Protective effect of quercetin against macrophage-mediated hepatocyte injury via anti-inflammation, anti-apoptosis and inhibition of ferroptosis.

Yinchenhao Decoction (YCHD) is a classic prescription in traditional Chinese medicine (TCM). It appears to play an important role in anti-inflammation and autoimmunity protection. As one of the key active ingredients in YCHD, quercetin is a novel anti-inflammatory metabolite that exerts protective effects in many autoimmune diseases. However, its role in autoimmune hepatitis (AIH)-related hepatic injury has not been studied. The aim of this study was to reveal the hepatocyte protective mechanism of quercetin. In this study, we used Concanavalin A (Con A) to establish an in vitro hepatocyte injury-associated AIH model. Brl3a hepatocyte injury was induced by the supernatant of J774A.1 cells treated with Con A. We found that quercetin mitigated Con A-induced via macrophage-mediated Brl3a hepatocyte injury. Quercetin administration reduced the levels of alanine transaminase (ALT) and aspartate transaminase (AST) in the supernatant of Con A-treated Brl3a cells and attenuated the infiltration of J774A.1 macrophages induced by Con A. Moreover, quercetin effectively inhibited the expression of proinflammatory cytokines including interleukin-1ß (IL-1ß) by Con A. Furthermore, quercetin decreased hepatocyte apoptosis and ferroptosis levels in the macrophage-induced hepatocyte injury model. In conclusion, our study indicates that quercetin alleviates macrophage-induced hepatocyte damage by reducing the inflammatory response, apoptosis and ferroptosis. Our work suggests that quercetin might be a potential therapeutic strategy for AIH.

Patients with autoimmune liver disease have glucose disturbances that mechanistically differ from steatotic liver disease.

Autoimmune liver diseases are associated with an increased risk of diabetes, yet the underlying mechanisms remain unknown. In this cross-sectional study, we investigated the glucose-regulatory disturbances in patients with autoimmune hepatitis (AIH, n = 19), primary biliary cholangitis (PBC, n = 15), and primary sclerosing cholangitis (PSC, n = 6). Healthy individuals (n = 24) and patients with metabolic dysfunction-associated steatotic liver disease (MASLD, n = 18) were included as controls. Blood samples were collected during a 120-min oral glucose tolerance test. We measured the concentrations of glucose, C-peptide, insulin, glucagon, and the two incretin hormones, glucose insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1). We calculated the homeostasis model assessment of insulin resistance (HOMA-IR), whole body insulin resistance (Matsuda index), insulin clearance, and insulinogenic index. All patient groups had increased fasting plasma glucose and impaired glucose responses compared with healthy controls. Beta-cell secretion was increased in AIH, PBC, and MASLD but not in PSC. Patients with AIH and MASLD had hyperglucagonemia and hepatic, as well as peripheral, insulin resistance and decreased insulin clearance, resulting in hyperinsulinemia. Patients with autoimmune liver disease had an increased GIP response, and those with AIH or PBC had an increased GLP-1 response. Our data demonstrate that the mechanism underlying glucose disturbances in patients with autoimmune liver disease differs from that underlying MASLD, including compensatory incretin responses in patients with autoimmune liver disease. Our results suggest that glucose disturbances are present at an early stage of the disease.NEW & NOTEWORTHY Patients with autoimmune liver disease but without overt diabetes display glucose disturbances early on in their disease course. We identified pathophysiological traits specific to these patients including altered incretin responses.

6-Formylindolo[3,2-b]carbazole, a potent ligand for the aryl hydrocarbon receptor, attenuates concanavalin-induced hepatitis by limiting T-cell activation and infiltration of proinflammatory CD11b+ Kupffer cells.

FICZ (6-formylindolo[3,2-b]carbazole) is a potent aryl hydrocarbon receptor agonist that has a poorly understood function in the regulation of inflammation. In this study, we investigated the effect of aryl hydrocarbon receptor activation by FICZ in a murine model of autoimmune hepatitis induced by concanavalin A. High-throughput sequencing techniques such as single-cell RNA sequencing and assay for transposase accessible chromatin sequencing were used to explore the mechanisms through which FICZ induces its effects. FICZ treatment attenuated concanavalin A-induced hepatitis, evidenced by decreased T-cell infiltration, decreased circulating alanine transaminase levels, and suppression of proinflammatory cytokines. Concanavalin A revealed an increase in natural killer T cells, T cells, and mature B cells upon concanavalin A injection while FICZ treatment reversed the presence of these subsets. Surprisingly, concanavalin A depleted a subset of CD55+ B cells, while FICZ partially protected this subset. The immune cells showed significant dysregulation in the gene expression profiles, including diverse expression of migratory markers such as CCL4, CCL5, and CXCL2 and critical regulatory markers such as Junb. Assay for transposase accessible chromatin sequencing showed more accessible chromatin in the CD3e promoter in the concanavalin A-only group as compared to the naive and concanavalin A-exposed, FICZ-treated group. While there was overall more accessible chromatin of the Adgre1 (F4/80) promoter in the FICZ-treated group, we observed less open chromatin in the Itgam (CD11b) promoter in Kupffer cells, supporting the ability of FICZ to reduce the infiltration of proinflammatory cytokine producing CD11b+ Kupffer cells. Taken together, these data demonstrate that aryl hydrocarbon receptor activation by FICZ suppresses liver injury through the limitation of CD3+ T-cell activation and CD11b+ Kupffer cell infiltration.

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

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

PD-L1 expression levels in mesenchymal stromal cells predict their therapeutic values for autoimmune hepatitis.

BACKGROUND: Autoimmune hepatitis is a chronic inflammatory hepatic disorder with no effective treatment. Mesenchymal stromal cells (MSCs) have emerged as a promising treatment owing to their unique advantages. However, their heterogeneity is hampering use in clinical applications. METHODS: Wharton's jelly derived MSCs (WJ-MSCs) were isolated from 58 human donors using current good manufacturing practice conditions. Gene expression profiles of the WJ-MSCs were analyzed by transcriptome and single-cell RNA-sequencing (scRNA-seq), and subsequent functional differences were assessed. Expression levels of programmed death-ligand 1 (PD-L1) were used as an indicator to screen WJ-MSCs with varied immunomodulation activities and assessed their corresponding therapeutic effects in a mouse model of concanavalin A-induced autoimmune hepatitis. RESULTS: The 58 different donor-derived WJ-MSCs were grouped into six gene expression profile clusters. The gene in different clusters displayed obvious variations in cell proliferation, differentiation bias, trophic factor secretion, and immunoregulation. Data of scRNA-seq revealed four distinct WJ-MSCs subpopulations. Notably, the different immunosuppression capacities of WJ-MSCs were positively correlated with PD-L1 expression. WJ-MSCs with high expression of PD-L1 were therapeutically superior to WJ-MSCs with low PD-L1 expression in treating autoimmune hepatitis. CONCLUSION: PD-L1 expression levels of WJ-MSCs could be regarded as an indicator to choose optimal MSCs for treating autoimmune disease. These findings provided novel insights into the quality control of MSCs and will inform improvements in the therapeutic benefits of MSCs.

Human menstrual blood-derived stem cells alleviate autoimmune hepatitis via JNK/MAPK signaling pathway in vivo and in vitro.

Autoimmune hepatitis (AIH) is a severe globally distributed liver disease that could occur at any age. Human menstrual blood-derived stem cells (MenSCs) have shown therapeutic effect in acute lung injury and liver failure. However, their role in the curative effect of AIH remains unclear. Here, a classic AIH mouse model was constructed through intravenous injection with concanavalin A (Con A). MenSCs were intravenously injected while Con A injection in the treatment groups. The results showed that the mortality by Con A injection was significantly decreased by MenSCs treatment and liver function tests and histological analysis were also ameliorated. The results of phosphoproteomic analysis and RNA-seq revealed that MenSCs improved AIH, mainly by apoptosis and c-Jun N-terminal kinase/mitogen-activated protein signaling pathways. Apoptosis analysis demonstrated that the protein expression of cleaved caspase 3 was increased by Con A injection and reduced by MenSCs transplantation, consistent with the TUNEL staining results. An AML12 co-culture system and JNK inhibitor (SP600125) were used to verify the JNK/MAPK and apoptosis signaling pathways. These findings suggested that MenSCs could be a promising strategy for AIH.

Spontaneous development of an autoimmune hepatitis - primary biliary cholangitis overlap syndrome in dnTGFßRII Aire-/- mice.

Autoimmune regulator (Aire) and TGF-ß signaling play important roles in central tolerance and peripheral tolerance, respectively, by eliminating or suppressing the activity of autoreactive T cells. We previously demonstrated that dnTGFßRII mice develop a defect in peripheral tolerance and a primary biliary cholangitis (PBC)-like disease. We hypothesized that by introducing the Aire gene to this model, we would observe a more severe PBC phenotype. Interestingly, however, we demonstrated that, while dnTGFßRII Aire-/- mice do manifest key histological and serological features of autoimmune cholangitis, they also develop mild to moderate interface hepatitis and show high levels of alanine transaminase (ALT) and antinuclear antibodies (ANA), characteristics of autoimmune hepatitis (AIH). To further understand this unique phenotype, we performed RNA sequencing (RNA-seq) and flow cytometry to explore the functional pathways and immune cell pathways in the liver of dnTGFßRII Aire-/- mice. Our data revealed enrichments of programmed cell death pathways and predominant CD8+ T cell infiltrates. Depleting CD8+ T cells using an anti-CD8α antibody significantly alleviated hepatic inflammation and prolonged the life span of these mice. Finally, RNA-seq data indicated the clonal expansion of hepatic CD8+ T cells. In conclusion, these mice developed an autoreactive CD8+ T-cell-mediated autoimmune cholangitis with concurrent hepatitis that exhibited key histological and serological features of the AIH-PBC overlap syndrome, representing a novel model for the study of tolerance and autoimmune liver disease. © 2023 The Pathological Society of Great Britain and Ireland.

Metabolic heterogeneity caused by HLA-DRB1*04:05 and protective effect of inosine on autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an autoimmune disease caused by disruption of liver immune homeostasis. Genetic studies have revealed the predisposition of AIH with the human leukocyte antigen (HLA) region. Recently, metabolomics integrated with genomics has identified many genetic loci of biomedical interest. However, there is no related report in AIH. In the present study, we found that HLA-DRB1*04:05 was linked to the clinical features and prognosis of AIH in Chinese patients. Furthermore, our patients were divided into DRB1*04:05 positive and DRB1*04:05 negative groups and the metabolic profiling was done by HPLC/MS. We chose inosine, one of the highly altered metabolites, to explore the effect on an acute severe hepatitis murine model. The results showed that inosine treatment attenuated hepatocyte apoptosis, enhanced antioxidant ability and inhibited the activation and glycolysis of CD4+ T cell. We propose that inosine participates in the regulation of AIH through its protective effect on hepatocytes and inhibition of overactivated immune cells, which might provide a potential novel approach in treating acute form of AIH.

Lamina propria interleukin 17 A aggravates natural killer T-cell activation in autoimmune hepatitis.

Autoimmune hepatitis is an interface hepatitis characterized by the progressive destruction of the liver parenchyma, the cause of which is still obscure. Interleukin (IL)-17A is a major driver of autoimmunity, which can be produced by innate immune cells against several intracellular pathogens. Here, we investigated the involvement of IL-17A in a mice model of immune-mediated hepatitis with the intestine exposed to Salmonella typhimurium. Our results showed more severe Concanavalin (Con) A-induced liver injury and gut microbiome dysbiosis when the mice were treated with a gavage of S. typhimurium. Then, the natural killer (NK) T cells were overactivated by the accumulated IL-17A in the liver in the Con A and S. typhimurium administration group. IL-17A could activate NKT cells by inducing CD178 expression via IL-4/STAT6 signaling. Furthermore, via the portal tract, the laminae propria mucosal-associated invariant T (MAIT)-cell-derived IL-17A could be the original driver of NKT cell overactivation in intragastric administration of S. typhimurium and Con A injection. In IL-17A-deficient mice, Con A-induced liver injury and NKT cell activation were alleviated. However, when AAV-sh-mIL-17a was used to specifically knock down IL-17A in liver, it seemed that hepatic IL-17a knock down did not significantly influence the liver injury. Our results suggested that, under Con A-induction, laminae propria MAIT-derived IL-17A activated hepatic NKT, and this axis could be a therapeutic target in autoimmune liver disease.

Patients with treated autoimmune hepatitis and persistent suppression of plasmacytoid dendritic cells: A different point of view.

Objectives: Plasmacytoid dendritic cells (pDCs) have been shown to have a role in autoimmune diseases, but their role in Autoimmune Hepatitis (AIH) is not completely clear. In the present study, we assessed the frequency of pDCs in peripheral blood of AIH patients under long-term standard immunosuppressive therapy. Methods: This cross-sectional analysis enrolled 27 AIH patients and 27 healthy controls. We analyzed and compared their proportion of pDCs, CD4+, CD8+, γδ T cells, CD25+ regulatory T (Treg) cells, FoxP3+, Foxp3+CD39+ Treg cells, total B (CD19+) cells, and plasma cells (CD38+) in peripheral blood using flow cytometry immunophenotyping. Results: AIH patients had a lower percentage of pDCs (median frequencies of 0.2% vs. 0.4%; p = .002) and higher expression of CD8 T cells (32.5% vs 28.6%; p = 0.008) in peripheral blood, when compared to healthy controls. We did not find statistically significant differences between the groups regarding the other cell subtypes.Conclusion: Our data suggest a persistent suppression of pDCs in AIH patients, along with increased CD8 T cell activity, years after AIH diagnosis and despite of good clinical response to treatment, thus pointing to a role of pDCs in the AIH pathogenesis.

RIP3 blockade prevents immune-mediated hepatitis through a myeloid-derived suppressor cell dependent mechanism.

Autoimmune hepatitis (AIH) is an immune-mediated chronic inflammatory liver disease, and its pathogenesis is not fully understood. Our previous study discovered that receptor interacting protein kinase 3 (RIP3) is correlated with serum transaminase levels in AIH patients. However, its role and underlying mechanism in AIH are poorly understood. Here, we detected the increased expression and activation of RIP3 in livers of patients and animal models with AIH. The inhibition of RIP3 kinase by GSK872 prevented concanavalin A (ConA)-induced immune-mediated hepatitis (IMH) by reduced hepatic proinflammatory cytokines and immune cells including Th17 cells and macrophages. Further experiments revealed that RIP3 inhibition resulted in an increase in CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) with immunoregulatory properties in the liver, spleen, and peripheral blood. Moreover, the depletion of Gr-1+ MDSCs abrogated the protective effect and immune suppression function of GSK872 in ConA-induced IMH. Altogether, our data demonstrate that RIP3 blockade prevents ConA-induced IMH through promoting MDSCs infiltration. Inhibition of RIP3 kinase may be a novel therapeutic avenue for AIH treatment.

Diagnostic Value of Serum Golgi Protein 73 for Liver Inflammation in Patients with Autoimmune Hepatitis and Primary Biliary Cholangitis.

BACKGROUND: There is lack of reliable serum biomarkers to reflect the severity of liver necroinflammation for those who suffer autoimmune liver diseases (AILDs). In this study, a previously established patient cohort was used to explore the potential of serum Golgi protein 73 (GP73) as a noninvasive marker of AILD-related liver necroinflammation. METHODS: Serum GP73 concentration was measured in a retrospective cohort of 168 AILD patients, which included 74 patients with autoimmune hepatitis (AIH) and 94 with primary biliary cholangitis (PBC) who had undergone liver biopsy. Spearman's correlation and multivariate analysis were used to evaluate the relationship between serum GP73 and liver necroinflammation. A receiver operating characteristic curve was constructed to evaluate the value of GP73 for the prediction of moderate or severe liver necroinflammation. The diagnostic value of serum GP73 was also compared with that of alkaline phosphatase (ALP) in patients with PBC. Histologically, immunohistochemical analysis was performed to assess hepatic GP73 expression. RESULTS: Both the serum level and hepatic tissue expression of GP73 protein were aberrantly elevated and correlated well with the severity of necroinflammation in both AIH (rho = 0.655, P < 0.001) and PBC (rho = 0.547, P < 0.001) patients. The results here suggested that serum GP73 could be an independent biomarker to reflect the severity of liver necroinflammation. The AUROCs for GP73 to predict moderate necroinflammation (≥G2) and severe necroinflammation (≥G3) in patients with AIH were 0.828 and 0.832, respectively. Moreover, the AUROCs of serum GP73 for the identification of moderate necroinflammation (≥G2) (AUROC = 0.820, P < 0.001) and severe necroinflammation (≥G3) (AUROC = 0.803, P < 0.001) were superior to those of ALP (≥G2: AUROC = 0.607, P = 0.028 and ≥G3: AUROC = 0.559, P = 0.357) in patients with PBC. Mechanically, interlukin-6 (IL-6), the proinflammatory and prohepatic regenerating cytokine, could transcriptionally upregulate GP73 gene expression. CONCLUSION: Serum GP73 is a potential noninvasive biomarker to evaluate the severity of liver necroinflammation in patients with AILDs.

Pivotal Role of Signal-Transducing Adaptor Protein-2 in Pathogenesis of Autoimmune Hepatitis.

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein involved in inflammatory and immune responses, such as inflammatory bowel disease and allergic responses. In this study, we investigated the role of STAP-2 in the pathogenesis of autoimmune hepatitis. After intravenous injection of concanavalin A (ConA), STAP-2 knock out (KO) mice showed more severe liver necrosis along with substantial lymphocyte infiltration compared to wild type (WT) mice. Serum alanine aminotransferase levels were significantly higher in ConA-injected STAP-2 KO mice than in WT mice. Levels of interferon-γ (IFN-γ), an important factor for liver necrosis, were also significantly increased in sera of STAP-2 KO mice compared to WT mice after ConA injection. Statistically significant upregulation of Fas ligand (FasL) expression was observed in the livers of ConA-injected STAP-2 KO mice compared to WT mice. In accordance with these results, apoptotic signals were facilitated in STAP-2 KO mice compared to WT mice after ConA injection. Correctively, these results suggest that STAP-2 is involved in the pathogenesis of autoimmune hepatitis by regulating the expression of FasL and the production of IFN-γ.

MARC1 p.A165T variant is associated with decreased markers of liver injury and enhanced antioxidant capacity in autoimmune hepatitis.

The clinical picture of autoimmune hepatitis (AIH) varies markedly between patients, potentially due to genetic modifiers. The aim of this study was to evaluate genetic variants previously associated with fatty liver as potential modulators of the AIH phenotype. The study cohort comprised 313 non-transplanted adults with AIH. In all patients, the MARC1 (rs2642438), HSD17B13 (rs72613567), PNPLA3 (rs738409), TM6SF2 (rs58542926), and MBOAT7 (rs641738) variants were genotyped using TaqMan assays. Mitochondrial damage markers in serum were analyzed in relation to the MARC1 variant. Carriers of the protective MARC1 allele had lower ALT and AST (both P < 0.05). In patients treated for AIH for ≥ 6 months, MARC1 correlated with reduced AST, ALP, GGT (all P ≤ 0.01), and lower APRI (P = 0.02). Patients carrying the protective MARC1 genotype had higher total antioxidant activity (P < 0.01) and catalase levels (P = 0.02) in serum. The PNPLA3 risk variant was associated with higher MELD (P = 0.02) in treated patients, whereas MBOAT7 increased the odds for liver cancer (OR = 3.71). None of the variants modulated the risk of death or transplantation. In conclusion, the MARC1 polymorphism has protective effects in AIH. Genotyping of MARC1, PNPLA3, and MBOAT7 polymorphisms might help to stratify patients with AIH.

Myeloid-Derived Suppressive Cells Deficient in Liver X Receptor α Protected From Autoimmune Hepatitis.

Myeloid-derived suppressor cells (MDSCs) emerge as a promising candidate for the immunotherapy of autoimmune hepatitis (AIH). However, targets for modulating MDSC in AIH are still being searched. Liver X receptors (LXRs) are important nuclear receptors linking lipid metabolism and immune responses. Despite the extensive studies of LXR in myeloid compartment, its role in MDSCs is currently less understood. Herein, expression of LXRα was found to be upregulated in AIH patients and colocalized with hepatic MDSCs. In ConA-induced hepatitis, deletion of LXRα led to increased expansion of MDSCs in the liver and alleviated the hepatic injury. MDSCs in LXRα-/- mice exhibited enhanced proliferation and survival comparing with WT mice. T-cell proliferation assay and adoptive cell transfer experiment validated the potent immunoregulatory role of MDSCs in vitro and in vivo. Mechanistically, MDSCs from LXRα-/- mice possessed significantly lower expression of interferon regulatory factor 8 (IRF-8), a key negative regulator of MDSC differentiation. Transcriptional activation of IRF-8 by LXRα was further demonstrated. Conclusion: We reported that abrogation of LXRα facilitated the expansion of MDSCs via downregulating IRF-8, and thereby ameliorated hepatic immune injury profoundly. Our work highlights the therapeutic potential of targeting LXRα in AIH.

Probiotics alleviate autoimmune hepatitis in mice through modulation of gut microbiota and intestinal permeability.

Autoimmune hepatitis (AIH) is an immune-mediated type of chronic liver inflammation accompanied by intestinal flora imbalance. Probiotics have been reported to ameliorate imbalances in the intestinal flora. This study aimed to investigate the effects of compound probiotic in the AIH mouse model. AIH mice were gavaged with compound probiotic and injected intraperitoneally with dexamethasone (dex) for 42 days. The results showed that these treatments suppressed hepatic inflammatory cell infiltration, serum transaminase, and Th1 and Th17 cells. However, Treg cells were increased only in the probiotics group, which indicates an immunomodulatory role of the compound probiotic. The compound probiotic maintained intestinal barrier integrity, blocked lipopolysaccharide (LPS) translocation, and inhibited the activation of the TLR4/NF-κB pathway and the production of inflammatory factors in the liver and ileum. Moreover, the compound probiotic treatment increased the abundance of beneficial bacteria and reduced the abundance of potentially harmful bacteria in gut. Compound probiotic may improve ileal barrier function while increasing the diversity of the intestinal flora, blocking the translocation of gut-derived LPS to the liver and therefore preventing activation of the TLR4/NF-κB pathway. The resulting inhibition of pro-inflammatory factor production facilitates AIH remission.

Ultrastructural Profile Combined with Immunohistochemistry of a Hepatic Progenitor Cell Line in Pediatric Autoimmune Hepatitis: New Insights into the Morphological Pattern of the Disease.

Considering that the heterogenic population of a hepatic progenitor cell line (HPCL) can play a vital role in autoimmune hepatitis (AIH), we decided to conduct pioneering retrospective evaluation of these cells in pediatric AIH by means of transmission electron microscopy (TEM). The aim of the study was to assess the ultrastructure of the HPCL in children with untreated AIH. Ultrastructural analysis of the HPCL population, preceded by immunohistochemical staining for cytokeratin 7 (CK7), was performed using pretreatment liver biopsies from 23 children with clinicopathologically diagnosed AIH. Immunohistochemical assessment for CK7 allowed detection of proliferating immature epithelial cells differentiating towards periportal and intralobular intermediate hepatocytes without marked formation of ductular reactions in AIH children. Using TEM, we distinguished three morphological types of HPCs: I-the most undifferentiated progenitor cells; III-intermediate hepatocyte-like cells; II-intermediate bile duct cells. Most frequent were the cells differentiating towards hepatocytes, most rare-those differentiating towards cholangiocytes. The results indicate that an HPCL may be an important source of hepatocyte regeneration. Ultrastructural analyses of the HPCL population, combined with immunohistochemistry for CK7, might be a useful tool to evaluate liver cell regeneration, including fibrogenesis, and may help better understand the morphological pattern of the disease, in pediatric AIH. Frequent appearance of an HPCL in the vicinity of fibrotic foci, often accompanied by hyperactive Kupffer cells and transitional hepatic stellate cells, may indicate their significant involvement in liver fibrogenesis.

Anti-CD20 Therapy Alters the Protein Signature in Experimental Murine AIH, but Not Exclusively towards Regeneration.

BACKGROUND: Autoimmune hepatitis (AIH) is a chronic autoimmune inflammatory disease that usually requires lifelong immunosuppression. Frequent recurrences after the discontinuation of therapy indicate that intrahepatic immune regulation is not restored by current treatments. Studies of other autoimmune diseases suggest that temporary depletion of B cells can improve disease progression in the long term. METHODS: We tested a single administration of anti-CD20 antibodies to reduce B cells and the amount of IgG to induce intrahepatic immune tolerance. We used our experimental murine AIH (emAIH) model and treated the mice with anti-CD20 during the late stage of the disease. RESULTS: After treatment, the mice showed the expected reductions in B cells and serum IgGs, but no improvements in pathology. However, all treated animals showed a highly altered serum protein expression pattern, which was a balance between inflammation and regeneration. CONCLUSIONS: In conclusion, anti-CD20 therapy did not produce clinically measurable results because it triggered inflammation, as well as regeneration, at the proteomic level. This finding suggests that anti-CD20 is ineffective as a sole treatment for AIH or emAIH.

Erythropoietin administration expands regulatory T cells in patients with autoimmune hepatitis.

OBJECTIVE: Autoimmune hepatitis (AIH) is a chronic liver disease associated with impaired regulatory T cell (Treg) number and function. Erythropoietin (EPO) is a kidney-produced erythropoietic hormone that has known immune-modulating effects, including Treg induction. Whether EPO administration increases Treg in patients with AIH is unknown. METHODS: We treated six stable AIH patients with a single 1000 IU dose of EPO and comprehensively characterized changes in Treg overall and in Treg subsets before and at 4 and 12 weeks after treatment using mass cytometry (CyTOF) combined with an unbiased clustering approach (Phenograph) based on 22 Treg-relevant cell-surface markers. RESULTS: EPO was well-tolerated and no patients showed significant changes in hematological parameters, liver enzymes, or IgG levels from baseline to 12 weeks following EPO administration. Total Treg and Treg/CD8+ T cell ratios significantly increased at 4 weeks and returned to baseline levels at 12 weeks after EPO injection. We identified 17 Treg subsets of which CD4+CD25HICD127NEG HLADR+ Treg had the highest increase and the most favorable Treg/CD8+ ratio upon EPO treatment. At 12 weeks after EPO administration, the HLADR+ Treg subset also returned to values comparable to those at baseline. Ex vivo assays documented that Treg were functional and the ones isolated at 12 weeks after EPO injection were significantly more suppressive than the ones isolated at baseline. In Treg-depleted assays, EPO did not show a significant effect on IFN-γ+, IL-2+, and IL-17+ CD4+ T cells. CONCLUSION: In stable AIH patients, EPO increases overall Treg and particularly those expressing the high function marker HLA-DR. These results provide the rationale for future studies testing the hypothesis that EPO or EPO analogues improve outcomes of AIH patients by increasing Treg.

Regulatory T cells in autoimmune hepatitis: an updated overview.

Regulatory T-cells (Tregs) are key players in the maintenance of immune homeostasis by preventing immune responses to self-antigens. Defects in Treg frequency and/or function result in overwhelming CD4 and CD8 T cell immune responses participating in the autoimmune attack. Perpetuation of autoimmune damage is also favored by Treg predisposition to acquire effector cell features upon exposure to a proinflammatory challenge. Treg impairment plays a permissive role in the initiation and perpetuation of autoimmune liver diseases, namely autoimmune hepatitis, primary biliary cholangitis and primary sclerosing cholangitis. In this Review, we outline studies reporting the role of Treg impairment in the pathogenesis of these conditions and discuss methods to restore Treg number and function either by generation/expansion in the test tube or through in vivo expansion upon administration of low dose IL-2. Challenges and caveats of these potential therapeutic strategies are also reviewed and discussed.