Comprehensive immune cell analysis of human menstrual-blood-derived stem cells therapy to concanavalin A hepatitis.

PKCzeta is required for nuclear factor kappa-B (NF-kappaB) activation in several cell systems. NF-kappaB is a suppressor of liver apoptosis during development and in concanavalin A (ConA)-induced T-cell-mediated hepatitis. Here we show that PKCzeta-/- mice display inhibited ConA-induced NF-kappaB activation and reduced damage in liver. As the IL-4/Stat6 pathway is necessary for ConA-induced hepatitis, we addressed here the potential role of PKCzeta in this cascade. Interestingly, the loss of PKCzeta severely attenuated serum IL-5 and liver eotaxin-1 levels, two critical mediators of liver damage. Stat6 tyrosine phosphorylation and Jak1 activation were ablated in the liver of ConA-injected PKCzeta-/- mice and in IL-4-stimulated PKCzeta-/- fibroblasts. PKCzeta interacts with and phosphorylates Jak1 and PKCzeta activity is required for Jak1 function. In contrast, Par-4-/- mice have increased sensitivity to ConA-induced liver damage and IL-4 signaling. This unveils a novel and critical involvement of PKCzeta in the IL-4/Stat6 signaling pathway in vitro and in vivo.

ObjectiveAstaxanthin, a potent antioxidant, exhibits a wide range of biological activities, including antioxidant, atherosclerosis and antitumor activities. However, its effect on concanavalin A (ConA)-induced autoimmune hepatitis remains unclear. The aim of this study was to investigate the protective effects of astaxanthin on ConA-induced hepatitis in mice, and to elucidate the mechanisms of regulation.Materials and MethodsAutoimmune hepatitis was induced in in Balb/C mice using ConA (25 mg/kg), and astaxanthin was orally administered daily at two doses (20 mg/kg and 40 mg/kg) for 14 days before ConA injection. Levels of serum liver enzymes and the histopathology of inflammatory cytokines and other maker proteins were determined at three time points (2, 8 and 24 h). Primary hepatocytes were pretreated with astaxanthin (80 μM) in vitro 24 h before stimulation with TNF-α (10 ng/ml). The apoptosis rate and related protein expression were determined 24 h after the administration of TNF-α.ResultsAstaxanthin attenuated serum liver enzymes and pathological damage by reducing the release of inflammatory factors. It performed anti-apoptotic effects via the descending phosphorylation of Bcl-2 through the down-regulation of the JNK/p-JNK pathway.ConclusionThis research firstly expounded that astaxanthin reduced immune liver injury in ConA-induced autoimmune hepatitis. The mode of action appears to be downregulation of JNK/p-JNK-mediated apoptosis and autophagy.

Hepatoprotective effect of quercetin via TRAF6/JNK pathway in acute hepatitis

Objective. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) reduces inflammation and has been identified as an anti-inflammatory prostaglandin in numerous animal models. In this study, we investigated both effects of 15d-PGJ2 and its protection mechanism in concanavalin A- (ConA-) induced autoimmune hepatitis in mice. Materials and Methods. In vivo, Balb/C mice were injected with ConA (25 mg/kg) to induce acute autoimmune hepatitis, and 15d-PGJ2 (10 μg or 25 μg) was administered 1 h before the ConA injection. The histological grade, proinflammatory cytokine levels, and NF-κB and PPARγ activity were determined 6, 12, and 24 h after the ConA injection. In vitro, LO2 cells and RAW264.7 cells were pretreated with 15d-PGJ2 (2 μM) 1 h before the stimulation with ConA (30 μg/mL). The NF-κB and PPARγ activity were determined 30 min after the ConA administration. Results. Pretreatment with 15d-PGJ2 reduced the pathological effects of ConA-induced autoimmune hepatitis and significantly reduced the levels of cytokines after injection. 15d-PGJ2 activated PPARγ, blocked the degradation of IκBα, and inhibited the translocation of NF-κB into the nucleus. Conclusion. These results indicate that 15d-PGJ2 protects against ConA-induced autoimmune hepatitis by reducing proinflammatory cytokines. This reduction in inflammation may correlate with the activation of PPARγ and the reduction in NF-κB activity.

Liver is an immunologically tolerogenic organ, while, autoreactive immune cells mediated autoimmune hepatitis (AIH) is observed. Suppression of the hyper-reactive immune responses may restore the tolerance of liver. IL-37, an emerging IL-1 family cytokine with anti-inflammatory effects on innate and adaptive immunity, shows benefit on many autoimmune diseases. We proposed IL-37 could inhibit immune responses and improve AIH. IL-37 expressing adeno-associated virus (AAV-IL-37) successfully dampened the activation of macrophages in vitro. We next analyzed the effect of IL-37 in Con A-induced autoimmune hepatitis mouse model. Surprisingly, the liver histopathology showed more necrotic hepatocytes and infiltrating immune cells in AAV-IL-37 treated Con A mice than in control mice. Higher levels of serum IFN-γ in IL-37-expressing Con A mice were observed. Moreover, expression of inflammation-related mRNA in the liver were also highly induced with AAV-IL-37 treatment. Among hepatic lymphoid cells, NK and NKT cells were particularly increased in IL-37 treated Con A mice. The frequency of IFN-γ-secreting NK cells was also significantly increased. Further, CCL5, a chemoattractant for NK cells, was significantly elevated in the serum of AAV-IL-37 injected Con A mice. These results suggest that IL-37 worsen liver inflammation instead of relieving the disease. Of note, there was no obvious inflammation in AAV-IL-37 treated naïve mice, suggesting the proinflammatory effect of IL-37 in Con A treated mice resulted from the inflamed liver microenvironment. In conclusion, IL-37 aggravated liver inflammation in Con A induced hepatitis through recruiting NK cells and elevating the IFN-γ secretion of NK cells.

ABSTRACTAutoimmune hepatitis (AIH) is a progressive inflammation-associated liver injury. Pyroptosis is a novel inflammatory programmed cell death wherein gasdermin D (GSDMD) serves as the executioner. Our work challenged Gsdmd−/− mice with concanavalin A (ConA) to try to unveil the actual role of GSDMD in AIH. After ConA injection, Gsdmd−/− mice exhibited more severe liver damage characterized by a lower survival rate, more extensive hepatocyte necrosis and apoptosis, and higher serum transaminase levels, indicating the protection of GSDMD in ConA-induced AIH. Furthermore, the Gsdmd−/− mice exhibited higher hepatic expression and serum levels of inflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-17A [IL-17A]) and more infiltration of macrophages and neutrophils after ConA treatment than did wild-type (WT) mice. Gsdmd−/− mice with AIH showed increased hepatic l-glutamine levels but decreased glycerophospholipid metabolites levels. L-glutamine levels showed positive correlations while glycerophospholipid metabolites showed negative associations with liver injury indexes and inflammation markers. We further observed a destroyed intestinal barrier in Gsdmd−/− mice after ConA injection as indicated by decreased transcriptional expressions of Tjp1, Ocln, Reg3g, and Muc2. ConA-treated Gsdmd−/− mice also exhibited higher serum LPS binding protein (LBP) concentrations and hepatic Tlr4 and Cd14 mRNA levels. Further fecal 16S rRNA gene sequencing demonstrated decreased relative abundances of Lactobacillus and Roseburia but increased relative abundances of Allobaculum and Dubosiella in Gsdmd−/− mice with AIH. Lactobacillus was negatively correlated with liver injury and inflammation indexes and positively associated with Ocln, Muc2, and Reg3g levels. Allobaculum was positively related to liver injury and inflammatory cytokines and negatively correlated with gut barrier indexes.IMPORTANCE Our study provides the first direct clues to the protective role of gasdermin D (GSDMD) in autoimmune hepatitis (AIH). We demonstrated that Gsdmd knockout exacerbated concanavalin A (ConA)-induced AIH in mice. It may be due to the destroyed intestinal barrier and changes in certain intestinal microbes and hepatic metabolites resulting in increased liver injury and inflammation in ConA-treated Gsdmd−/− mice. This finding suggested a nonnegligible role of GSDMD in AIH and also confirmed its physiological nonpyroptosis effects on the host. The role of GSDMD in autoimmune liver diseases or other liver diseases is complex and intriguing, deserving deep investigation.

Novel Model of Antigen-Specific Induction of Bile Duct Injury

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.

Natural killer group 2D (NKG2D) is a well-characterized activating receptor expressed on many immune cells, including invariant natural killer T (iNKT) cells. These cells were shown to be responsible of liver injury in the model of concanavalin A (Con A)-induced hepatitis, considered to be an experimental model of human autoimmune hepatitis. In this study, we investigated whether NKG2D plays a role in the hepatitis induced by iNKT cell-mediated immune response to Con A. By using killer cell lectin-like receptor subfamily K, member 1 deficient (Klrk1−/−) mice, we found that the absence of NKG2D reduced the hepatic injury upon Con A administration. This was not due to an intrinsic functional defect of NKG2D-deficient iNKT cells as mice missing NKG2D have normal distribution and function of iNKT cells. Furthermore, increased resistance to Con A-induced hepatitis was confirmed using neutralizing anti-NKG2D antibodies. The reduced pathogenic effect of Con A in the absence of NKG2D correlates with a reduction in pathogenic cytokine production and FAS-Ligand (FAS-L) expression by iNKT cells. We also found that Con A administration led to an increase in the retinoic acid early inducible (RAE-1) surface expression on wild-type hepatocytes. Finally, we found that Con A has no direct action on FAS-L expression or cytokine production by iNKT cells and thus propose that NKG2D-L expression on stressed hepatocytes promote cytotoxic activity of iNKT cells via its interaction with NKG2D contributing to hepatic injury. In conclusion, our results highlight NKG2D as an essential receptor required for the activation of iNKT cells in Con A-induced hepatitis and indicate that it represents a potential drug target for prevention of autoimmune hepatitis.

Aims Levo-tetrahydropalmatine (L-THP) is an active ingredient of Corydalis yanhusuo W. T. Wang, which has many bioactive properties. Herein, we investigated the protective effects of L-THP on concanavalin A- (ConA-) induced hepatitis in mice and explored its possible mechanisms of these effects. Main Methods Balb/c mice were intravenously injected with 25 mg/kg ConA to generate a model of acute autoimmune hepatitis, and L-THP (20 or 40 mg/kg) was administered orally once daily for 5 d before the ConA injection. The liver enzyme levels, proinflammatory cytokine levels, and other marker protein levels were determined 2, 8, and 24 h after ConA injection. Results L-THP could decrease serum liver enzymes and pathological damage by reducing the release of inflammatory factors like IL-6 and TNF-α. The results of Western Blot and PCR indicated that L-THP could ameliorate liver cell apoptosis and autophagy. L-THP could suppress T lymphocyte proliferation and the production of TNF-α and IL-6 induced by ConA in a dose-dependent manner in vitro. Additionally, the protective functions of L-THP depended on downregulating TRAF6/JNK signaling. Conclusion. The present study indicated that L-THP attenuated acute liver injury in ConA-induced autoimmune hepatitis by inhibiting apoptosis and autophagy via the TRAF6/JNK pathway.

Natural killer T (NKT) cells constitute a distinct subpopulation of T cells with a unique antigen specificity, prompt effector functions, and an unusual tissue distribution. NKT cells are especially abundant in the liver, but their physiological function in this organ remains unclear. In the present study, we examined the possible contribution of NKT cells to a murine model of hepatitis induced by i.v. injection of Con A. CD1-deficient mice lacking NKT cells were highly resistant to Con A-induced hepatitis. Adoptive transfer of hepatic NKT cells isolated from wild-type mice, but not from FasL-deficient gld mice, sensitized CD1-deficient mice to Con A-induced hepatitis. Furthermore, adoptive transfer of hepatic mononuclear cells from wild-type mice, but not from CD1-deficient mice, sensitized gld mice to Con A-induced hepatitis. Upon Con A administration, hepatic NKT cells rapidly up-regulated cell surface FasL expression and FasL-mediated cytotoxicity. At the same time, NKT cells underwent apoptosis leading to their rapid disappearance in the liver. These results implicated FasL expression on liver NKT cells in the pathogenesis of Con A-induced hepatitis, suggesting a similar pathogenic role in human liver diseases such as autoimmune hepatitis.

Pharmacologic Relevance. Resveratrol, an antioxidant derived from grapes, has been reported to modulate the inflammatory process. In this study, we investigated the effects of resveratrol and its mechanism of protection on concanavalin-A- (ConA-) induced liver injury in mice. Materials and Methods. Acute autoimmune hepatitis was induced by ConA (20 mg/kg) in Balb/C mice; mice were treated with resveratrol (10, 20, and 30 mg/kg) daily by oral gavage for fourteen days prior to a single intravenous injection of ConA. Eight hours after injection, histologic grading, proinflammatory cytokine levels, and hedgehog pathway activity were determined. Results. After ConA injection, the cytokines IL-2, IL-6, and TNF-α were increased, and Sonic hedgehog (Shh), Glioblastoma- (Gli-) 1, and Patched (Ptc) levels significantly increased. Pretreatment with resveratrol ameliorated the pathologic effects of ConA-induced autoimmune hepatitis and significantly inhibited IL-2, IL-6, TNF-α, Shh, Gli-1, and Ptc. The effects of resveratrol on the hedgehog pathway were studied by western blotting and immunohistochemistry. Resveratrol decreased Shh expression, possibly by inhibiting Shh expression in order to reduce Gli-1 and Ptc expression. Conclusion. Resveratrol protects against ConA-induced autoimmune hepatitis by decreasing cytokines expression in mice. The decreases seen in Gli-1 and Ptc may correlate with the amelioration of hedgehog pathway activity.

Background Interleukin (IL)-39 is a novel member of IL-12 family and has been reported to play a pro-inflammatory role in lupus-like mice, but its function in concanavalin A (ConA)-induced liver injury is currently unclear. Materials and methods In this study, we investigated the effects of IL-39 expression in a mouse model of ConA induced-hepatitis. We first showed that delivery of plasmid DNA encoding mouse IL-39 using the hydrodynamic tail vein injection method increased IL-39 mRNA and protein levels in the liver. We then administrated mice with IL-39 plasmid before ConA injection and measured serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, inflammatory infiltration, and hepatocyte necrosis in the liver. Additionally, we further explored the potential mechanism of IL-39 in ConA-induced liver injury by measuring several inflammatory mediators. Results We found that ectopic IL-39 expression promoted the ConA-induced increase in serum ALT and AST levels, inflammatory infiltration, and hepatocyte necrosis in the liver. We also observed that IL-39 plasmid administration significantly increased serum and liver interferon-γ, tumor necrosis factor-α, and IL-17A levels, but did not affect serum and liver IL-10 levels in ConA-induced hepatitis. Conclusion Our results suggest that IL-39 can exacerbate ConA-induced hepatitis and may be a therapeutic target in inflammatory liver disease.

Administration of Con A induces liver injury that is considered to be an experimental model for human autoimmune or viral hepatitis, where immunopathology plays roles mediated by activated lymphocytes, especially NK1.1+ CD3+ NKT cells, and inflammatory cytokines, including IFN-gamma and IL-4. In the present study we investigated the role of WSX-1, a component of IL-27R, in Con A-induced hepatitis by taking advantage of WSX-1 knockout mice. WSX-1-deficient mice were more susceptible to Con A treatment than wild-type mice, showing serum alanine aminotransferase elevation and massive necrosis in the liver. Although the development of NKT cells appeared normal in WSX-1 knockout mice, purified NKT cells from the knockout mice produced more IFN-gamma and IL-4 than those from wild-type mice in response to stimulation with Con A both in vitro and in vivo. In addition, hyperproduction of proinflammatory cytokines, including IL-1, IL-6, and TNF-alpha, was observed in the knockout mice after Con A administration. These data revealed a novel role for WSX-1 as an inhibitory regulator of cytokine production and inflammation in Con A-induced hepatitis.

<b>Abstract ID 16563</b> <b>Poster Board 387</b> <b>Background &amp; Aims:</b> 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 female preference of AIH. <b>Methods:</b> Mice that bear genetic or pharmacological inhibition of Est were subjected to Concanavalin A (ConA)-induced T cell-mediated hepatitis. The AIH phenotypes were characterized by the serum ALT and AST, liver histology, and the levels of cytokines and hepatic immune cells by qPCR, ELISA, and flow cytometry. <b>Results:</b> Est was highly induced in the liver of ConA-treated mice. Systemic (EstKO) or hepatocyte-specific ablation, or pharmacological inhibition of Est protected female mice from ConA-induced hepatitis regardless of ovariectomy. In contrast, hepatocyte-specific transgenic reconstitution of Estin EstKO mice abolished the protective phenotype. ConA-treated EstKO mice exhibited a more robust inflammatory response with elevated pro-inflammatory cytokines but less toxic hepatic immune profile. Mechanistically, ablation of Est led to the hepatic induction of lipocalin 2 (Lcn2), whereas ablation of Lcn2 abolished the protective phenotype of EstKO females. <b>Conclusions:</b> Hepatocyte Est is required for female sensitivity 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.