Con A-induced Hepatitis Research Articles

A non-catalytic role of IPMK is required for PLCγ1 activation in T cell receptor signaling by stabilizing the PLCγ1-Sam68 complex

BackgroundPhospholipase C gamma 1 (PLCγ1) is an important mediator of the T cell receptor (TCR) and growth factor signaling. PLCγ1 is activated by Src family kinases (SFKs) and produces inositol 1,4,5-triphosphate (InsP3) from phosphatidylinositol 4,5-bisphosphate (PIP2). Inositol polyphosphate multikinase (IPMK) is a pleiotropic enzyme with broad substrate specificity and non-catalytic activities that mediate various functional protein-protein interactions. Therefore, IPMK plays critical functions in key biological events such as cell growth. However, the contribution of IPMK to the activation of PLCγ1 in TCR signaling remains mostly unelucidated. The current study aimed to elucidate the functions of IPMK in TCR signaling and to uncover the mode of IPMK-mediated signaling action in PLCγ1 activation.MethodsConcanavalin A (ConA)-induced acute hepatitis model was established in CD4+ T cell-specific IPMK knockout mice (IPMKΔCD4). Histological analysis was performed to assess hepatic injury. Primary cultures of naïve CD4+ T cells were used to uncover the role of mechanisms of IPMK in vitro. Western blot analysis, quantitative real-time PCR, and flow cytometry were performed to analyze the TCR-stimulation-induced PLCγ1 activation and the downstream signaling pathway in naïve CD4+ T cells. Yeast two-hybrid screening and co-immunoprecipitation were conducted to identify the IPMK-binding proteins and protein complexes.ResultsIPMKΔCD4 mice showed alleviated ConA-induced acute hepatitis. CD4+ helper T cells in these mice showed reduced PLCγ1 Y783 phosphorylation, which subsequently dampens calcium signaling and IL-2 production. IPMK was found to contribute to PLCγ1 activation via the direct binding of IPMK to Src-associated substrate during mitosis of 68 kDa (Sam68). Mechanistically, IPMK stabilizes the interaction between Sam68 and to PLCγ1, thereby promoting PLCγ1 phosphorylation. Interfering this IPMK-Sam68 binding interaction with IPMK dominant-negative peptides impaired PLCγ1 phosphorylation.ConclusionsOur results demonstrate that IPMK non-catalytically promotes PLCγ1 phosphorylation by stabilizing the PLCγ1–Sam68 complex. Targeting IPMK in CD4+ T cells may be a promising strategy for managing immune diseases caused by excessive stimulation of TCR.

TIPE2 protein restrains invariant NKT activation and protects against immune-mediated hepatitis in mice.

Concanavalin A (ConA) administration induces a rapid and severe liver injury in mice, and invariant natural killer T (iNKT) cells are recognized to be the key effector cells in this process. However, the underlying regulatory mechanisms are not well defined. We found that iNKT cells constitutively expressed TIPE2 (Tumor necrosis factor-α-induced protein 8-like 2, or TNFAIPL2). Genetic TIPE2 ablation strongly sensitized mice to ConA-induced hepatitis, accompanied with hyperactivation of iNKT cells. Moreover, Tipe2-/- mice were also more susceptible to α-galactosylceramide (αGalCer)-induced liver injury, with elevated serum ALT level and enhanced proinflammatory cytokine production. CD1d signaling blockade or iNKT cell elimination through antibodies reduced the effect of TIPE2 deficiency on liver injury. Mechanistic studies revealed that TIPE2 in iNKT cells functioned as a negative regulator, limiting iNKT cell activity and cytokine production through PIP3- AKT/mTOR pathway. TIPE2-mediated protection from liver injury was further validated by the administration of adeno-associated viruses expressing TIPE2, which effectively ameliorated ConA-induced hepatic injury. However, TIPE2 was dispensable in two other liver injury models, including D-GalN/LPS and APAP-induced hepatitis. Our findings reveal a new role of TIPE2 in the attenuation of iNKT cell-mediated hepatic injury. We propose that TIPE2 serves as an important regulator of immune homeostasis in the liver, and might be exploited for the therapeutic treatment of autoimmune liver diseases.

Knockout of C6orf120 in Rats Alleviates Concanavalin A-induced Autoimmune Hepatitis by Regulating Macrophage Polarization

ObjectiveThe effect of the functionally unknown gene C6orf120 on autoimmune hepatitis was investigated on C6orf120 knockout rats (C6orf120-/-) and THP-1 cells. MethodSix–eight-week-old C6orf120-/- and wild-type (WT) SD rats were injected with Con A (16 mg/kg), and euthanized after 24 h. The sera, livers, and spleens were collected. THP-1 cells and the recombinant protein (rC6ORF120) were used to explore the mechanism in vitro. The frequency of M1 and M2 macrophages was analyzed using flow cytometry. Western blotting and PCR were used to detect macrophage polarization-associated factors. ResultsC6orf120 knockout attenuated Con A-induced autoimmune hepatitis. Flow cytometry indicated that the proportion of CD68+CD86+M1 macrophages from the liver and spleen in the C6orf120-/- rats decreased. C6orf120 knockout induced downregulation of CD86 protein and the mRNA levels of related inflammatory factors TNF-α, IL-1β, and IL-6 in the liver. C6orf120 knockout did not affect the polarization of THP-1 cells. However, rC6ORF120 promoted the THP-1 cells toward CD68+CD80+M1 macrophages and inhibited the CD68+CD206+M2 phenotype. ConclusionC6orf120 knockout alleviates Con A-induced autoimmune hepatitis by inhibiting macrophage polarization toward M1 macrophages and reducing the expression of related inflammatory factors in C6orf120-/- rats.

FKBP38 deletion exacerbates ConA-induced hepatitis by promoting the immune response through the MCP-1/p38 pathway

Autoimmune hepatitis (AIH) is a chronic liver disease characterized by immune dysregulation and hepatocyte damage. FKBP38, a member of the immunophilin family, has been implicated in immune regulation and the modulation of intracellular signaling pathways; however, its role in AIH pathogenesis remains poorly understood. In this study, we aimed to investigate the effects of hepatic FKBP38 deletion on AIH using a hepatic FKBP38 knockout (LKO) mouse model created via cre-loxP technology. We compared the survival rates, incidence, and severity of AIH in LKO mice with those in control mice. Our findings revealed that hepatic FKBP38 deletion resulted in an unfavorable prognosis in LKO mice with AIH. Specifically, LKO mice exhibited heightened liver inflammation and extensive hepatocyte damage compared to control mice, with a significant decrease in anti-apoptotic proteins and a marked increase in pro-apoptotic proteins. Additionally, transcriptional and translational levels of pro-inflammatory cytokines and chemokines were significantly increased in LKO mice compared to control mice. Immunoblot analysis showed that MCP-1 expression was significantly elevated in LKO mice. Furthermore, the phosphorylation of p38 was increased in LKO mice with AIH, indicating that FKBP38 deletion promotes liver injury in AIH by upregulating p38 phosphorylation and increasing MCP-1 expression. Immune cell profiling demonstrated elevated populations of T, NK, and B cells, suggesting a dysregulated immune response in LKO mice with AIH. Overall, our findings suggest that FKBP38 disruption exacerbates AIH severity by augmenting the immune response by activating the MCP-1/p38 signaling pathway.

Glycyrrhizin attenuates renal inflammation in a mouse Con A-hepatitis model via the IL-25/M2 axis

Glycyrrhizin (GL) has immunoregulatory effects on various inflammatory diseases including hepatitis and nephritis. However, the mechanisms underlying the anti-inflammatory effect of GL on renal inflammation are not fully understood. Hepatorenal syndrome (HRS) is a functional acute renal impairment that occurs in severe liver disease, and we found that kidney injury also occurs in Con A-induced experimental hepatitis in mice. We previously found that GL can alleviate Con A-induced hepatitis by regulating the expression of IL-25 in the liver. We wanted to investigate whether GL can alleviate Con A-induced nephritis by regulating IL-25. IL-25 regulates inflammation by modulating type 2 immune responses, but the mechanism by which IL-25 affects kidney disease remains unclear. In this study, we found that the administration of GL enhanced the expression of IL-25 in renal tissues; the latter promoted the generation of type 2 macrophages (M2), which inhibited inflammation in the kidney caused by Con A challenge. IL-25 promoted the secretion of the inhibitory cytokine IL-10 by macrophages but inhibited the expression of the inflammatory cytokine IL-1β by macrophages. Moreover, IL-25 downregulated the Con A-mediated expression of Toll-like receptor (TLR) 4 on macrophages. By comparing the roles of TLR2 and TLR4, we found that TLR4 is required for the immunoregulatory effect of IL-25 on macrophages. Our data revealed that GL has anti-inflammatory effects on Con A-induced kidney injury and that the GL/IL-25/M2 axis participates in the anti-inflammatory process. This study suggested that GL is a potential therapeutic for protecting against acute kidney injury.

YTHDF1 shapes immune-mediated hepatitis via regulating inflammatory cell recruitment and response

Severe immune responses regulate the various clinical hepatic injuries, including autoimmune hepatitis and acute viral hepatitis. N6-methyladenosine (m6A) modification is a crucial regulator of immunity and inflammation. However, the precise role of YTHDF1 in T cell-mediated hepatitis remains incompletely characterized. To address this, we utilized Concanavalin A (ConA)-induced mouse liver damage as an experimental model for T cell-mediated hepatitis. Our findings found that hepatic YTHDF1 protein rapidly decreased during ConA-induced hepatitis, and YTHDF1-deficient (Ythdf1−/−) mice showed more susceptibility to ConA-induced liver injury, along with an intensified inflammatory storm accompanied by aggravated hepatic inflammatory response via ERK and NF-κB pathways. Interestingly, hepatic-specific over-expression or deletion of YTHDF1 exhibited redundancy in ConA-induced liver injury. Validation in bone marrow chimeric mice confirmed the necessity of YTHDF1 in hematopoietic cells for controlling the response to ConA-induced hepatitis. Additionally, our data revealed that YTHDF1 deletion in macrophages exacerbated the inflammatory response induced by lipopolysaccharide. In summary, our study uncovered that YTHDF1 deficiency exacerbates the immune response in ConA-induced hepatitis by modulating the expression of inflammatory mediators, highlighting the potential of YTHDF1 as a therapeutic target for clinical hepatitis.

PIM1 inhibitor SMI-4a attenuated concanavalin A-induced acute hepatitis through suppressing inflammatory responses.

Serine/threonine kinase 1 (PIM1) plays a crucial role in cell growth, differentiation, and apoptosis. However, its role in the pathogenesis of concanavalin A (ConA)-induced acute hepatitis is not well understood. PIM1 kinase inhibitor can reduce the expression of PIM1. This study aims to investigate the effects of PIM1 kinase inhibitor and its protective mechanism in ConA-induced acute hepatitis. C57/BL six mice were injected with ConA (20, 15, and 12 mg/kg) to induce acute hepatitis, and PIM1 kinase inhibitor SMI-4a (60 mg/kg) was administered orally 24 h before ConA injection. The survival rate of the mice was observed after ConA injection. The levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Serum inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was performed on liver tissue collected at different time points. The major cytokines expression in liver tissue was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The number of macrophages, T-cell and neutrophils in liver tissue were detected by flow cytometry (FCM). PIM1 in liver tissue was detected by western blot (WB) and qRT-PCR. SMI-4a (80 µM) was pretreated for 24 h and ConA (400 µg/mL) was stimulated for 12 h in RAW264.7 cell model. Phosphorylated p65 (p-p65) and cleaved caspase-3 (c-caspase-3) in liver tissue and macrophages were detected by WB. Different concentrations of ConA caused different acute hepatitis mortality, 12 mg/kg concentration within 24 h of the mortality showed a gradient increase. The levels of AST and ALT increased significantly at 12 h after ConA injection. PIM1 expression was upregulated at 12 h. SMI-4a can suppress the PIM1 expression. SMI-4a suppressed cytokines production, AST, and ALT in ConA-treated serum. SMI-4a suppressed the major cytokines in liver tissue. Tests in liver tissue showed that SMI-4a reduced the number of T cells, neutrophils, and macrophages. SMI-4a inhibited the inflammatory response by downregulating the expression of p-p65. Meanwhile, apoptosis was decreased by decreasing the expression of c-caspase-3. In conclusion, the protective effect of SMI-4a against acute hepatitis is by reducing the inflammatory response and apoptosis. These findings suggest that SMI-4a may have therapeutic potential in the treatment of autoimmune hepatitis.

Cornuside improves murine autoimmune hepatitis through inhibition of inflammatory responses.

Autoimmune hepatitis (AIH) poses an important public health concern worldwide, with few therapeutic options available. Cornuside, a primary cornel iridoid glycoside present in Cornus officinalis Sieb. et Zucc., is a well-known traditional Chinese medicine that possesses anti-inflammatory, antioxidant and anti-apoptotic properties. However, the effects of cornuside on autoimmune diseases including AIH is still not defined, neither is clear on the mechanisms of cornuside in the suppression of inflammatory responses. The study was aimed to investigate the therapeutic effects of cornuside on AIH using murine models. A murine model of AIH induced by concanavalin A (Con A) was used to examine the pharmacological activity of cornuside in suppressing the inflammatory responses in vivo. C57BL/6J mice were intravenously with different doses of cornuside and challenged with 18mg/kg Con A 3h later. Network pharmacological analysis was performed to identify the potential target genes and signaling pathways by cornuside in AIH. Next serum and liver tissues were collected 12h after Con A injection to analyze the levels of markers for hepatic injury, apoptosis, oxidative stress, immune responses, and inflammation. Network pharmacological analysis revealed that cornuside may modulate oxidative stress and apoptosis in AIH. Compared with the Con A group, cornuside pretreatment significantly reduced the serum levels of alanine aminotransferase and aspartate aminotransferase, improving histopathological damage and apoptosis in the livers. In addition, cornuside decreased the levels of malondialdehyde, myeloperoxidase, but increased superoxide dismutase levels, suggesting the relieving of oxidative stress. Furthermore, cornuside suppressed the activation of T and natural killer T cells, whereas the proportion of myeloid-derived suppressor cells was significantly increased. The production of proinflammatory cytokines, including interleukin (IL)-6, IL-12, IL-1β, and tumor necrosis factor-alpha (TNF-α), was also clearly decreased. Finally, western blot analysis displayed that cornuside inhibited the phosphorylation of extracellular receptor kinase (ERK) and c-Jun N-terminal kinase (JNK). We demonstrated that cornuside has protective effects for Con A-induced immune-mediated hepatitis by suppressing the oxidative stress, apoptosis, and the inflammatory responses through the ERK and JNK signaling pathways, as well as by modulating the activation and recruitment of immune cells.

Monocyte-derived macrophages contribute to the deterioration of immunological liver injury in mice

Background & aimsAutoimmune hepatitis (AIH) is characterized by hepatocyte destruction, leading to lymphocyte and macrophage accumulation in the liver. However, the specific mechanisms of how macrophages participate in the occurrence and development of AIH are still unclear. In this study, we investigated the effect of monocyte-derived macrophages on Con A-induced immunological liver injury in mice and we hypothesized that inhibition of CCR2 with the dual CCR2/5 inhibitor, cenicriviroc (CVC), would attenuate Con A-induced hepatitis in mice by reducing the recruitment of monocytes into the liver. MethodsMurine experimental AIH was established by concanavalin A (Con A) injection intravenously. Macrophages were depleted by injection of clodronate liposomes in Con A-treated mice. Moreover, inhibition of the CCR2/5 signaling pathway in Con A mice is achieved by CVC. Liver injury and infiltration of monocyte-derived macrophages were assessed by serum transaminase levels, histopathology, immunohistochemistry, flow cytometry, RT–qPCR, ELISA, TUNEL assay and dihydroethidium staining. ResultsThe number of macrophages in the mouse livers increased in the Con A-induced hepatitis mouse model, and flow cytometry showed a significant increase in the proportion of F4/80loCD11bhi monocyte-derived macrophages, while there was no significant change in the proportion of F4/80hiCD11blo Kupffer cells. After the depletion of liver macrophages by clodronate liposomes, the levels of serum ALT and AST, and the degree of liver tissue damage were alleviated in Con A-treated mice. Furthermore, Con A leaded an increase in the expression of a group of CC chemokines in mouse livers, and the elevation of CCL2 was prevented with the depletion of macrophages. Additionally, CVC reduced macrophage infiltration in the liver and ameliorated Con A-induced liver injury. Meanwhile, CVC reduced the apoptosis and oxidative damage of hepatocytes caused by Con A. ConclusionsOur research demonstrates that there is an increase in monocyte-derived macrophages in the livers due to the monocyte infiltration resulted from the activation of the CCL2-CCR2 axis in Con A-induced liver injury mouse model. Pharmacological inhibition of CCR2 monocyte recruitment by CVC efficiently ameliorates the hepatic inflammation, indicating the therapeutic potential of CVC in patients with AIH.

CD73 mediates the therapeutic effects of endometrial regenerative cells in concanavalin A-induced hepatitis by regulating CD4+ T cells

BackgroundAs a kind of mesenchymal-like stromal cells, endometrial regenerative cells (ERCs) have been demonstrated effective in the treatment of Concanavalin A (Con A)-induced hepatitis. However, the therapeutic mechanism of ERCs is not fully understood. Ecto-5`-nucleotidase (CD73), an enzyme that could convert immune-stimulative adenosine monophosphate (AMP) to immune-suppressive adenosine (ADO), was identified highly expressed on ERCs. The present study was conducted to investigate whether the expression of CD73 on ERCs is critical for its therapeutic effects in Con A-induced hepatitis.MethodsERCs knocking out CD73 were generated with lentivirus-mediated CRISPR-Cas9 technology and identified by flow cytometry, western blot and AMPase activity assay. CD73-mediated immunomodulatory effects of ERCs were investigated by CD4+ T cell co-culture assay in vitro. Besides, Con A-induced hepatitis mice were randomly assigned to the phosphate-buffered saline treated (untreated), ERC-treated, negative lentiviral control ERC (NC-ERC)-treated, and CD73-knockout-ERC (CD73-KO-ERC)-treated groups, and used to assess the CD73-mediated therapeutic efficiency of ERCs. Hepatic histopathological analysis, serum transaminase concentrations, and the proportion of CD4+ T cell subsets in the liver and spleen were performed to assess the progression degree of hepatitis.ResultsExpression of CD73 on ERCs could effectively metabolize AMP to ADO, thereby inhibiting the activation and function of conventional CD4+ T cells was identified in vitro. In addition, ERCs could markedly reduce levels of serum and liver transaminase and attenuate liver damage, while the deletion of CD73 on ERCs dampens these effects. Furthermore, ERC-based treatment achieved less infiltration of CD4+ T and Th1 cells in the liver and reduced the population of systemic Th1 and Th17 cells and the levels of pro-inflammatory cytokines such as IFN-γ and TNF-α, while promoting the generation of Tregs in the liver and spleen, while deletion of CD73 on ERCs significantly impaired their immunomodulatory effects locally and systemically.ConclusionTaken together, it is concluded that CD73 is critical for the therapeutic efficiency of ERCs in the treatment of Con A-induced hepatitis.

A low dose of naloxone mitigates autoimmune hepatitis by regulating TLR4/NF-κB and Nrf2/HO-1 signaling pathways.

Naloxone is a non-selective opiate receptor antagonist that is mainly used in the management of acute opioid overdose or intoxication. Previously, naloxone has been shown to have anti-inflammatory and antioxidant properties. Concanavalin A (Con A) model is a common and well established animal model of autoimmune hepatitis that closely resembles the pathological alterations that occur in human. The present study demonstrates that a low dose of naloxone (LD NX) has the ability to improve hepatic function and attenuate hepatic damage induced by Con A as indicated by a clear reduction in serum aminotransferase, bilirubin and enhancement of albumin production as well as liver pathological changes. Also, The proinflammatory cytokines, tumor necrosis factor-α (TNF-α), interferon- γ (IFN-γ), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were highly suppressed in animals pretreated with LD NX via interference with TLR4/NF-κB as well as JNK signaling pathways. Furthermore, oxidative stress was highly attenuated in animals pretreated with LD NX as indicated by high reduction in hepatic MDA and an increase in Nrf2, HO-1 expression and subsequent production of the endogenous antioxidants, SOD, CAT and GSH. Collectively, this study demonstrates that LD NX has the ability to mitigate Con A-induced autoimmune hepatitis via modulation of inflammatory cytokines secretion and interference with reactive oxygen species generation.

Bioactivity of Eriocephalus africanus essential oil against concanavalin A-induced hepatitis via suppressing immune cell infiltration, inhibiting TNF-α/NF-κB and IFN-γ/STAT1 signaling pathways

Ethnopharmacological relevanceEriocephalus africanus infusion is used as a diuretic and a diaphoretic and is also used in the treatment of gastrointestinal disorders and gynaecological conditions, inflammation and dermal disorders, asthma, coughs, fevers, and painful ailments. The plant has been used traditionally as a medication to cure inflammation and skin problems. Aim of the studyStudying E. africanus essential oil (EAEO) as a potential hepatoprotective measure against concanavalin (Con) A-induced hepatitis in mice and investigating its underlying mechanism. Materials and methodsHydro-distilled oil of the fresh plant aerial shoots is subjected to GC/MS analysis. Autoimmune hepatitis (AIH) was induced in mice by intravenous injection of Con A (15 mg/kg). EAEO was administered orally before Con A injection to test its hepatoprotective activity. ResultsGC/MS analysis revealed the presence of 22 compounds representing 99.43% of the oil components. The monoterpene artemisia ketone (41.02%) and the sesquiterpene juniper camphor (14.17%) are the major components. The in vivo study showed that the oil suppressed Con A-induced neutrophil and CD4+T cell infiltration into the liver, restored hepatic redox balance, inhibited Con A-induced elevation of tumor necrosis factor-alpha (TNF-α), interleukin (IL-6), and interferon-gamma (IFN-γ) hepatic levels which were correlated with its ability to suppress nuclear factor kappa B (NF-κB) and Signal Transducer and Activator of Transcription (STAT1) activation in the liver. ConclusionEAEO showed hepatoprotective potential against Con A-induced hepatitis in mice collectively through selective anti-oxidant, anti-inflammatory, and anti-necrotic effects.

Celecoxib abrogates concanavalin A-induced hepatitis in mice: Possible involvement of Nrf2/HO-1, JNK signaling pathways and COX-2 expression.

Concanavalin A (ConA) is an established model for inducing autoimmune hepatitis (AIH) in mice, mimicking clinical features in human. The aimof the current study is to explore the possible protective effect of celecoxib, a cyclooxygenase-2 inhibitor,on immunological responses elicited in the ConA model of acute hepatitis. ConA (20mg/kg) was administered intravenously to adult male mice for 6h. Prior to ConA intoxication, mice in the treatedgroups received daily doses of celecoxib (30 and 60mg/kg in CMC) for 7days. Results revealed that administration of celecoxib 60mg/kg for 7days significantly protected the liver from ConA-induced liver damage revealed by significant decrease in ALT and AST serum levels. Celecoxib 30 and 60mg/kg pretreatment enhanced oxidant/antioxidant hemostasis by significantreduction of MDA and NO content and increase hepatic GSH contents and SOD activity. In addition, celecoxib 30 and 60mg/kg caused significant increase in hepatic nuclear factor erythroid 2-related factor 2 (Nrf2) and the stress protein heme oxygenase-1 (HO-1) levels. Moreover, celecoxib 30 and 60mg/kg inhibited the release of proinflammatory markers including IL-1β and TNF-α along with significant decrease in p-JNK, AKT phosphorylation ratio and caspase-3 expression. Besides, Con A was correlated to high expression of cyclooxygenase COX-2 and this increasing was improved by administration of celecoxib. These changes were in good agreement with improvement in histological deterioration. The protective effect of celecoxib was also associated with significant reduction of autophagy biomarkers (Beclin-1 and LC3II). In conclusion, celecoxib showed antioxidant, anti-inflammatory, anti-apoptotic and anti-autophagy activity against Con A-induced immune-mediated hepatitis. These effects could be produced by modulation of Nrf2/HO-1, IL-1B /p-JNK/p-AKT, JNK/caspase-3, and Beclin-1/LC3II signaling pathways.

Circadian control of ConA-induced acute liver injury and inflammatory response via Bmal1 regulation of Junb

Circadian rhythms play significant roles in immune responses, and many inflammatory processes in liver diseases are associated with malfunctioning molecular clocks. However, the significance of the circadian clock in autoimmune hepatitis (AIH), which is characterised by immune-mediated hepatocyte destruction and extensive inflammatory cytokine production, remains unclear. We tested the difference in susceptibility to the immune-mediated liver injury induced by concanavalin A (ConA) at various time points throughout a day in mice and analysed the effects of global, hepatocyte, or myeloid cell deletion of the core clock gene, Bmal1 (basic helix-loop-helix ARNT-like 1), on liver injury and inflammatory responses. Multiple molecular biology techniques and mice with macrophage-specific knockdown of Junb, a Bmal1 target gene, were used to investigate the involvement of Junb in the circadian control of ConA-induced hepatitis. The susceptibility to ConA-induced liver injury is highly dependent on the timing of ConA injection. The treatment at Zeitgeber time 0 (lights on) triggers the highest mortality as well as the severest liver injury and inflammatory responses. Further study revealed that this timing effect was driven by macrophage, but not hepatocyte, Bmal1. Mechanistically, Bmal1 controls the diurnal variation of ConA-induced hepatitis by directly regulating the circadian transcription of Junb and promoting M1 macrophage activation. Inhibition of Junb in macrophages blunts the administration time-dependent effect of ConA and attenuates liver injury. Moreover, we demonstrated that Junb promotes macrophage inflammation by regulating AKT and extracellular signal-regulated kinase (ERK) signalling pathways. Our findings uncover a critical role of the Bmal1-Junb-AKT/ERK axis in the circadian control of ConA-induced hepatitis and provide new insights into the prevention and treatment of AIH. This study unveils a critical role of the Bmal1-Junb-AKT/ERK axis in the circadian control of ConA-induced liver injury, providing new insights into the prevention and treatment of immune-mediated hepatitis, including autoimmune hepatitis (AIH). The findings have scientific implications as they enhance our understanding of the circadian regulation of immune responses in liver diseases. Furthermore, clinically, this research offers opportunities for optimising treatment strategies in immune-mediated hepatitis by considering the timing of therapeutic interventions.

Oral administration of ovalbumin protects mice from concanavalin A-induced hepatitis through suppression of interferon-gamma responses

The liver is a tolerogenic organ that exhibits hypo-responsiveness to antigens circulating in the portal vein. Antigens that are orally administered at high doses reach the liver. In our previous study, we demonstrated that administering ovalbumin (OVA) orally at high doses generates unique CD4+ T cells and tolerogenic dendritic cells, both of which can suppress T helper type 1 (Th1) responses, in the livers of two groups of mice: DO11.10 mice with transgenic CD4+ T cell receptors for OVA and BALB/c mice that received OVA-specific CD4+ T cells through adoptive transfer. This study aimed to investigate whether oral administration of OVA at high doses inhibits the development of hepatitis in the presence of OVA-specific CD4+ T cells. Oral administration of OVA at high doses inhibited the development of OVA-specific and concanavalin A (Con A)-induced hepatitis in DO11.10 mice, and these effects were associated with the downregulation of Th1 responses. Furthermore, the adoptive transfer of CD4+ T cells from the liver of OVA-fed DO11.10 mice inhibited the development of Con A-induced hepatitis in recipient BALB/c mice through the downregulation of Th1 responses. Finally, oral administration of OVA at high doses inhibited the development of Con A-induced hepatitis in BALB/c mice bearing naïve OVA-specific CD4+ T cells. These results suggest that the oral administration of antigens at high doses suppresses Th1-mediated hepatitis in an antigen-non-specific manner in the presence of antigen-specific CD4+ T cells.

M1 macrophage-derived exosomes promote autoimmune liver injury by transferring long noncoding RNA H19 to hepatocytes.

Exosomes mediate intercellular communication by transmitting active molecules. The function of long noncoding RNA (lncRNA) H19 in autoimmune liver injury is unclear. Concanavalin A (ConA)-induced liver injury is well-characterized immune-mediated hepatitis. Here, we showed that lncRNA H19 expression was increased in the liver after ConA treatment, accompanied by increased exosome secretion. Moreover, injection of AAV-H19 aggravated ConA-induced hepatitis, with an increase in hepatocyte apoptosis. However, GW4869, an exosome inhibitor, alleviated ConA-induced liver injury and inhibited the upregulation of lncRNA H19. Intriguingly, lncRNA H19 expression in the liver was significantly downregulated, after macrophage depletion. Importantly, the lncRNA H19 was primarily expressed in type I macrophage (M1) and encapsulated in M1-derived exosomes. Furthermore, H19 was transported from M1 to hepatocytes via exosomes, and exosomal H19 dramatically induced hepatocytes apoptosis both in vitro and vivo. Mechanistically, H19 upregulated the transcription of hypoxia-inducible factor-1 alpha (HIF-1α), which accumulated in the cytoplasm and mediated hepatocyte apoptosis by upregulating p53. M1-derived exosomal lncRNA H19 plays a pivotal role in ConA-induced hepatitis through the HIF-1α-p53 signaling pathway. These findings identify M1 macrophage-derived exosomal H19 as a novel target for the treatment of autoimmune liver diseases.

Tripterygium wilfordii protects against an animal model of autoimmune hepatitis.

Tripterygium wilfordii tablets (TWT) is widely used to treat autoimmune diseases such as rheumatoid arthritis. Celastrol, one main active ingredient in TWT, has been shown to produce a variety of beneficial effects, including anti-inflammatory, anti-obesity, anti-cancer, and immunomodulatory. However, whether TWT could protect against Concanavalin A (Con A)-induced hepatitis remains unclear. This study aims to investigate the protective effect of TWT against Con A-induced hepatitis and elucidate the underlying mechanism. Metabolomic analysis, pathological analysis, biochemical analysis, qPCR and Western blot analysis and the Pxr-null mice were used in this study. The results indicated that TWT and its active ingredient celastrol could protect against Con A-induced acute hepatitis. Plasma metabolomics analysis revealed that metabolic perturbations related to bile acid and fatty acid metabolism induced by Con A were reversed by celastrol. The level of itaconate in the liver was increased by celastrol and speculated as an active endogenous compound mediating the protective effect of celastrol. Administration of 4-octanyl itaconate (4-OI) as a cell-permeable itaconate mimicker was found to attenuate Con A-induced liver injury through activation of the pregnane X receptor (PXR) and enhancement of the transcription factor EB (TFEB)-mediated autophagy. Celastrol increased itaconate and 4-OI promoted activation of TFEB-mediated lysosomal autophagy to protect against Con A-induced liver injury in a PXR-dependent manner. Our study reported a protective effect of celastrol against Con A-induced AIH via an increased production of itaconate and upregulation of TFEB. The results highlighted that PXR and TFEB-mediated lysosomal autophagic pathway may offer promising therapeutic target for the treatment of autoimmune hepatitis.

Ginseng polysaccharide reduces autoimmune hepatitis inflammatory response by inhibiting PI3K/AKT and TLRs/NF-κB signaling pathways

BackgroundGinseng polysaccharides (GP) have been found to exhibit significant immune regulatory effects, making them a promising candidate for treating immune-related diseases. However, their mechanism of action in immune liver injury is not yet clear. The innovation of this study lies in exploring the mechanism of action of ginseng polysaccharides (GP) in immune liver injury. While GP has been previously identified for its immune regulatory effects, this study aims to provide a clearer understanding of its therapeutic potential for immune-related liver diseases. PurposeThe purpose of this study is to characterize low molecular weight gingeng polysaccharides (LGP), investigate their effect on ConA-induced autoimmune hepatitis (AIH), and identify their potential molecular mechanisms. MethodsLGP was extracted and purified using water-alcohol precipitation, DEAE-52 cellulose column, and Sephadex G200. And its structure was analyzed. It was then evaluated for anti-inflammatory and hepatoprotective effects in ConA-induced cells and mice, assessing cellular viability and inflammation with Cell Counting Kit-8 (CCK-8), Reverse Transcription-polymerase Chain Reaction (RT-PCR), and Western Blot, and hepatic injury, inflammation, and apoptosis with various biochemical and staining methods. ResultsLGP is a polysaccharide composed of glucose (Glu), galactose (Gal), and arabinose (Ara), with a molar ratio of 12.9:1.6:1.0. LGP has a low crystallinity amorphous powder structure and is free from impurities. LGP enhances cell viability and reduces inflammatory factors in ConA-induced RAW264.7 cells and inhibits inflammation and hepatocyte apoptosis in ConA-induced mice. LGP inhibits Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Toll-like receptors/Nuclear factor kappa B (TLRs/NF-κB) signaling pathways in vitro and in vivo to treat AIH. ConclusionsLGP was successfully extracted and purified, exhibiting potential as a treatment for ConA-induced autoimmune hepatitis due to its ability to inhibit the PI3K/AKT and TLRs/NF-κB signaling pathways and protect liver cells from damage.

Celastrol pretreatment attenuates concanavalin A-induced hepatitis in mice by suppressing interleukin-6/STAT3-interleukin-17 signaling.

Celastrol is extracted from Tripterygium wilfordii Hook F. It has been reported to have protective effects against various liver diseases and immune regulation of autoimmune diseases. However, little is known about whether celastrol protects against immune-mediated hepatitis. This study aimed to investigate the effect of celastrol on liver injury induced by concanavalin A (ConA) and the potential mechanisms. Intravenous administration of ConA was applied to induce acute liver injury in mice with or without pretreatment of celastrol. The effects of celastrol on ConA-induced liver injury were further demonstrated by biochemical and histopathological assessments, immunoblotting, and flow cytometry analysis. Both biochemical and histopathological observations showed that pretreatment of celastrol significantly ameliorated liver injury induced by ConA. Moreover, the hepatocyte apoptosis and inflammatory responses induced by ConA were also improved in celastrol-pretreated mice. Further studies revealed that these improvements were characterized as the celastrol-mediated suppression of total interleukin (IL)-17 from liver mononuclear cells in ConA-treated mice. Flow cytometry analysis suggested that celastrol specifically decreased IL-17 production by CD4+ T cells but not by CD8+ T cells. Fundamentally, pretreatment with celastrol inhibited both the IL-6 produced by F4/80+ macrophages and the IL-6 receptor on Th17 cells in the liver, which further led to the downregulated activation of STAT3, thus accounting for blocked Th17 signaling. Celastrol may exhibit immune regulatory effects by regulating IL-6/STAT3-IL-17 signaling in ConA-induced hepatitis, which suggested new potentials for celastrol to be applied in treating immune-mediated liver diseases.

Hepatocyte estrogen sulfotransferase inhibition protects female mice from concanavalin A–induced T cell–mediated hepatitis independent of estrogens

Autoimmune hepatitis (AIH) is a typical T cell-mediated chronic liver disease with a higher incidence in females. However, the molecular mechanism for the female predisposition is poorly understood. Estrogen sulfotransferase (Est) is a conjugating enzyme best known for its function in sulfonating and deactivating estrogens. The goal of this study is to investigate whether and how Est plays a role in the higher incidence of AIH in females. Concanavalin A (ConA) was used to induce T cell-mediated hepatitis in female mice. We first showed that Est was highly induced in the liver of ConA-treated mice. Systemic or hepatocyte-specific ablation of Est, or pharmacological inhibition of Est, protected female mice from ConA-induced hepatitis regardless of ovariectomy, suggesting the effect of Est inhibition was estrogen independent. In contrast, we found that hepatocyte-specific transgenic reconstitution of Est in the whole-body Est knockout (EstKO) mice abolished the protective phenotype. Upon the ConA challenge, EstKO mice exhibited a more robust inflammatory response with elevated production of proinflammatory cytokines and changed liver infiltration of immune cells. Mechanistically, we determined that ablation of Est led to the hepatic induction of lipocalin 2 (Lcn2), whereas ablation of Lcn2 abolished the protective phenotype of EstKO females. Our findings demonstrate that hepatocyte Est is required for the sensitivity of female mice to ConA-induced and T cell-mediated hepatitis in an estrogen-independent manner. Est ablation may have protected female mice from ConA-induced hepatitis by upregulating Lcn2. Pharmacological inhibition of Est might be a potential strategy for the treatment of AIH.