Immune-mediated Hepatic Injury Research Articles

Curdlan-Decorated Fullerenes Mitigate Immune-Mediated Hepatic Injury for Autoimmune Hepatitis Therapeutics via Reducing Macrophage Infiltration.

Autoimmune hepatitis (AIH) is a severe immune-mediated inflammatory liver disease whose standard of care is immunosuppressive treatment with inevitable undesired outcomes. Macrophage is acknowledged to aggravate liver damage, providing a promising AIH therapeutic target. Accordingly, in this study, a kind of curdlan-decorated fullerene nanoparticle (Cur-F) is fabricated to alleviate immune-mediated hepatic injury for treating AIH via reducing macrophage infiltration in a concanavalin A (Con A)-induced AIH mouse model. After intravenous administration, Cur-F primarily distributes in liver tissues, efficiently eliminates the excessive reactive oxygen species, significantly attenuates oxidative stress, and subsequently suppresses the nuclear factor kappa-B-gene binding (NF-κB) signal pathway, resulting in the lowered production of pro-inflammatory cytokines and the balancing of the immune homeostasis with the prevention of macrophage infiltration in the liver. The regulation of hepatic inflammation contributes to inhibiting inflammatory cytokines-induced hepatocyte apoptosis, decreasing the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) contents and thus ameliorating immune-mediated hepatic injury. Notably, there is no detectable toxicity to the body. Our findings may open up novel avenues for AIH based on curdlan and fullerene materials.

Natural Killer T (NKT) Cells in Autoimmune Hepatitis: Current Evidence from Basic and Clinical Research.

Natural killer T (NKT) cells are unconventional T cells that are activated by glycolipid antigens. They can produce a variety of inflammatory and regulatory cytokines and, therefore, modulate multiple aspects of the immune response in different pathological settings, including autoimmunity. NKT cells have also been implicated in the immunopathogenesis of autoimmune hepatitis, and in this review we summarize and analyze the main studies investigating the involvement and/or homeostasis of NKT cells in this disease. In detail, the evidence from both basic and clinical research has been specifically analyzed. Even though the experimental murine models supported a relevant role of NKT cells in immune-mediated hepatic injury, very few studies specifically investigated NKT cell homeostasis in patients with autoimmune hepatitis; however, these initial studies reported some alterations of NKT cells in these patients, which may also correlate with the disease activity to some extent. Further clinical studies are needed to investigate the potential role and use of NKT cell analysis as a disease marker of clinical relevance, and to better understand the precise cellular and molecular mechanisms by which NKT cells contribute to the pathogenesis of autoimmune hepatitis.

Corilagin Ameliorates Con A-Induced Hepatic Injury by Restricting M1 Macrophage Polarization.

Immune-mediated hepatic injury plays a key role in the initiation and pathogenesis of diverse liver diseases. However, treatment choice for immune-mediated hepatic injury remains limited. Corilagin, a natural ellagitannin extracted from various traditional Chinese medicines, has been demonstrated to exhibit multiple pharmacological activities, such as anti-inflammatory, anti-tumor, and hepatoprotective properties. The present study aimed to investigate the effects of corilagin on immune-mediated hepatic injury using a murine model of concanavalin A (Con A)-induced hepatitis, which is well-characterized to study acute immune-mediated hepatitis. Herein, mice were administered corilagin (25 mg/kg) intraperitoneally twice at 12 h intervals, and 1 h later, the mice were challenged with Con A (20 mg/kg body weight); serum and liver samples were collected after 12 h. The results showed that corilagin significantly increased the survival of mice and reduced serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels. In addition, corilagin markedly improved histopathological damage, hepatocyte apoptosis, and oxidative stress in the liver. The activation of M1 macrophages in the hepatic mononuclear cells was also significantly reduced compared with that in the control group. The expression of M1 macrophage-associated proinflammatory cytokines and genes, including interleukin (IL)-6, IL-12, and inducible nitric oxide synthase (iNOS), was also decreased after corilagin treatment. Finally, the results demonstrated that corilagin regulated macrophage polarization by modulating the mitogen-activated protein kinases (MAPK), nuclear factor (NF)-κB, and interferon regulatory factor (IRF) signaling pathways. Thus, the findings indicate that corilagin protects mice from Con A-induced immune-mediated hepatic injury by limiting M1 macrophage activation via the MAPK, NF-κB, and IRF signaling pathways, suggesting corilagin as a possible treatment choice for immune-mediated hepatic injury.

Overexpression of KLF14 protects against immune-mediated hepatic injury in mice

The underlying immunopathogenic mechanisms of autoimmune hepatitis (AIH) have not yet been well elucidated. An impairment in regulatory T cells (Tregs) is key to the development of AIH. Krüppel-like factors (KLFs) regulate a broad of cellular processes including immunocyte maturation. KLF14 may regulate Treg differentiation, but the biological functions remain far from elucidated. In this study, we identified the hepatic expression of KLF14 in human and murine liver diseases. Immune-mediated hepatitis was induced by concanavalin A (Con A). A KLF14 recombinant adenoviruses plasmid (Ad-KLF14) was constructed and injected into mice. Tregs were assessed by flow cytometry analysis; inflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), were tested by enzyme-linked immunosorbent assay (ELISA). The expression of KLF14 was suppressed in a time-and dose-dependent manner. Changes in cytokine levels were consistent with the degree of hepatic injury. Overexpression of KLF14 protected the liver from immune-mediated damage in vivo. Ad-KLF14 transfection before Con A challenge increased the frequency of Tregs in liver mononuclear cells (MNCs), and suppressed the expression of cytokines. All of these improvements were completely abrogated after Treg deletion in vivo by intraperitoneal injection of a CD25 antibody. In conclusion, these data suggest that KLF14 plays an as-yet unrecognized role in immune-mediated hepatitis mainly via induced Treg differentiation. Our findings extend the knowledge of the biological function of KLF14 to the autoimmune disease field, and indicate the possibility of KLF14 as a therapeutic target in AIH patients.The underlying immunopathogenic mechanisms of autoimmune hepatitis (AIH) have not been well elucidated. Kruppel-like factors 14 (KLF14) may regulate Treg differentiation, but the biological functions remain unclear. In this study, the authors reveal that KLF14 plays an as yet unrecognized role in immune- mediated hepatitis via induced Treg differentiation and inflammatory cytokine suppression. These findings indicate the possibility of KLF14 as a therapeutic target in AIH patients.

MTOR signaling disruption from myeloid-derived suppressive cells protects against immune-mediated hepatic injury through the HIF1α-dependent glycolytic pathway.

The mechanistic target of rapamycin (mTOR) pathway integrates diverse environmental inputs, including immune signals and metabolic cues, to direct innate and adaptive immune responses. Myeloid-derived suppressive cells (MDSCs) are a heterogeneous cell population that plays a crucial regulatory effect in immune-related diseases. However, whether mTOR signaling affects the functions of MDSCs remains largely unexplored. Here, we show that mTOR signaling is a pivotal, negative determinant of MDSC function in immune-mediated hepatic injury (IMH) diseases. In the context of IMH, the blocking of mTOR with rapamycin or mTOR-deficient CD11b+Gr1+ MDSCs mediates the protection against IMH; mTOR with rapamycin and mTOR-deficient CD11b+Gr1+ MDSCs are suppressive immune modulators that result in less IFN-γ-producing TH1 cells and more Foxp3+ Tregs Mechanistically, mTOR activity down-regulation in MDSCs induced iNOS expressions and NO productions. Pharmacologic inhibitions of iNOS completely eliminate MDSC-suppressive function and lose their inducible effects on T cell differentiation. Importantly, HIF1α-dependent glycolytic activity is responsible for mTOR-deficient, increased MDSC functional changes in IMH inflammation. Thus, these data demonstrate that mTOR acts as a fundamental "rheostat" in MDSCs to link immunologic signals to glycolytic pathways and functional fitness and highlights a central role of metabolic programming of MDSC-suppressive activity in protecting against immune hepatic injuries.

Immunogenomics reveal molecular circuits of diclofenac induced liver injury in mice.

Diclofenac is a non-steroidal anti-inflammatory drug and its use can be associated with severe adverse reactions, notably myocardial infarction, stroke and drug-induced liver injury (DILI). In pursue of immune-mediated DILI mechanisms an immunogenomic study was carried out. Diclofenac treatment of mice at 30 mg/kg for 3 days caused significant serum ALT and AST elevations, hepatomegaly and degenerative changes including hepatic glycogen depletion, hydropic swelling, cholesterolosis and eosinophilic hepatocytes with one animal presenting subsegmental infarction due to portal vein thrombosis. Furthermore, portal/periportal induction of the rate limiting enzyme in ammonia detoxification, i.e. carbamoyl phosphate synthetase 1 was observed. The performed microarray studies informed on > 600 differential expressed genes of which 35, 37 and 50 coded for inflammation, 51, 44 and 61 for immune and 116, 129 and 169 for stress response, respectively after single and repeated dosing for 3 and 14 days. Bioinformatic analysis defined molecular circuits of hepatic inflammation with the growth hormone (Ghr)− and leptin receptor, the protein-tyrosine-phosphatase, selectin and the suppressor-of-cytokine-signaling (Socs) to function as key nodes in gene regulatory networks. Western blotting confirmed induction of fibronectin and M-CSF to hallmark tissue repair and differentiation of monocytes and macrophages. Transcript expression of the macrophage receptor with collagenous structure increased > 7-fold and immunohistochemistry of CD68 evidenced activation of tissue-resident macrophages. Importantly, diclofenac treatment prompted strong expression of phosphorylated Stat3 amongst individual animals and the associated 8- and 4-fold Soc3 and Il-6 induction reinforced Ghr degradation as evidenced by immunoblotting. Moreover, immunohistochemistry confirmed regulation of master regulatory proteins of diclofenac treated mice to suggest complex pro-and anti-inflammatory reactions in immune-mediated hepatic injury. The findings encourage translational research.

Imaging of reactive oxygen species generated in vivo.

We sought to image the biodistribution of reactive oxygen species (ROS) within the living body using an in vivo electron spin resonance (ESR) imaging system using a spin probe, 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrroline (ACP) that produces ESR-detectable nitroxide upon reaction with ROS. Acute hepatic injury was induced in mice by priming with heat-killed Corynebacterium parvum followed by injection of a low dose of lipopolysaccharide. ACP was administered intravenously and an in vivo ESR imaging system was used to visualize hepatic oxidative stress. In this immune-mediated hepatic injury model, significant oxidative stress was evident at 3 h after lipopolysaccharide administration before the onset of massive hepatic injury. ACP was administered intravenously at 3 h after lipopolysaccharide injection when significant hepatic oxidative stress had been observed, and the ESR imaging system detected a high signal for 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (carbamoyl-PROXYL), which had originated from the ACP-derived hydroxylamine and produced large amount of ROS within the living body. Using the ESR imaging system with ACP, we were able to visualize ROS in the abdomen before onset of hepatic injury. We have succeeded in visualizing ROS within the body before onset of organ damage, representing a significant development in imaging for toxic molecules.

Tonsil-derived mesenchymal stem cells alleviate concanavalin A-induced acute liver injury

Acute liver failure, the fatal deterioration of liver function, is the most common indication for emergency liver transplantation, and drug-induced liver injury and viral hepatitis are frequent in young adults. Stem cell therapy has come into the limelight as a potential therapeutic approach for various diseases, including liver failure and cirrhosis. In this study, we investigated therapeutic effects of tonsil-derived mesenchymal stem cells (T-MSCs) in concanavalin A (ConA)- and acetaminophen-induced acute liver injury. ConA-induced hepatitis resembles viral and immune-mediated hepatic injury, and acetaminophen overdose is the most frequent cause of acute liver failure in the United States and Europe. Intravenous administration of T-MSCs significantly reduced ConA-induced hepatic toxicity, but not acetaminophen-induced liver injury, affirming the immunoregulatory capacity of T-MSCs. T-MSCs were successfully recruited to damaged liver and suppressed inflammatory cytokine secretion. T-MSCs expressed high levels of galectin-1 and -3, and galectin-1 knockdown which partially diminished interleukin-2 and tumor necrosis factor α secretion from cultured T-cells. Galectin-1 knockdown in T-MSCs also reversed the protective effect of T-MSCs on ConA-induced hepatitis. These results suggest that galectin-1 plays an important role in immunoregulation of T-MSCs, which contributes to their protective effect in immune-mediated hepatitis. Further, suppression of T-cell activation by frozen and thawed T-MSCs implies great potential of T-MSC banking for clinical utilization in immune-mediated disease.

Targeting S1P1 Receptor Protects against Murine Immunological Hepatic Injury through Myeloid-Derived Suppressor Cells

Although FTY720 may alter migration and homing of lymphocytes via sphingosine-1-phosphate (S1P) receptors, our recent studies indicated that FTY720 directly controls the differentiation of Th1 cells to regulatory T cells (Tregs) by targeting S1P1. However, the pharmacological function of FTY720 in immunological hepatic injury remains unknown. In this study, the role and regulatory signaling pathway of S1P receptor were investigated using a pharmacological approach in immune-mediated hepatic injury (IMH). In the context of IMH, FTY720 significantly ameliorated mortality and hepatic pathology. In FTY720-treated mice, recruited CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs) mediate protection against IMH and are functional suppressive immune modulators that result in fewer IFN-γ-producing Th1 cells and more Foxp3(+) Tregs. In agreement, FTY720-treated MDSCs promote the reciprocal differentiation between Th1 cells and Tregs in vitro and in vivo. Mechanistically, FTY720 treatment induced inducible NO synthase expression and NO production in MDSCs. Pharmacologic inhibition of inducible NO synthase completely eliminates MDSC suppressive function and eradicates their inducible effects on T cell differentiation. Finally, the mTOR inhibitor, rapamycin, photocopies the effects of FTY720 on MDSCs, implicating mTOR as a downstream effector of S1P1 signaling. This study identifies MDSCs as an essential component that provides protection against IMH following FTY720 or rapamycin treatment, validating the S1P1-mTOR signaling axis as a potential therapeutic target in hepatic injury.

Complement Activation and Liver Impairment in Trichloroethylene-Sensitized BALB/c Mice

Our recent studies have shown that trichloroethylene (TCE) was able to induce multisystem injuries in the form of occupational medicamentosa-like dermatitis, including skin, kidney, and liver damages. However, the role of complement activation in the immune-mediated liver injury is not known. This study examined the role of complement activation in the liver injury in a mouse model of TCE-induced sensitization. Treatment of female BALB/c mice with TCE under specific dosing protocols resulted in skin inflammation and sensitization. Skin edema and erythema occurred in TCE-sensitized groups. Trichloroethylene sensitization produced liver histopathological lesions, increased serum alanine aminotransferase, aspartate transaminase activities, and the relative liver weight. The concentrations of serum complement components C3a-desArg, C5a-desArg, and C5b-9 were significantly increased in 24-hour, 48-hour, and 72-hour sensitization-positive groups treated with TCE and peaked in the 72-hour sensitization-positive group. Depositions of C3a, C5a, and C5b-9 into the liver tissue were also revealed by immunohistochemistry. Immunofluorescence further verified high C5b-9 expression in 24-hour, 48-hour, and 72-hour sensitization-positive groups in response to TCE treatment. Reverse transcription-polymerase chain reaction detected C3 messenger RNA expression in the liver, and this was significantly increased in 24-hour and 48-hour sensitization-positive groups with a transient reduction at 72 hours. These results provide the first experimental evidence that complement activation may play a key role in the generation and progression of immune-mediated hepatic injury by exposure to TCE.

Induction of Regulatory T Cells by High-Dose gp96 Suppresses Murine Liver Immune Hyperactivation

Immunization with high-dose heat shock protein gp96, an endoplasmic reticulum counterpart of the Hsp90 family, significantly enhances regulatory T cell (Treg) frequency and suppressive function. Here, we examined the potential role and mechanism of gp96 in regulating immune-mediated hepatic injury in mice. High-dose gp96 immunization elicited rapid and long-lasting protection of mice against concanavalin A (Con A)-and anti-CD137-induced liver injury, as evidenced by decreased alanine aminotransaminase (ALT) levels, hepatic necrosis, serum pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-6), and number of IFN-γ + CD4+ and IFN-γ + CD8+ T cells in the spleen and liver. In contrast, CD4+CD25+Foxp3+ Treg frequency and suppressive function were both increased, and the protective effect of gp96 could be generated by adoptive transfer of Treg cells from gp96-immunized mice. In vitro co-culture experiments demonstrated that gp96 stimulation enhanced Treg proliferation and suppressive function, and up-regulation of Foxp3, IL-10, and TGF-β1 induced by gp96 was dependent on TLR2- and TLR4-mediated NF-κB activation. Our work shows that activation of Tregs by high-dose gp96 immunization protects against Con A- and anti-CD137-induced T cell-hepatitis and provides therapeutic potential for the development of a gp96-based anti-immune hyperactivation vaccine against immune-mediated liver destruction.

Substance P participates in immune-mediated hepatic injury induced by concanavalin A in mice and stimulates cytokine synthesis in Kupffer cells

Studies have indicated that the immune system plays a pivotal role in hepatitis. Substance P (SP) has been shown to modulate the immune response. In order to investigate the role of SP in liver injury and to determine whether it leads to pro-inflammatory signaling, we established a mouse model of hepatic injury induced by concanavalin A (ConA). We also exposed mouse Kupffer cells (KCs) to SP in vitro. Cytokine and SP levels in liver homogenates were detected using enzyme-linked immunosorbent assay (ELISA) and the protective effects of L-703,606 were evaluated through serological and histological assessments. Neurokinin-1 receptor (NK-1R) expression was evaluated by immunofluorescence and quantitative polymerase chain reaction (PCR). The levels of SP, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly increased in the ConA-treated mice and the levels of ALT and AST were markedly reduced by L-703,606-pretreatment. Liver injury was significantly reduced by treatment with L-703,606. The mouse KCs expressed NK-1R and SP increased NK-1R mRNA expression. Furthermore, NK-1R blockade eliminated the effect of SP on NK-1R mRNA expression. The cytokine levels exhibited a substantial increase in the SP-pretreated KCs compared with the KCs that were cultured in control medium. The inter-leukin (IL)-6 and tumor necrosis factor (TNF)-α levels in the L-703,606-pretreated KCs were significantly lower compared with those in the SP-pretreated KCs. Our study suggests that neurogenic inflammation induced by SP plays an important role in hepatitis. Mouse KCs express NK-1R and SP increases NK-1R mRNA expression. SP enhances IL-6 and TNF-α secretion and an NK-1R antagonist inhibits this secretion.

Differential Oxidation of Two Thiophene-Containing Regioisomers to Reactive Metabolites by Cytochrome P450 2C9

The uricosuric diuretic agent tienilic acid (TA) is a thiophene-containing compound that is metabolized by P450 2C9 to 5-OH-TA. A reactive metabolite of TA also forms a covalent adduct to P450 2C9 that inactivates the enzyme and initiates immune-mediated hepatic injury in humans, purportedly through a thiophene-S-oxide intermediate. The 3-thenoyl regioisomer of TA, tienilic acid isomer (TAI), is chemically very similar and is reported to be oxidized by P450 2C9 to a thiophene-S-oxide, yet it is not a mechanism-based inactivator (MBI) of P450 2C9 and is reported to be an intrinsic hepatotoxin in rats. The goal of the work presented in this article was to identify the reactive metabolites of TA and TAI by the characterization of products derived from P450 2C9-mediated oxidation. In addition, in silico approaches were used to better understand both the mechanisms of oxidation of TA and TAI and/or the structural rearrangements of oxidized thiophene compounds. Incubation of TA with P450 2C9 and NADPH yielded the well-characterized 5-OH-TA metabolite as the major product. However, contrary to previous reports, it was found that TAI was oxidized to two different types of reactive intermediates that ultimately lead to two types of products, a pair of hydroxythiophene/thiolactone tautomers and an S-oxide dimer. Both TA and TAI incorporated ¹⁸O from ¹⁸O₂ into their respective hydroxythiophene/thiolactone metabolites indicating that these products are derived from an arene oxide pathway. Intrinsic reaction coordinate calculations of the rearrangement reactions of the model compound 2-acetylthiophene-S-oxide showed that a 1,5-oxygen migration mechanism is energetically unfavorable and does not yield the 5-OH product but instead yields a six-membered oxathiine ring. Therefore, arene oxide formation and subsequent NIH-shift rearrangement remains the favored mechanism for formation of 5-OH-TA. This also implicates the arene oxide as the initiating factor in TA induced liver injury via covalent modification of P450 2C9. Finally, in silico modeling of P450 2C9 active site ligand interactions with TA using the catalytically active iron-oxo species revealed significant differences in the orientations of TA and TAI in the active site, which correlated well with experimental results showing that TA was oxidized only to a ring carbon hydroxylated product, whereas TAI formed both ring carbon hydroxylated products and an S-oxide.

Allergic hepatitis induced by drugs

To examine recent advances in our understanding of how drugs can trigger a hypersensitivity reaction in the liver, how tolerance is lost, the mechanisms of damage to hepatocytes and the strategies towards a better assessment of an idiosyncratic drug liver reaction. Formation and presentation of drug-protein adducts, or a direct interaction with the major histocompatibility complex/T-cell receptor complex is a necessary but not sufficient stimulus to trigger a hypersensitivity reaction. Liver shows considerable tolerogenic potential towards drug adducts. Recent studies highlight allergic hepatitis as a loss of liver tolerance towards drug antigens, the mechanisms of which are beginning to be unravelled. Cell injury caused by the drug itself, a concomitant inflammatory process, or a coincidental stimulus probably represents the additional signal needed to initiate the allergic process. Drug-induced liver injury is of concern due to its unpredictable nature and serious clinical implications. Clinically, both hepatocellular injury and cholestasis can occur and most episodes have good clinical prognoses upon drug discontinuation. In a few cases, damage to the liver cells may continue in the form of an autoimmune hepatitis. The available diagnostic tools to confirm an immune-mediated hepatic injury are still very limited, and rely on the lymphocyte transformation test.

Recurrent Toxic Hepatitis in a Caucasian Girl Related to the Use of Shou‐Wu‐Pian, a Chinese Herbal Preparation

Recurrent Toxic Hepatitis in a Caucasian Girl Related to the Use of Shou‐Wu‐Pian, a Chinese Herbal Preparation