed bioinformatics analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blotting to detect mitochondrial apoptosis-induced changes in proteins from the B-cell lymphoma 2(Bcl-2) family. Succinic acid ameliorated ConA-induced AIH in a concentration-dependent manner, as reflected in the survival curve. HE and TUNEL staining and terminal deoxynucleotidyl transferase dUTP nick end labeling revealed decreased alanine transaminase and aspartate aminotransferase levels, and reduced liver inflammation and apoptosis. RT-qPCR and enzyme-linked immunosorbent assay revealed that succinic acid significantly reduced liver pro-inflammatory cytokine levels. Flow cytometry revealed significantly decreased levels of liver neutrophils. Moreover, the protective effect of succinic acid disappeared after the Kupffer cells were eliminated, confirming their important role in the effect. Bioinformatics analysis, RT-qPCR, and western blotting showed that succinic acid-induced changes in proteins from the Bcl-2 family involved mitochondrial apoptosis, indicating the molecular mechanism underlying the protective effect of succinic acid. Succinic acid ameliorated ConA-induced liver injury by regulating immune balance, inhibiting pro-inflammatory factors, and promoting anti-apoptotic proteins in the liver. This study provides novel insights into the biological functions and therapeutic potential of succinic acid in the treatment of autoimmune liver injury.

PPARß/δ as a promising molecular drug target for liver diseases: A focused review.

Various liver diseases pose great threats to humans. Although the etiologies of these liver diseases are quite diverse, they share similar pathologic phenotypes and molecular mechanisms such as oxidative stress, lipid and glucose metabolism disturbance, hepatic Kupffer cell (KC) proinflammatory polarization and inflammation, insulin resistance, and hepatic stellate cell (HSC) activation and proliferation. Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is expressed in various types of liver cells with relatively higher expression in KCs and HSCs. Accumulating evidence has revealed the versatile functions of PPARß/δ such as controlling lipid homeostasis, inhibiting inflammation, regulating glucose metabolism, and restoring insulin sensitivity, suggesting that PPARß/δ may serve as a potential molecular drug target for various liver diseases. This article aims to provide a concise review of the structure, expression pattern and biological functions of PPARß/δ in the liver and its roles in various liver diseases, and to discuss potential future research perspectives.

Integration of Kupffer cells into human iPSC-derived liver organoids for modeling liver dysfunction in sepsis.

Maximizing the potential of human liver organoids (LOs) for modeling human septic liver requires the integration of innate immune cells, particularly resident macrophage Kupffer cells. In this study, we present a strategy to generate LOs containing Kupffer cells (KuLOs) by recapitulating fetal liver hematopoiesis using human induced pluripotent stem cell (hiPSC)-derived erythro-myeloid progenitors (EMPs), the origin of tissue-resident macrophages, and hiPSC-derived LOs. Remarkably, LOs actively promote EMP hematopoiesis toward myeloid and erythroid lineages. Moreover, supplementing with macrophage colony-stimulating factor (M-CSF) proves crucial in sustaining the hematopoietic population during the establishment of KuLOs. Exposing KuLOs to sepsis-like endotoxins leads to significant organoid dysfunction that closely resembles the pathological characteristics of the human septic liver. Furthermore, we observe a notable functional recovery in KuLOs upon endotoxin elimination, which is accelerated by using Toll-like receptor-4-directed endotoxin antagonist. Our study represents a comprehensive framework for integrating hematopoietic cells into organoids, facilitating in-depth investigations into inflammation-mediated liver pathologies.

Effect of Zinc Oxide Nanoparticles on Liver Functions in Albino Mice.

BACKGROUND: An alarming number of zinc oxide nanoparticles (ZnO-NPs) have leaked into the environment, endangering the tissues of many living creatures, due to the recent surge in their use in several items. Through intra-peritoneal injection, this research intends to examine the impact of ZnO-NPs on the hepatic and gastrointestinal structures of male albino mice. METHOD: For seven and 14 days, animals were given 0.1 ml of 100 and 200 mg kg-1 of 50 nm-size ZnO-NPs, respectively. In contrast, those in the control group were given only water and food. RESULT: The results demonstrated that the treated mice's livers underwent functional changes and histological damage. After seven and 14 days, there was a notable rise in the average levels of the glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase enzymes in comparison to the control group (p≤0.05). Concentration time determines the magnitude of this impact. When enzyme levels vary, it means the liver isn't working properly. Histological changes in the liver, such as necrosis, destruction of hepatocyte membranes, widening of sinusoidal spaces and vacuolation of their cytoplasm, vascular congestion, and an increased number of Kupffer cells, were induced in mice treated with ZnO-NPs at two studied concentrations (100 and 200 mg/kg) for seven and 14 days, respectively. These effects were time-dose-dependent, according to the results of hematoxylin-eosin staining of liver tissue images.

Cells in the liver microenvironment regulate the process of liver metastasis.

The research of liver metastasis is a developing field. The ability of tumor cells to invade the liver depends on the complicated interactions between metastatic cells and local subpopulations in the liver (including Kupffer cells, hepatic stellate cells, liver sinusoidal endothelial cells, and immune-related cells). These interactions are mainly mediated by intercellular adhesion and the release of cytokines. Cell populations in the liver microenvironment can play a dual role in the progression of liver metastasis through different mechanisms. At the same time, we can see the participation of liver parenchymal cells and nonparenchymal cells in the process of liver metastasis of different tumors. Therefore, the purpose of this article is to summarize the relationship between cellular components of liver microenvironment and metastasis and emphasize the importance of different cells in the occurrence or potential regression of liver metastasis.

MicroRNA-7 deficiency ameliorates d-galactose-induced aging in mice by regulating senescence of Kupffer cells.

Aging is intricately linked to immune system dysfunction. Recent studies have highlighted the biological function of microRNA-7 (miR-7) as a novel regulator of immune cell function and related diseases. However, the potential role of miR-7 in aging remains unexplored. Here, we investigated the contribution of miR-7 to d-gal-induced aging in mice, focusing on its regulation of senescent Kupffer cells. Our findings revealed that miR-7 deficiency significantly ameliorated the aging process, characterized by enhanced CD4+ T-cell activation. However, the adoptive transfer of miR-7-deficient CD4+T cells failed to improve the age-related phenotype. Further analysis showed that miR-7 deficiency significantly reduced IL-1ß production in liver tissue, and inhibiting IL-1ß in vivo slowed down the aging process in mice. Notably, IL-1ß is mainly produced by senescent Kupffer cells in the liver tissue of aging mice, and miR-7 expression was significantly up-regulated in these cells. Mechanistically, KLF4, a target of miR-7, was down-regulated in senescent Kupffer cells in aging mouse model. Furthermore, miR-7 deficiency also modulated the NF-κB activation and IL-1ß production in senescent Kupffer cells through KLF4. In conclusion, our findings unveil the role of miR-7 in d-gal-induced aging in mice, highlighting its regulation of KLF4/NF-κB/IL-1ß pathways in senescent Kupffer cells. This research may enhance our understanding of miRNA-based aging immune cells and offer new avenues for new intervention strategies in aging process.

Immunomodulation in non-alcoholic fatty liver disease: exploring mechanisms and applications.

Non-alcoholic fatty liver disease (NAFLD) exhibits increased lipid enrichment in hepatocytes. The spectrum of this disease includes stages such as nonalcoholic simple fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), and liver fibrosis. Changes in lifestyle behaviors have been a major factor contributing to the increased cases of NAFLD patients globally. Therefore, it is imperative to explore the pathogenesis of NAFLD, identify therapeutic targets, and develop new strategies to improve the clinical management of the disease. Immunoregulation is a strategy through which the organism recognizes and eliminates antigenic foreign bodies to maintain physiological homeostasis. In this process, multiple factors, including immune cells, signaling molecules, and cytokines, play a role in governing the evolution of NAFLD. This review seeks to encapsulate the advancements in research regarding immune regulation in NAFLD, spanning from underlying mechanisms to practical applications.

Mouse Type-I Interferon-Mannosylated Albumin Fusion Protein for the Treatment of Chronic Hepatitis.

Although a lot of effort has been put into creating drugs and combination therapies against chronic hepatitis, no effective treatment has been established. Type-I interferon is a promising therapeutic for chronic hepatitis due to its excellent anti-inflammatory effects through interferon receptors on hepatic macrophages. To develop a type-I IFN equipped with the ability to target hepatic macrophages through the macrophage mannose receptor, the present study designed a mouse type-I interferon-mannosylated albumin fusion protein using site-specific mutagenesis and albumin fusion technology. This fusion protein exhibited the induction of anti-inflammatory molecules, such as IL-10, IL-1Ra, and PD-1, in RAW264.7 cells, or hepatoprotective effects on carbon tetrachloride-induced chronic hepatitis mice. As expected, such biological and hepatoprotective actions were significantly superior to those of human fusion proteins. Furthermore, the repeated administration of mouse fusion protein to carbon tetrachloride-induced chronic hepatitis mice clearly suppressed the area of liver fibrosis and hepatic hydroxyproline contents, not only with a reduction in the levels of inflammatory cytokine (TNF-α) and fibrosis-related genes (TGF-ß, Fibronectin, Snail, and Collagen 1α2), but also with a shift in the hepatic macrophage phenotype from inflammatory to anti-inflammatory. Therefore, type-I interferon-mannosylated albumin fusion protein has the potential as a new therapeutic agent for chronic hepatitis.