Hepatic Immune Cells Research Articles

Extrahepatic factors in hepatic immune regulation.

The liver is a site of complex immune activity. The hepatic immune system tolerates harmless immunogenic loads in homeostasis status, shelters liver function, while maintaining vigilance against possible infectious agents or tissue damage and providing immune surveillance at the same time. Activation of the hepatic immunity is initiated by a diverse repertoire of hepatic resident immune cells as well as non-hematopoietic cells, which can sense “danger signals” and trigger robust immune response. Factors that mediate the regulation of hepatic immunity are elicited not only in liver, but also in other organs, given the dual blood supply of the liver via both portal vein blood and arterial blood. Emerging evidence indicates that inter-organ crosstalk between the liver and other organs such as spleen, gut, lung, adipose tissue, and brain is involved in the pathogenesis of liver diseases. In this review, we present the features of hepatic immune regulation, with particular attention to the correlation with factors from extrahepatic organ. We describe the mechanisms by which other organs establish an immune association with the liver and then modulate the hepatic immune response. We discuss their roles and distinct mechanisms in liver homeostasis and pathological conditions from the cellular and molecular perspective, highlighting their potential for liver disease intervention. Moreover, we review the available animal models and methods for revealing the regulatory mechanisms of these extrahepatic factors. With the increasing understanding of the mechanisms by which extrahepatic factors regulate liver immunity, we believe that this will provide promising targets for liver disease therapy.

Liposome-encapsulated curcumin attenuates HMGB1-mediated hepatic inflammation and fibrosis in a murine model of Wilson's disease.

Wilson's disease (WD) is an inherited disorder of copper metabolism with predominant hepatic manifestations. Left untreated, it can be fatal. Current therapies focus on treating copper overload rather than targeting the pathophysiology of copper-induced liver injuries. We sought to investigate whether liposome-encapsulated curcumin (LEC) could attenuate the underlying pathophysiology of WD in a mouse model of WD. Subcutaneous administration in a WD mouse model with ATP7B knockout (Atp7b-/-) resulted in robust delivery of LEC to the liver as determined by in-vitro and in-vivo imaging. Treatment with LEC attenuated hepatic injuries, restored lipid metabolism and decreased hepatic inflammation and fibrosis, and thus hepatosplenomegaly in Atp7b-/- mice. Mechanistically, LEC decreased hepatic immune cell and macrophage infiltration and attenuated the hepatic up-regulation of p65 by preventing cellular translocation of high-mobility group box-1 (HMGB-1). Moreover, decreased translocation of HMGB1 was associated with reduced splenic CD11b+/CD43+/Ly6CHi inflammatory monocyte expansion and circulating level of proinflammatory cytokines. Nevertheless there was no change in expression of oxidative stress-related genes or significant copper chelation effect of LEC in Atp7b-/- mice. Our results indicate that treatment with subcutaneous LEC can attenuate copper-induced liver injury in an animal model of WD via suppression of HMGB1-mediated hepatic and systemic inflammation. These findings provide important proof-of-principle data to develop LEC as a novel therapy for WD as well as other inflammatory liver diseases.

Chemotactic cytokines secreted from Kupffer cells contribute to the sex-dependent susceptibility to non-alcoholic fatty liver diseases in mice

AimsThe global prevalence of non-alcoholic fatty liver disease (NAFLD) has rapidly increased over the last decade due to an elevated occurrence of metabolic syndromes. Importantly, the prevalence and severity of NAFLD is higher in men than in women. Therefore, in the present study we endeavored to identify the mechanistic disparity between male and female mice. Main methodsGlobal gene transcriptomics analysis was done with the high-fat diet (HFD)-induced NAFLD model of male, female, and ovariectomized (OVX) female mice. The expression of CCL2, CXCL2, and CXCL10 in mRNA level and serum protein level was done by qPCR and ELISA each. Immunohistochemistry staining was used to observe hepatic immune cell infiltration. To analyzing portion of immune cells, flow cytometry was done with isolated liver cells from HFD-fed male and female mice. Primary mouse liver cells were isolated from male and female mice for in vitro studies. Key findingsWe identified sex differences in inflammatory chemokines, CCL2, CXCL2, and CXCL10, with the expression of these chemokines enhanced in male and OVX, but not in female, mice after HFD feeding. Resident Kupffer cells (KCs) were identified as the major source of production of CCL2, CXCL2, and CXCL10 in the mouse NAFLD model. Notably, KCs obtained from male mice expressed higher levels of chemokines than those from female mice, indicating that KCs may mediate the sex discrepancy in NAFLD progression. SignificanceOur findings offer new insights into the pathology of sex-specific differences in NAFLD, involving chemokines and KCs.

Macrophage-derived MLKL in alcohol-associated liver disease: Regulation of phagocytosis.

Mixed lineage kinase domain-like pseudokinase (MLKL), a key terminal effector of necroptosis, also plays a role in intracellular vesicle trafficking that is critical for regulating liver inflammation and injury in alcohol-associated liver disease (ALD). Although receptor interacting protein kinase 3 (Rip3)-/- mice are completely protected from ethanol-induced liver injury, Mlkl-/- mice are only partially protected. Therefore, we hypothesized that cell-specific functions of MLKL may contribute to ethanol-induced injury. Bone marrow transplants between Mlkl-/- mice and littermates were conducted to distinguish the role of myeloid versus nonmyeloid Mlkl in the Gao-binge model of ALD. Ethanol-induced hepatic injury, steatosis, and inflammation were exacerbated in Mlkl-/- →wild-type (WT) mice, whereas Mlkl deficiency in nonmyeloid cells (WT→ Mlkl-/- ) had no effect on Gao-binge ethanol-induced injury. Importantly, Mlkl deficiency in myeloid cells exacerbated ethanol-mediated bacterial burden and accumulation of immune cells in livers. Mechanistically, challenging macrophages with lipopolysaccharide (LPS) induced signal transducer and activator of transcription 1-mediated expression and phosphorylation of MLKL, as well as translocation and oligomerization of MLKL to intracellular compartments, including phagosomes and lysosomes but not plasma membrane. Importantly, pharmacological or genetic inhibition of MLKL suppressed the phagocytic capability of primary mouse Kupffer cells (KCs) at baseline and in response to LPS with/without ethanol as well as peripheral monocytes isolated from both healthy controls and patients with alcohol-associated hepatitis. Further, in vivo studies revealed that KCs of Mlkl-/- mice phagocytosed fewer bioparticles than KCs of WT mice. Together, these data indicate that myeloid MLKL restricts ethanol-induced liver inflammation and injury by regulating hepatic immune cell homeostasis and macrophage phagocytosis.

Tetrahydroxylated bile acids improve cholestatic liver and bile duct injury in the Mdr2-/- mouse model of sclerosing cholangitis via immunomodulatory effects.

Bile salt export pump (Bsep) (Abcb11) −/− mice are protected from acquired cholestatic injury due to metabolic preconditioning with a hydrophilic bile acid (BA) pool with formation of tetrahydroxylated bile acids (THBAs). We aimed to explore whether loss of Bsep and subsequent elevation of THBA levels may have immunomodulatory effects, thus improving liver injury in the multidrug resistance protein 2 (Mdr2) (Abcb4) −/− mouse. Cholestatic liver injury in Mdr2 −/− Bsep −/− double knockout (DKO), Mdr2 −/− , Bsep −/− , and wild‐type mice was studied for comparison. Mdr2 −/− mice were treated with a THBA (3α,6α,7α,12α‐Tetrahydroxycholanoic acid). RNA/protein expression of inflammatory/fibrotic markers were investigated. Serum BA‐profiling was assessed by ultra‐performance liquid chromatography tandem mass spectrometry. Hepatic immune cell profile was quantified by flow cytometric analysis (FACS). In vitro, the THBA effect on chenodeoxycholic acid (CDCA)–induced inflammatory signaling in hepatocyte and cholangiocytes as well as lipopolysaccharide (LPS)/interferon‐γ (IFN‐γ)–induced macrophage activation was analyzed. In contrast to Mdr2 −/− , DKO mice showed no features of sclerosing cholangitis. Sixty‐seven percent of serum BAs in DKO mice were polyhydroxylated (mostly THBAs), whereas Mdr2 −/− mice did not have these BAs. Compared with Mdr2 −/−, DKO animals were protected from hepatic inflammation/fibrosis. THBA feeding in Mdr2 −/− mice improved liver injury. FACS analysis in DKO and Mdr2 −/− THBA‐fed mice showed changes of the hepatic immune cell profile towards an anti‐inflammatory pattern. Early growth response 1 (EGR1) protein expression was reduced in DKO and in Mdr2 −/− THBA‐fed mice compared with Mdr2 −/− control mice. In vitro, THBA‐reduced CDCA induced EGR1 protein and mRNA expression of inflammatory markers in hepatocytes and cholangiocytes. LPS/IFN‐γ–induced macrophage activation was ameliorated by THBA. THBAs repress EGR1‐related key pro‐inflammatory pathways. Conclusion: THBA and their downstream targets may represent a potential treatment strategy for cholestatic liver diseases.

839-P: Pharmacological Inhibition of Mammalian INDY Ameliorates Western Diet–Induced NASH in Mice: Possible Implication of Fgf21-AMPK Signaling

Background: Recent data proposed a role of the citrate transporter INDY (I’m Not Dead Yet) in the development of obesity, insulin resistance and NAFLD suggesting its potential as a therapeutic target for metabolic-related disorders. The aim of our study is to determine if mammalian INDY (mINDY) inhibitors can be introduced as a new therapeutic option for NAFLD/NASH. Methods: Six-week-old C57Bl/6N mice were fed a western diet (WD) for 18 weeks to induce NASH. After 12 weeks of feeding, mice received mINDY inhibitor (PF-06649298, mINDYi; 100 mg/kg) or vehicle bid until end of week 18. During the treatment period, body weight and composition were monitored. ipGTT and FACS analysis of liver inflammatory cells were performed. Results: mINDYi-treated mice exhibited lower body weight, fat mass and higher lean mass compared to vehicle-treated mice. Results from ipGTT revealed improved glucose tolerance and insulin sensitivity. The latter was further confirmed through increased insulin-stimulated Akt phosphorylation in liver, gonadal WAT and BAT. Treatment with mINDYi attenuated WD-induced hepatic injury, steatosis and inflammation as shown histologically and biochemically by reduction in plasma ALT and AST, decrease in hepatic triglyceride accumulation and reduced hepatic lymphoid and myeloid immune cell populations. Interestingly, these effects were associated with a significant rise in plasma and hepatic Fgf21 together with increased hepatic phosphorylation of LKB1 and AMPK and decreased mTOR. Results from primary mouse hepatocytes revealed that mINDYi-induction of Fgf21 is partially dependent on AMPK activation. Conclusion: Our study shows for the first time that mINDYi attenuates diet-induced steatohepatitis; offering a promising novel treatment strategy for NAFLD. Further investigations are currently ongoing to elucidate the molecular mechanisms underlying mINDY inhibition and whether it is dependent on Fgf21-AMPK signaling. Disclosure N.El-agroudy: None. G.Zahn: Employee; Eternygen GmbH. C.Herrmann: None. G.Mingrone: Advisory Panel; Fractyl Health, Inc., Novo Nordisk, Consultant; ReCor Medical, Inc. T.C.Alves: None. A.L.Birkenfeld: None.

Crosstalk Between Autophagy and Innate Immunity: A Pivotal Role in Hepatic Fibrosis.

Liver fibrosis is a repair process of chronic liver injuries induced by toxic substances, pathogens, and inflammation, which exhibits a feature such as deposition of the extracellular matrix. The initiation and progression of liver fibrosis heavily relies on excessive activation of hepatic stellate cells (HSCs). The activated HSCs express different kinds of chemokine receptors to further promote matrix remodulation. The long-term progression of liver fibrosis will contribute to dysfunction of the liver and ultimately cause hepatocellular carcinoma. The liver also has abundant innate immune cells, including DCs, NK cells, NKT cells, neutrophils, and Kupffer cells, which conduct complicated functions to activation and expansion of HSCs and liver fibrosis. Autophagy is one specific type of cell death, by which the aberrantly expressed protein and damaged organelles are transferred to lysosomes for further degradation, playing a crucial role in cellular homeostasis. Autophagy is also important to innate immune cells in various aspects. The previous studies have shown that dysfunction of autophagy in hepatic immune cells can result in the initiation and progression of inflammation in the liver, directly or indirectly causing activation of HSCs, which ultimately accelerate liver fibrosis. Given the crosstalk between innate immune cells, autophagy, and fibrosis progression is complicated, and the therapeutic options for liver fibrosis are quite limited, the exploration is essential. Herein, we review the previous studies about the influence of autophagy and innate immunity on liver fibrosis and the molecular mechanism to provide novel insight into the prevention and treatment of liver fibrosis.

The Role of Interferon Regulatory Factors in Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease with many metabolic comorbidities, such as obesity, diabetes, and cardiovascular diseases. Non-alcoholic steatohepatitis (NASH), an advanced form of NAFLD, accompanies the progression of hepatic steatosis, inflammation, cell death, and varying degree of liver fibrosis. Interferons (IFNs) have been shown to play important roles in the pathogenesis of NAFLD and NASH. Their regulating transcriptional factors such as interferon regulatory factors (IRFs) can regulate IFN expression, as well as genes involved in macrophage polarization, which are implicated in the pathogenesis of NAFLD and advanced liver disease. In this review, the roles of IRF-involved signaling pathways in hepatic inflammation, insulin resistance, and immune cell activation are reviewed. IRFs such as IRF1 and IRF4 are also involved in the polarization of macrophages that contribute to critical roles in NAFLD or NASH pathogenesis. In addition, IRFs have been shown to be regulated by treatments including microRNAs, PPAR modulators, anti-inflammatory agents, and TLR agonists or antagonists. Modulating IRF-mediated factors through these treatments in chronic liver disease can ameliorate the progression of NAFLD to NASH. Furthermore, adenoviruses and CRISPR activation plasmids can also be applied to regulate IRF-mediated effects in chronic liver disease. Pre-clinical and clinical trials for evaluating IRF regulators in NAFLD treatment are essential in the future direction.

Modulation of the Gut Microbiome to Improve Clinical Outcomes in Hepatocellular Carcinoma.

Simple SummaryHepatocellular carcinoma (HCC), the most common form of liver cancer, is the third leading cause of cancer-related mortality worldwide. Recent research indicates that altering the gut environment affects liver immune response and cancer progression, through what is known as the ‘gut–liver axis’. HCC patients have dampened anticancer responses, but through modulating the gut environment, there is potential to reinvigorate these ‘exhausted’ immune cells to target tumors. In promising research on melanoma, transplanting stool from healthy donors through faecal microbiota transplantation (FMT) resulted in increased response to immunotherapy treatment in patients who were previously nonresponding. However, manipulating the gut environment as a therapeutic option in HCC remains to be explored. In this review, we explore the mechanisms through which this occurs, including how the gut environment affects gut barrier function, bacterial sensing, and liver immune responses, and how FMT may be a potential therapy for HCC patients nonresponsive to immunotherapy.Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Recently, the gut microbiota has been shown to be closely linked to modulation of the immune and inflammatory responses, hence its potential as a therapeutic target. Although still under intense investigation, there exists a ‘gut–liver axis’ that links changes in the gut to the liver. In this regard, composition of gut microbiota and related metabolites, such as bile acids and short-chain fatty acids, have been shown to orchestrate key immune–metabolic events in liver disease and liver cancer. As hepatic immune cells are important determinants of antitumor responses, it is now increasingly recognized that the gut–liver axis plays a key role in influencing the intrahepatic immune response in HCC to favor a pro- or antitumor immune milieu. Hence, modulation of gut microbiota is potentially an attractive option to reinvigorate the antitumor responses. In this regard, promising evidence from melanoma preclinical and clinical studies has demonstrated the efficacy of gut-based intervention in reinvigorating the antitumor responses and improving responses to immunotherapy. However, the role of gut-based interventions as a therapeutic option in HCC remains to be elucidated. This review details how the gut microbiota and bacterial metabolites affect gut barrier function and ultimately immune response in HCC and raises the question of the potential of gut-based interventions as an adjunct therapy for patients with HCC receiving immunotherapy.

Intrahepatic microbes govern liver immunity by programming NKT cells.

The gut microbiome shapes local and systemic immunity. The liver is presumed to be a protected sterile site. As such, a hepatic microbiome has not been examined. Here, we showed a liver microbiome in mice and humans that is distinct from that of the gut and is enriched in Proteobacteria. It undergoes dynamic alterations with age and is influenced by the environment and host physiology. Fecal microbial transfer experiments revealed that the liver microbiome is populated from the gut in a highly selective manner. Hepatic immunity is dependent on the microbiome, specifically the bacteroidetes species. Targeting bacteroidetes with oral antibiotics reduced hepatic immune cells by approximately 90%, prevented antigen-presenting cell (APC) maturation, and mitigated adaptive immunity. Mechanistically, our findings are consistent with presentation of bacteroidetes-derived glycosphingolipids to NKT cells promoting CCL5 signaling, which drives hepatic leukocyte expansion and activation, among other possible host-microbe interactions. Collectively, we reveal a microbial/glycosphingolipid/NKT/CCL5 axis that underlies hepatic immunity.

Role of Immune Cells in Biliary Repair.

The biliary system is comprised of cholangiocytes and plays an important role in maintaining liver function. Under normal conditions, cholangiocytes remain in the stationary phase and maintain a very low turnover rate. However, the robust biliary repair is initiated in disease conditions, and different repair mechanisms can be activated depending on the pathological changes. During biliary disease, immune cells including monocytes, lymphocytes, neutrophils, and mast cells are recruited to the liver. The cellular interactions between cholangiocytes and these recruited immune cells as well as hepatic resident immune cells, including Kupffer cells, determine disease outcomes. However, the role of immune cells in the initiation, regulation, and suspension of biliary repair remains elusive. The cellular processes of cholangiocyte proliferation, progenitor cell differentiation, and hepatocyte-cholangiocyte transdifferentiation during biliary diseases are reviewed to manifest the underlying mechanism of biliary repair. Furthermore, the potential role of immune cells in crucial biliary repair mechanisms is highlighted. The mechanisms of biliary repair in immune-mediated cholangiopathies, inherited cholangiopathies, obstructive cholangiopathies, and cholangiocarcinoma are also summarized. Additionally, novel techniques that could clarify the underlying mechanisms of biliary repair are displayed. Collectively, this review aims to deepen the understanding of the mechanisms of biliary repair and contributes potential novel therapeutic methods for treating biliary diseases.

Loss of bile salt export pump aggravates lipopolysaccharide-induced liver injury in mice due to impaired hepatic endotoxin clearance.

Background and AimsLipopolysaccharide (LPS) clearance is delayed in cholestatic liver diseases. While compromised clearance by Kupffer cells (KCs) is involved, the role of LPS uptake into hepatocytes and canalicular excretion remains unclear.Approach and ResultsWild‐type (WT) and bile salt export pump (Bsep) knockout (KO) mice were challenged i.p. with LPS. Liver injury was assessed by serum biochemistry, histology, molecular inflammation markers, and immune cell infiltration. LPS concentrations were determined in liver tissue and bile. Subcellular kinetics of fluorescently labeled LPS was visualized by intravital two‐photon microscopy, and the findings in Bsep KO mice were compared to common bile duct–ligated (BDL) and multidrug resistance protein 2 (Mdr2) KO mice. Changes in gut microbiota composition were evaluated by 16S ribosomal RNA gene amplicon sequencing analysis. Bsep KO mice developed more pronounced LPS‐induced liver injury and inflammatory signaling, with subsequently enhanced production of proinflammatory cytokines and aggravated hepatic immune cell infiltration. After LPS administration, its concentrations were higher in liver but lower in bile of Bsep KO compared to WT mice. Intravital imaging of LPS showed a delayed clearance from sinusoidal blood with a basolateral uptake block into hepatocytes and reduced canalicular secretion. Moreover, LPS uptake into KCs was reduced. Similar findings with respect to hepatic LPS clearance were obtained in BDL and Mdr2 KO mice. Pretreatment with the microtubule inhibitor colchicine inhibited biliary excretion of LPS in WT mice, indicating that LPS clearance is microtubule‐dependent. Microbiota analysis showed no change of the gut microbiome between WT and Bsep KO mice at baseline but major changes upon LPS challenge in WT mice.ConclusionsAbsence of Bsep and cholestasis in general impair LPS clearance by a basolateral uptake block into hepatocytes and consequently less secretion into canaliculi. Impaired LPS removal aggravates hepatic inflammation in cholestasis.

Mechanisms of liver injury in high fat sugar diet fed mice that lack hepatocyte X-box binding protein 1.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of liver diseases in the United States and can progress to cirrhosis, end-stage liver disease and need for liver transplantation. There are limited therapies for NAFLD, in part, due to incomplete understanding of the disease pathogenesis, which involves different cell populations in the liver. Endoplasmic reticulum stress and its adaptative unfolded protein response (UPR) signaling pathway have been implicated in the progression from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH). We have previously shown that mice lacking the UPR protein X-box binding protein 1 (XBP1) in the liver demonstrated enhanced liver injury and fibrosis in a high fat sugar (HFS) dietary model of NAFLD. In this study, to better understand the role of liver XBP1 in the pathobiology of NAFLD, we fed hepatocyte XBP1 deficient mice a HFS diet or chow and investigated UPR and other cell signaling pathways in hepatocytes, hepatic stellate cells and immune cells. We demonstrate that loss of XBP1 in hepatocytes increased inflammatory pathway expression and altered expression of the UPR signaling in hepatocytes and was associated with enhanced hepatic stellate cell activation after HFS feeding. We believe that a better understanding of liver cell-specific signaling in the pathogenesis of NASH may allow us to identify new therapeutic targets.

Maternal regulation of biliary disease in neonates via gut microbial metabolites

Maternal seeding of the microbiome in neonates promotes a long-lasting biological footprint, but how it impacts disease susceptibility in early life remains unknown. We hypothesized that feeding butyrate to pregnant mice influences the newborn’s susceptibility to biliary atresia, a severe cholangiopathy of neonates. Here, we show that butyrate administration to mothers renders newborn mice resistant to inflammation and injury of bile ducts and improves survival. The prevention of hepatic immune cell activation and survival trait is linked to fecal signatures of Bacteroidetes and Clostridia and increases glutamate/glutamine and hypoxanthine in stool metabolites of newborn mice. In human neonates with biliary atresia, the fecal microbiome signature of these bacteria is under-represented, with suppression of glutamate/glutamine and increased hypoxanthine pathways. The direct administration of butyrate or glutamine to newborn mice attenuates the disease phenotype, but only glutamine renders bile duct epithelial cells resistant to cytotoxicity by natural killer cells. Thus, maternal intake of butyrate influences the fecal microbial population and metabolites in newborn mice and the phenotypic expression of experimental biliary atresia, with glutamine promoting survival of bile duct epithelial cells.

NLRP6 Inflammasome Modulates Disease Progression in a Chronic-Plus-Binge Mouse Model of Alcoholic Liver Disease.

A considerable percentage of the population is affected by alcoholic liver disease (ALD). It is characterized by inflammatory signals from the liver and other organs, such as the intestine. The NLR family pyrin domain containing 6 (NLRP6) inflammasome complex is one of the most important inflammatory mediators. The aim of this study was to evaluate a novel mouse model for ALD characterized by 8-week chronic-plus-binge ethanol administration and to investigate the role of NLRP6 inflammasome for intestinal homeostasis and ALD progression using Nlrp6-/- mice. We showed that chronic-plus-binge ethanol administration triggers hepatic steatosis, injury, and neutrophil infiltration. Furthermore, we discovered significant changes of intestinal microbial communities, including increased relative abundances of bacteria within the phyla Bacteroidota and Campilobacterota, as well as reduced Firmicutes. In this ALD model, inhibiting NLRP6 signaling had no effect on liver steatosis or damage, but had a minor impact on intestinal homeostasis via affecting intestinal epithelium function and gut microbiota. Surprisingly, Nlrp6 loss resulted in significantly decreased hepatic immune cell infiltration. As a result, our novel mouse model encompasses several aspects of human ALD, such as intestinal dysbiosis. Interfering with NLRP6 inflammasome activity reduced hepatic immune cell recruitment, indicating a disease-aggravating role of NLRP6 during ALD.

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.

Serum Proteomics Uncovers Biomarkers of Clinical Portal Hypertension in Children With Biliary Atresia.

Children with biliary atresia (BA) often develop portal hypertension (PHT) and its complications, which are associated with high morbidity and mortality. The goal of this study was to identify serum biomarkers of PHT by using large‐scale proteomics. We applied the slow off‐rate modified aptamer scan (SOMAscan) to measure 1,305 proteins in serum samples of children with BA with and without clinical evidence of PHT in validation and discovery cohorts enrolled in the Biliary Atresia Study of Infants and Children. Serum proteomics data was analyzed using logistic regression to identify protein(s) with an area under the receiver operating characteristic curve (AUROC) ≥ 0.90. Immunostaining was used to characterize the cellular localization of the new biomarker proteins in liver tissues. We identified nine proteins in the discovery cohort (n = 40 subjects) and five proteins in the validation cohort (n = 80 subjects) that individually or in combination predicted clinical PHT with AUROCs ≥ 0.90. Merging the two cohorts, we found that semaphorin 6B (SEMA6B) alone and three other protein combinations (SEMA6B+secreted frizzle protein 3 [SFRP3], SEMA6B+COMM domain containing 7 [COMMD7], and vascular cell adhesion molecule 1 [VCAM1]+BMX nonreceptor tyrosine kinase [BMX]) had AUROCs ≥ 0.90 in both cohorts, with high positive‐ and negative‐predictive values. Immunostaining of the new protein biomarkers showed increased expression in hepatic endothelial cells, cholangiocytes, and immune cells within portal triads in BA livers with clinical PHT compared to healthy livers. Conclusion: Large‐scale proteomics identified SEMA6B, SFRP3, COMMD7, BMX, and VCAM1 as biomarkers highly associated with clinical PHT in BA. The expression of the biomarkers in hepatic epithelial, endothelial, and immune cells support their potential role in the pathophysiology of PHT.

The Liver and Heart: How the Two Most Beloved “Babies” in the Human Body Communicate

The Liver and Heart: How the Two Most Beloved “Babies” in the Human Body Communicate

Immune cell-mediated features of non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) includes a range of hepatic manifestations, starting with liver steatosis and potentially evolving towards non-alcoholic steatohepatitis (NASH), cirrhosis or even hepatocellular carcinoma. NAFLD is a major health burden, and its incidence is increasing worldwide. Although it is primarily a disease of disturbed metabolism, NAFLD involves several immune cell-mediated inflammatory processes, particularly when reaching the stage of NASH, at which point inflammation becomes integral to the progression of the disease. The hepatic immune cell landscape is diverse at steady state and it further evolves during NASH with direct consequences for disease severity. In this Review, we discuss current concepts related to the role of immune cells in the onset and progression of NASH. A better understanding of the mechanisms by which immune cells contribute to NASH pathogenesis should aid the design of innovative drugs to target NASH, for which current therapeutic options are limited.

Interleukin 23 Produced by Hepatic Monocyte-Derived Macrophages Is Essential for the Development of Murine Primary Biliary Cholangitis.

Background and AimsPrimary Biliary Cholangitis (PBC) is an organ-specific autoimmune liver disease. Mononuclear phagocytes (MNPs), comprise of monocyte, dendritic cells and monocyte-derived macrophages, constitute major arm of the innate immune system known to be involved in the pathogenesis of autoimmune disorders. MNPs were shown to accumulate around intra-hepatic bile ducts in livers of PBC patients. Interleukin 23 (IL-23) is a pro-inflammatory cytokine. IL-23-positive cells were detected in livers of patients with advanced stage PBC and IL-23 serum levels found to be in correlation with PBC disease severity. Our overall goal was to assess the importance of IL-23 derived from MNPs in PBC pathogenesis.MethodsWe utilized an inducible murine model of PBC and took advantage of transgenic mice targeting expression of IL-23 by specific MNP populations. Analysis included liver histology assessment, flow cytometry of hepatic immune cells and hepatic cytokine profile evaluation. Specific MNPs sub-populations were sorted and assessed for IL-23 expression levels.ResultsFlow cytometry analysis of non-parenchymal liver cells in autoimmune cholangitis revealed massive infiltration of the liver by MNPs and neutrophils and a decrease in Kupffer cells numbers. In addition, a 4-fold increase in the incidence of hepatic IL-17A producing CD4+ T cells was found to be associated with an increase in hepatic IL23-p19 and IL17A expression levels. Disease severity was significantly ameliorated in both CD11ccreP19flox/flox and CX3CR1creP19 flox/flox mice as assessed by reduced portal inflammation and decreased hepatic expression of various inflammatory cytokines. Amelioration of disease severity was associated with reduction in IL-17A producing CD4+ T cells percentages and decreased hepatic IL23-p19 and IL17A expression levels. qRT-PCR analysis of sorted hepatic MNPs demonstrated high expression levels of IL-23 mRNA specifically by CX3CR1hiCD11c+ monocyte-derived macrophages.ConclusionOur results indicate a major role for IL-23 produced by hepatic monocyte-derived macrophages in the pathogenesis of PBC. These results may pave the road for the development of new immune-based and cell specific therapeutic modalities for PBC patients not responding to current therapies.