Con A-induced Hepatitis Research Articles

New Insight into the Concanavalin A-Induced Apoptosis in Hepatocyte of an Animal Model: Possible Involvement of Caspase-Independent Pathway.

Concanavalin A (Con A) is known to be a T-cell mitogen and has been shown to induce hepatitis in mice through the triggering of conventional T cells and NKT cells. However, it remains unknown whether Con A itself can directly induce rapid hepatocyte death in the absence of a functional immune system. Here, by using an immunodeficient mouse model, we found Con A rapidly induced liver injury in vivo despite a lack of immunocyte involvement. We further observed in vitro that hepatocytes underwent a dose-dependent but caspase-independent apoptosis in response to Con A stimulation in vitro. Moreover, transcriptome RNA-sequencing analysis revealed that apoptosis pathways were activated in both our in vivo and in vitro models. We conclude that Con A can directly induce rapid but non-classical apoptosis in hepatocytes without the participation of immunocytes. These findings provide new insights into the mechanism of Con A-induced hepatitis.

RAGE deficiency ameliorates autoimmune hepatitis involving inhibition of IL-6 production via suppressing protein Arid5a in mice.

Activation of T cells and pro-inflammatory cytokines are essential for human autoimmune hepatitis. RAGE is one of the receptors for the inflammatory alarm molecule high mobility group box 1 (HMGB1), and it is involved in autoimmune hepatitis. However, the molecular mechanism of RAGE in the context of autoimmune hepatitis remains elusive. This study aimed to identify the function and mechanism of RAGE in autoimmune hepatitis. The role and underlying mechanisms of RAGE signaling-driven immune inflammatory response in ConA-induced experimental hepatitis were examined using the RAGE-deficient mice. We found that the RAGE deficiency protected the mouse from liver inflammatory injury caused by the ConA challenge. mRNA expression of VCAM-1, IL-6, and TNF-α within the livers is markedly decreased in RAGE-deficient mice compared to wild-type mice. In parallel, RAGE deficiency leads to reduced levels of the serum pro-inflammatory cytokines IL-6 and TNF-α as compared with wild-type control mice. RAGE-deficient mice exhibit increased hepatic NK cells and decreased CD4+ T cells compared with wild-type control mice. Notably, in vivo blockade of IL-6 in wild-type mice significantly protected mice from ConA-induced hepatic injury. Furthermore, RAGE deficiency impaired IL-6 production and was associated with decreased expression of Arid5a in liver tissues, a half-life IL-6 mRNA regulator. RAGE signaling is important in regulating the development of autoimmune hepatitis. Immune regulation of RAGE may represent a novel therapeutic strategy to prevent immune-mediated liver injury.

CX3CR1 deficiency exacerbates immune-mediated hepatitis by increasing NF-κB-mediated cytokine production in macrophage and T cell.

Immune-mediated hepatitis is marked by liver inflammation characterized by immune cell infiltration, chemokine/cytokine production, and hepatocyte injury. C-X3C motif receptor 1 (CX3CR1), as the receptor of chemokine C-X3C motif ligand 1 (CX3CL1)/fractalkine, is mainly expressed on immune cells including monocytes and T cells. Previous studies have shown that CX3CR1 protects against liver fibrosis, but the exact role of CX3CL1/CX3CR1 in acute immune-mediated hepatitis remains unknown. Here, we investigate the role of the CX3CL1/CX3CR1 axis in immune-mediated hepatitis using concanavalin A (ConA)-induced liver injury model in CX3CR1-deficient (Cx3cr1-/-) mice. We observed that Cx3cr1-/- mice had severe liver injury and increased pro-inflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interferon-gamma [IFN-γ], interleukin-1 beta [IL-1β], and IL-6) in serum and liver compared to wild-type (Cx3cr1+/+) mice after ConA injection. The deficiency of CX3CR1 did not affect ConA-induced immune cell infiltration in liver but led to elevated production of TNF-α in macrophages as well as IFN-γ in T cells after ConA treatment. On the contrary, exogenous CX3CL1 attenuated ConA-induced cytokine production in wild type, but not CX3CR1-deficient macrophages and T cells. Furthermore, in vitro results showed that CX3CR1 deficiency promoted the pro-inflammatory cytokine expression by increasing the phosphorylation of nuclear factor kappa B (NF-κB) p65 (p-NF-κB p65). Finally, pre-treatment of p-NF-κB p65 inhibitor, resveratrol, attenuated ConA-induced liver injury and inflammatory responses, especially in Cx3cr1-/- mice. In conclusion, our data show that the deficiency of CX3CR1 promotes pro-inflammatory cytokine production in macrophages and T cells by enhancing the phosphorylation of NF-κB p65, which exacerbates liver injury in ConA-induced hepatitis.

Inhibition of receptor-interacting protein 3 improves experimental autoimmune hepatitis

Objective: To evaluate the potential of receptor-interacting protein 3 (RIP3) as a therapeutic target for autoimmune hepatitis (AIH). Methods: Immunofluorescence assay was used to observe the activated expression levels of RIP3 and its downstream signal mixed lineage protein kinase domain-like protein (MLKL) in the liver tissues of patients with AIH and hepatic cyst. Concanavalin A (ConA) was injected into the tail vein to induce acute immune-mediated hepatitis in mice. Intervention was performed by intraperitoneal injection of RIP3 inhibitor GSK872 or solvent carrier. Peripheral blood and liver tissues were collected. Serum transaminases level, qPCR and flow cytometry were analyzed. The intergroup comparison was performed with an independent sample t-test. Results: The expression level of p-RIP3 (the activated forms of RIP3) and phosphorylated p-MLKL (MLKL after phosphorylation) downstream signal were significantly higher in the liver tissue of AIH patients than those of controls. Compared with the control group, the expression levels of RIP3 and MLKL mRNA were significantly increased in the liver tissue of AIH patients (relative expression levels 3.28±0.29 vs. 0.98±0.09, 4.55±0.51 vs. 1.06±0.11), and the differences were statistically significant (t=6.71 and 6.77, respectively, and P<0.01). The expression levels of RIP3 and MLKL mRNA were significantly higher in the mice liver tissue of ConA-induced immune hepatitis than those in the control group (relative expression levels 2.35±0.09 vs. 0.89±0.11,2.77±0.22 vs. 0.73±0.16,t=10.4,6.33, P<0.01). RIP3 inhibitor GSK872 had significantly attenuated ConA-induced immune liver injury and inhibited the expression of tumor necrosis factor-α, interleukin-6, interleukin-1β and NLRP3 in liver. Compared with the control group, the proportions of CD45+F4/80+ macrophages, CD4+ IL-17+ Th17 cells, CD4+ CD25+ regulatory T (Treg) cells and CD11b+ Gr-1+ myeloid derived suppressor cells (MDSCs) were significantly increased in the liver of ConA + Vehicle group. Compared with ConA + Vehicle group, the proportion of CD45+F4/80+ macrophages and CD4+ IL-17+ Th17 cells were significantly decreased, while the proportion of CD4+ CD25+Treg cells and CD11b+ Gr-1+ MDSCs with immunomodulatory functions were significantly increased in mice liver of ConA+GSK872 group. Conclusion: AIH patients and ConA-induced immune hepatitis mice have activated RIP3 signal in liver tissues. Inhibition of RIP3 reduces the expression and proportion of proinflammatory factors and cells, and promotes the accumulation of CD4+ CD25+ Treg cells and CD11b+ Gr-1+ MDSCs with immunomodulatory functions in the liver of mice with immune hepatitis, thereby alleviating liver inflammation and injury. Therefore, the inhibition of RIP3 is expected to be a new approach for the treatment of AIH.

CP-25 exerts a protective effect against ConA-induced hepatitis via regulating inflammation and immune response.

Hepatitis is a complex multifactorial pathological disorder, which can eventually lead to liver failure and even potentially be life threatening. Paeoniflorin-6'-O-benzene sulfonate (CP-25) has proven to have critical anti-inflammatory effects in arthritis. However, the effects of CP-25 in the pathogenesis of hepatitis remains unclear. In this experiment, mice were intragastrically administered with CP-25 (25, 50 and 100mg/kg), and then ConA (25mg/kg) was intravenous injected to establish hepatitis model in vivo. CP-25 administration attenuated liver damage and decreased ALT and AST activities in mice with hepatitis. Besides, CP-25 modulated immune responses including down-regulated the proportions of activated CD4+, activated CD8+ T cells, and ratio of Th1/Th2 in ConA-injected mice. Furthermore, ConA-mediated production of reactive oxygen species (ROS), release of inflammatory cytokines including IFN-γ, TNF-α, activation of MAPK pathways and nuclear translocation of nuclear factor-kappaB (NF-κB) were significantly decreased in CP-25 administrated mice. In ConA-stimulated RAW264.7 cells, CP-25 suppressed inflammatory cytokines secretion and reduced ROS level, which were consistent with animal experiments. Otherwise, the data showed that CP-25 restrained phosphorylation of ERK, JNK and p38 MAPK pathways influenced by ROS, accompanied with inhibiting NF-κB nuclear translocation. In conclusion, our findings indicated that CP-25 protected against ConA-induced hepatitis may through modulating immune responses and attenuating ROS-mediated inflammation via the MAPK/NF-κB signaling pathway.

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

Autoimmune hepatitis is an autoimmune disease with increasing occurrence worldwide. The most common and convenient mouse model is the concanavalin A (ConA) mouse model. Human menstrual-blood-derived stem cells (MenSCs) have shown great potential as a type of mesenchymal stem cell for treating various diseases. Time-of-flight mass cytometry was performed in phosphate-buffered saline control (NC) group and ConA injection with or without MenSCs treatment groups, and conventional flow cytometry was used for further validation. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and H&E staining depicted that MenSCs treatment could significantly alleviate ConA-induced hepatitis. The t-distributed stochastic neighbor embedding (t-SNE) analysis of nine liver samples displayed favorable cell clustering, and the NC group was significantly different from the other two groups. The proportions of CD69+ T cells, NKT cells, and PD-L1+ macrophages were notably increased by ConA injection, while MenSCs could decrease ConA-induced macrophage percentage and M1 polarization in the liver tissue. The analysis of proinflammatory factors carried out by cytometric bead array demonstrated that tumor necrosis factor alpha (TNF-α), interleukin (IL)-17A, IL-12p70, IL-6, IL-2, IL-1b, and interferon gamma (IFN-γ) were upregulated after ConA injection and then rapidly decreased at 12 h. MenSCs also played an important role in downregulating these cytokines. Here, we described the comprehensive changes in leukocytes in the liver tissue of ConA-induced hepatitis at 12 h after ConA injection and found that MenSCs rescued ConA-induced hepatitis mostly by inhibiting macrophages and M1 polarization in mouse liver.

Escin suppresses immune cell infiltration and selectively modulates Nrf2/HO-1, TNF-α/JNK, and IL-22/STAT3 signaling pathways in concanavalin A-induced autoimmune hepatitis in mice

The current study aims to investigate the possible protective effect of escin, the active constituent of a natural mixture of triterpene saponin glycoside, against immune-mediated hepatitis driven by concanavalin A (Con A) and to elucidate its possible underlying mechanisms. Adult male mice were administered Con A (15 mg/kg, intravenously) for 8 h. In the treated groups, mice were pretreated with escin daily (10 mg/kg in CMC, orally) for 4 days before Con A intoxication. In addition, escin was administered in a group to examine its effect on normal mice. Our results showed that escin inhibited Con A-induced elevation in liver enzymes (ALT, AST, and LDH) and curbed the Con A-induced hepatocyte necrosis and apoptosis together with abrogating the death pathway, JNK. Coincidentally, escin has shown a reduction in neutrophil, CD4+ T cell, and monocyte infiltration into the liver. In addition, escin modulated the cellular oxidant status by compensating for the Con A-depleted expression of the transcription factor Nrf2 and the stress protein hemeoxygenase-1. These effects were in good agreement with the restraining effect of escin on Con A-instigated overexpression of NF-κB and the pro-inflammatory cytokines TNF-α and IL-17A. Interestingly, Con A provoked the cellular protective pathway IL-22/STAT3, which was revoked by the escin pretreatment. In conclusion, escin shows extended antioxidant, anti-inflammatory, antinecrotic, and anti-apoptotic effects against Con A-induced immune-mediated hepatitis. These effects may collectively be via suppressing immune cell infiltration into the liver and selective modulation of Nrf2/HO-1, TNF-α/NF-κB, TNF-α/JNK, and IL-22/STAT3 signaling pathways.

Gastrodin ameliorates Concanavalin A-induced acute hepatitis via the IL6/JAK2/STAT3 pathway

Aims Gastrodin, the main active ingredient of Gastrodia elata Blume, has been shown to protect against many inflammatory diseases. Our study aimed to investigate the anti-inflammatory role of gastrodin in concanavalin A (ConA)-induced acute hepatitis in mice and to explore its precise mechanism. Methods C57BL/6 mice were administered with gastrodin (50 or 100mg/kg) for 3 days prior to intravenous injection of ConA to induce acute autoimmune hepatitis (AIH). Serum aminotransferases levels and cytokine levels were measured. Liver tissue histology was conducted to assess the degree of liver injury. Splenocytes pretreated with gastrodin were stimulated with ConA to observe splenocyte proliferation. Results Gastrodin greatly reduced the level of serum aminotransferases, inflammatory cytokine such as IL-6 and TNF-α and histopathological damage in ConA-induced hepatitis. Besides, gastrodin had an inhibitory effect on liver apoptosis, and autophagy. Furthermore, gastrodin inhibited the proliferation of splenocytes in vitro. The protein expression of p-JAK2 and p-STAT3 was markedly affected by gastrodin pretreatment. Conclusions The present study indicated that gastrodin pretreatment exerted protective effects against ConA-induced acute hepatitis, partly through the inhibition of the IL6/JAK2/STAT3 pathway. Further studies are recommended to determine the potential therapeutic role of gastrodin in acute AIH.

Methyl butyrate attenuates concanavalin A-induced autoimmune hepatitis by inhibiting Th1-cell activation and homing to the liver

Con A-induced hepatitis is the most commonly used animal model for simulating autoimmune hepatitis (AIH). In this study, we investigated whether methyl butyrate (MB) alleviates Con A-induced hepatitis and how it affects Con A-stimulated lymphocytes. MB improves liver function in AIH mice, reducing the expression of several inflammatory cytokines and Th1 cell-associated chemokines in the liver, while significantly inhibiting toll-like receptor signaling pathway. Also in the liver, we verified that infiltrating Th1 cells were fewer after MB treatment. In vitro, we found that the activation of both human and mouse Th1 cells by Con A were inhibited by MB and the human-derived cells were even more sensitive. And MB caused a reduction in IFN-γ secretion together with TNF-α and IL-6. The above findings suggest that MB inhibits the activation and homing of Th1 cells to the liver, thereby attenuating Con A-induced liver injury, and may be a potential therapeutic agent for AIH.

The Protective Role of IL-36/IL-36R Signal in Con A-Induced Acute Hepatitis

Abstract The IL-36 family, including IL-36α, IL-36β, IL-36γ, and IL-36 receptor antagonist (IL-36Ra), belongs to the IL-1 super-family. Although IL-36 is known to play a role in autoimmune diseases, it remains unclear as how IL-36 regulates inflammation in the liver. To determine the role of IL-36/IL-36R signaling pathways, we established an acute hepatitis mouse model by intravenous injection of the plant lectin concanavalin A (Con A), and found that the levels of IL-36 were increased in the liver following Con A injection. We also demonstrated that intrahepatic leukocytes, but not hepatocytes, were the main source of IL-36. Moreover, further research verified that infiltrated neutrophils were the main source of IL-36γ in the liver. Using the IL-36R knockout mouse model (H-2b), surprisingly, we found that the absence of IL-36 signals led to aggravated liver injuries, as evidenced by increased mortality, accompanied by elevated serum ALT and AST levels and severe pathological changes in the liver. Further investigations demonstrated that a lack of IL-36 signaling induced intrahepatic activation of CD4+ and CD8+T lymphocytes and increased the production of inflammatory cytokines. In addition, IL-36R knockout mice had reduced Treg cell functions and chemotaxis in the liver. Together, our results suggest a vital role of IL-36 in regulating T cell function and homeostasis during liver injury and inflammation. Supported by grants from NIH (R01 EY028773, AI132674, R21 AI153586)

Pharmacokinetic/Pharmacodynamic Assessment of Selective Phosphodiesterase Inhibitors in a Mouse Model of Autoimmune Hepatitis.

Autoimmune hepatitis (AIH) is a life-threatening disorder currently treated with nonspecific immunosuppressive drugs. It is postulated that phosphodiesterase (PDE) inhibitors, as agents exerting anti-inflammatory and immunomodulatory activities, may constitute a possible treatment of autoimmune disorders. This study develops a pharmacokinetic/pharmacodynamic (PK/PD) model to assess the effects of PDE-selective inhibitors, namely, cilostazol (PDE3), rolipram (PDE4), and BRL-50481 (PDE7), in a mouse model of AIH. The pharmacokinetics of the PDE inhibitors (PDEi) were assessed in male BALB/c mice after intraperitoneal administration. In pharmacodynamic studies, mice received PDEi and AIH was induced in these animals by intravenous injection of concanavalin A (ConA). Serum drug concentrations, tumor necrosis factor α (TNFα), interleukin 17 (IL-17), and aminotransferase activities were quantified. The PK/PD analysis was performed using ADAPT5 software. The PK/PD model assumes inhibition of cAMP hydrolysis in T cells by PDEi, ConA-triggered formation of TNFα and IL-17, suppression of TNFα and IL-17 production by cAMP, and stimulatory effects of TNFα and IL-17 on the hepatic release of aminotransferases. Selective blockage of PDE4 leads to the highest inhibition of cAMP degradation in T cells and amelioration of disease outcomes. However, inhibition of both PDE3 and PDE7 also contribute to this effect. The proposed PK/PD model may be used to assess and predict the activities of novel PDEi and their combinations in ConA-induced hepatitis. A balanced suppression of different types of PDE appears to be a promising treatment option for AIH; however, this hypothesis warrants testing in humans based on translation of the PK/PD model into clinical settings. SIGNIFICANCE STATEMENT: A novel PK/PD model of PDE inhibitor effects in mice with ConA-induced autoimmune hepatitis was developed involving a mechanistic component describing changes in cAMP concentrations in mouse T cells. According to model predictions, inhibition of PDE4 in T cells causes the highest cAMP elevation in T cells, but suppression of PDE3 and PDE7 also contribute to this effect. A balanced inhibition of PDE3, PDE4, and PDE7 appears to be a promising treatment strategy for AIH.

MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells.

Autoimmune hepatitis (AIH) is an inflammatory liver disease driven by the hyperactivation of various intrahepatic antigen‐specific T cells due to a breach of immune tolerance. Studies in immunometabolism demonstrate that activated T cells harbor increased levels of reactive oxygen species that cause oxidative DNA damage. In this study, we assessed the potential of DNA damage repair enzyme MutT homolog 1 (MTH1) as a therapeutic target in AIH and karonudib as a novel drug for patients with AIH. We report herein that MTH1 expression was significantly increased in liver samples from patients with AIH compared to patients with chronic hepatitis B and nonalcoholic fatty liver disease and from healthy controls. In addition, the expression of MTH1 was positively correlated with AIH disease severity. We further found abundant T cells that expressed MTH1 in AIH. Next, we found that karonudib significantly altered T‐cell receptor signaling in human T cells and robustly inhibited proliferation of human T cells in vitro. Interestingly, our data reflected a preferential inhibition of DNA damage repair in activated T cells by karonudib. Moreover, MTH1 was required to develop liver inflammation and damage because specific deletion of MTH1 in T cells ameliorated liver injury in the concanavalin A (Con A)‐induced hepatitis model by inhibiting T‐cell activation and proliferation. Lastly, we validated the protective effect of karonudib on the Con A‐induced hepatitis model. Conclusion: MTH1 functions as a critical regulator in the development of AIH, and its inhibition in activated T cells reduces liver inflammation and damage.

C6orf120 gene knockout in rats mitigates concanavalin A‑induced autoimmune hepatitis via regulating NKT cells

ObjectiveTo elucidate the role of the functional unknown gene C6orf120 in the pathogenesis of AIH and its mechanism of action, using C6orf120 knockout rats. MethodsAn autoimmune hepatitis model was established with 35 mg/kg intravenous injection of concanavalin A (Con A) in C6orf120-knockout (C6orf120-/-) and wild-type (WT) rats. Rats were sacrificed after administering Con A for 0, 12, and 24 h. The peripheral blood, liver, spleen, and mesenteric lymph nodes were collected for follow-up studies. ResultsC6orf120 knockout significantly decreased the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and improved the histological damage in Con A-induced autoimmune liver injury.Loss of C6orf120 function significantly increased the frequency of CD3+ CD161+ NKT cells in the peripheral blood, liver, and spleen; downregulated the expression of CD314 (NKG2D) in the liver, spleen, and mesenteric lymph nodes; reduced the expression of inflammatory cytokines and chemokines; and suppressed the mRNA and protein expression of Fas and FasL in the liver. Additionally, C6orf120 knockout significantly downregulated the expression of p-JAK1, p-JAK2, p-STAT1, and p-STAT3 in liver tissue. ConclusionThe protective effect of C6orf120 knockout against Con A-induced hepatitis may be due to the inhibition of NKT cell activation, restriction of cytokine and chemokine activities, inhibition of JAK-STAT and Fas/FasL signaling pathway activation, and reduction in liver inflammation and hepatocyte apoptosis.

Taraxasterol mitigates Con A-induced hepatitis in mice by suppressing interleukin-2 expression and its signaling in T lymphocytes

Discovery of anti-inflammatory drugs that can suppress T lymphocyte activation and proliferation by inhibiting TCR/CD3 and IL-2/IL-2R signaling is still needed in clinic, though rapamycin and other related reagents have made great success. Taraxasterol (TAS) is an active ingredient of dandelion, an anti-inflammatory medicinal herb with low in vivo toxicity that has long been used in China. Yet the action mechanism of TAS on lymphocytes remains elusive. The anti-inflammatory effects of TAS were evaluated in C57BL/6 mouse primary lymphocytes stimulated with concanavalin A (Con A) in vitro and in mouse model of Con A-induced acute hepatitis in vivo. Our results showed that TAS significantly suppressed Con A-induced acute hepatitis in a mouse model, reducing the hepatic necrosis areas, the release of aminotransferases, and the production of IL-2 and other inflammatory cytokines. Supporting this, in vitro study also showed that TAS reduced the production of IL-2 and the expression of IL-2 receptor subunit α (CD25) upon the stimulation of Con A, which was likely mediated by suppressing NF-κB activation. The downstream pathways of IL-2/IL-2R signaling, including the activation of PI3K/PDK1/mTOR, STAT3 and STAT5, were also suppressed by TAS. Consistently, Con A-induced T cell proliferation was also inhibited by TAS in vitro. Our data indicate that TAS can suppress both T lymphocyte activation and cell proliferation by down-regulating IL-2 expression and its signaling pathway thereby ameliorating Con A-induced acute hepatitis, highlighting TAS as a potential drug candidate for treating inflammatory diseases including autoimmune hepatitis.

Inflammatory arthritis increases the susceptibility to acute immune-mediated hepatitis in mice through enhancing leptin expression in T cells

Liver function abnormalities are common in patients with inflammatory arthritis. However, the precise mechanism is still unclear. In this study, inflammatory arthritis was established in mice by subcutaneous injection of complete Freund’s adjuvant, and the intravenous injection of concanavalin A (Con A) was employed to induce acute immune-mediated hepatitis in mice. The result showed that the arthritis mice were more susceptible to ConA-induced hepatitis than the control mice, as evidenced by increased hepatic necrosis, elevated serum alanine aminotransferase activity, and raised inflammatory cytokines. Besides, the in vitro assay demonstrated that the T cells from arthritis mice were more sensitive to the Con A stimulation than those from control mice. Moreover, we determined that the level of leptin, a kind of adipokine, was significantly increased in the serum and hepatic T cells of arthritis mice. Interestingly, the data indicated that the enhanced expression of leptin in hepatic T cells is responsible for the hypersensitivity of arthritis mice-derived T cells to Con A challenge. Collectively, our findings demonstrate an unexpected role of leptin in the connection between inflammatory arthritis and acute immune-mediated hepatitis, thus providing new insight into the clinical therapy of arthritis-related liver dysfunction.

Endometrial regenerative cells with galectin-9 high-expression attenuate experimental autoimmune hepatitis

BackgroundAutoimmune hepatitis (AIH) is a T cell-mediated immune disease that activates abnormally against hepatic antigens. We have previously reported that endometrial regenerative cells (ERCs) were a novel source of adult stem cells, which exhibiting with powerful immunomodulatory effects. Galectin-9 (Gal-9) is expressed in ERCs and plays an important role in regulating T cell response. This study aims to explore the role of ERCs in attenuation of AIH and to determine the potential mechanism of Gal-9 in ERC-mediated immune regulation.MethodsERCs were obtained from menstrual blood of healthy female volunteers. In vitro, ERCs were transfected with lentivirus vectors carrying LGALS9 gene and encoding green fluoresce protein (GFP-Gal-9-LVs) at a MOI 50, Gal-9 expression in ERCs was detected by ELISA and Q-PCR. CD4+ T cells isolated from C57BL/6 mouse spleen were co-cultured with ERCs. The proliferation of CD4+ T cells was detected by CCK-8 kit and the level of Lck/zap-70/LAT protein was measured by western blot. Furthermore, AIH was induced by ConA in C57BL/6 mice which were randomly assigned to untreated, unmodified ERC-treated and Gal-9 high-expressing ERC-treated groups. Histopathological score, liver function, CD4+/CD8+ cell infiltration in liver tissues, the proportion of immune cells in the spleen and liver, and ERC tracking were performed accordingly to assess the progression degree of AIH.ResultsAfter transfecting with GFP-Gal-9-LVs, Gal-9 expression in ERCs was significantly increased. Additionally, Gal-9 high-expressing ERCs effectively inhibited CD4+ T cell proliferation and downregulated CD4+ T cell active related proteins p-Lck/p-ZAP70/p-LAT in vitro. Furthermore, treatment with Gal-9 high-expressing ERCs restored liver function, ameliorated liver pathological damage, inhibit CD4+ and CD8+ T cell proliferation and suppress Th1 and Th17 cell response in the hepatitis mice. In addition, Gal-9 high-expressing ERCs further markedly enhanced the level of IL-10 but reduced the levels of IFN-γ, TNF-α, and IL-4 in mouse sera and liver. Cell tracking also showed that ERCs could migrate to the damaged liver organs.ConclusionsThe results suggested that Gal-9 was an essential modulator, which was required by ERCs in regulating T cell response and attenuating ConA-induced experimental hepatitis. And also, it provides a novel idea for the clinical treatment of AIH.

Human Wharton's jelly-derived mesenchymal stem cells alleviate concanavalin A-induced fulminant hepatitis by repressing NF-\u03baB signaling and glycolysis

BackgroundFulminant hepatitis is a severe life-threatening clinical condition with rapid progressive loss of liver function. It is characterized by massive activation and infiltration of immune cells into the liver and disturbance of inflammatory cytokine production. Mesenchymal stem cells (MSCs) showed potent immunomodulatory properties. Transplantation of MSCs is suggested as a promising therapeutic approach for a host of inflammatory conditions.MethodsIn the current study, a well-established concanavalin A (Con A)-induced fulminant hepatitis mouse model was used to investigate the effects of transplanting human umbilical cord Wharton's jelly-derived MSCs (hWJ-MSCs) on fulminant hepatitis.ResultsWe showed that hWJ-MSCs effectively alleviate fulminant hepatitis in mouse models, primarily through inhibiting T cell immunity. RNA sequencing of liver tissues and human T cells co-cultured with hWJ-MSCs showed that NF-κB signaling and glycolysis are two main pathways mediating the protective role of hWJ-MSCs on both Con A-induced hepatitis in vivo and T cell activation in vitro.ConclusionIn summary, our data confirmed the potent therapeutic role of MSCs-derived from Wharton's jelly of human umbilical cord on Con A-induced fulminant hepatitis, and uncovered new mechanisms that glycolysis metabolic shift mediates suppression of T cell immunity by hWJ-MSCs.

Glucose Metabolism Reprogramming of Regulatory T Cells in Concanavalin A-Induced Hepatitis.

Autoimmune hepatitis (AIH) is an inflammatory liver disease caused by a dysregulated immune response. Although the pathogenesis of AIH remains unclear, impaired regulatory T cells (Tregs) have been considered a driver of AIH development. Unlike autoreactive T cells, Tregs mainly utilize oxidative phosphorylation (OXPHOS) as their energy supply. Elevated glycolysis has been reported to limit the suppressive functions of Tregs. However, whether glucose metabolism reprogramming in Tregs is involved in AIH etiology remains unknown. The aim of this study was to examine alternations in Treg numbers and functions in AIH patients and concanavalin A (Con A)-induced hepatitis, while exploring associations between impaired Tregs and glucose metabolism. The frequency of Tregs was decreased in the peripheral blood but increased in liver biopsies of AIH patients. Moreover, immunosuppressive therapy rescued circulating Tregs in AIH. In Con A-induced immune hepatitis, enhanced intrahepatic Treg accumulation was observed over time, accompanied by reduced splenic Treg numbers. To investigate whether functional impairment of Tregs occurs in AIH, Tregs were isolated from experimental AIH (EAH) model mice and normal controls and the former displayed downregulated mRNA levels of FOXP3, CTLA4, CD103, TIGIT, CD39, and CD73. EAH model-derived Tregs also produced fewer anti-inflammatory mediators (TGF-β and IL-35) than control Tregs. Moreover, enhanced glycolysis and reduced OXPHOS were found in Tregs from EAH model mice, as reflected by elevated levels of key glycolytic enzymes (HK2, PK-M2, and LDH-A) and a decreased ATP concentration. This study revealed a decreased peripheral Treg frequency and abnormal intrahepatic Treg infiltration in AIH. It is first reported that glucose metabolism reprogramming is associated with decreases and functional impairments in the Treg population, promoting AIH development. Targeting glucose metabolism may provide novel insights for the treatment of AIH.

Anti-fibrotic activity of sitagliptin against concanavalin A-induced hepatic fibrosis. Role of Nrf2 activation/NF-κB inhibition

Sitagliptin is known for its anti-diabetic activity though it has other pleiotropic pharmacological actions. Its effect against concanavalin A (Con A)-induced hepatic fibrosis has not been investigated yet. Our target was to test whether sitagliptin can suppress the development of Con A-induced hepatic fibrosis and if so, what are the mechanisms involved? Con A (6 mg/kg) was injected once weekly to male Swiss albino mice for four weeks. Sitagliptin was daily administered concurrently with Con A. Results have shown the potent hepatoprotective activity of sitagliptin against Con A-induced hepatitis and fibrosis. That was evident through the amelioration of hepatotoxicity serum parameters (ALT, AST, ALP, and LDH) and the increase in the level of serum albumin in sitagliptin treated mice. Simultaneously, there was amendment of the Con A-induced hepatic lesions and repression of fibrosis in sitagliptin-treated animals. Hydroxyproline, collagen content and the immuno-expression of the fibrotic markers, TGF-β and α-SMA were depressed upon sitagliptin treatment. Sitagliptin suppressed Con A-induced oxidative stress and increased antioxidants. RT-PCR analysis showed enhancement of mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target genes (GCLc, GCLm, NQO-1, HO-1) by sitagliptin. Furthermore, sitagliptin ameliorated the level and immuno-expression of nuclear factor kappa-B (NF-κB) alongside the immuno-expression of the inflammatory cytokine, TNF-α. Taken together, this study demonstrates the hepatoprotective activity of sitagliptin which may be in part related to enhancement of Nrf2 signaling pathway and inhibition of NF-κB which interact inflammatory response in liver. Sitagliptin might be a new candidate to suppress hepatitis-associated fibrosis.

Tetrazole exerts anti-hepatitis effect in mice via activation of PI3K/Akt pathway, inhibition of cell autophagy and suppression of inflammatory cytokine expressions

Purpose: To investigate the effect of tetrazole on concanavalin A (Con A)-induced hepatitis in mice, and the underlying mechanism(s).&#x0D; Methods: Thirty 5-week-old, male BALB/c mice (mean weight, 30.5 ± 1.04 g) were used for this study. They were randomly assigned to six groups of five mice each: control group, hepatitis group and four treatment groups. With the exception of control group, hepatitis was induced in all mice with Con A (20 mg/kg) via their tail veins. The treatment groups received varied doses of tetrazole (1.0 - 6.0 mg/kg) within 1 h after hepatitis induction, while mice in the control group received an equivalent volume of normal saline in place of tetrazole. Serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined while expressions of interleukin-2 (IL-2), tumor necrosis factor ߙ) TNF-ߙ ,(and interferon gamma (IFN-ߛ (were evaluated by enzyme-linked immunosorbent assay (ELISA) kits. Expressions of protein kinase B (Akt), phosphoinositide 3-kinase (PI3K), nuclear transcription factor- ߢB (NF-ߢB), and autophagy-related genes were determined by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting.&#x0D; Results: Con A-induced hepatitis significantly increased the activities of serum ALT and AST in the mice. However, after treatment with tetrazole, the activities of these enzymes were significantly and dose-dependently reduced in the treatment groups, relative to hepatitis group (p &lt; 0.05). The levels of IL-2, IFN-ߛ and TNF-ߙ were significantly increased in hepatitis group when compared with the control group (p &lt; 0.05). However, treatment with tetrazole significantly inhibited the expressions of these parameters. There were no significant differences in the levels of expressions of Akt mRNAs among the treatment groups (p &gt; 0.05). The levels of expressions of LC3II and Beclin 1 were also significantly upregulated in hepatitis group, when compared with control group (p &lt; 0.05). However, expression levels of LC3II and Beclin 1 were significantly and dose-dependently reduced by tetrazole treatment&#x0D; Conclusion: Tetrazole is effective in the treatment of hepatitis via mechanisms involving the activation of PI3K/Akt pathway, inhibition of cell autophagy and suppression of inflammatory cytokines expressions.