Mesenchymal stromal cell-dependent immunoregulation in chemically-induced acute liver failure

Drug-induced liver injury(DILI),which refers to liver damage caused by a drug or its metabolites,has emerged as an important cause of acute liver failure(ALF)in recent years.Chemically-induced ALF in animal models mimics the pathology of DILI in humans;thus,these models are used to study the mechanism of potentially effective treatment strategies.Mesenchymal stromal cells(MSCs)possess immunomodulatory properties,and they alleviate acute liver injury and decrease the mortality of animals with chemically-induced ALF.Here,we summarize some of the existing research on the interaction between MSCs and immune cells,and discuss the possible mechanisms underlying the immunomodulatory activity of MSCs in chemically-induced ALF.We conclude that MSCs can impact the phenotype and function of macrophages,as well as the differentiation and maturation of dendritic cells,and inhibit the proliferation and activation of T lymphocytes or B lymphocytes.MSCs also have immunomodulatory effects on the production of cytokines,such as prostaglandin E2 and tumor necrosis factor-alpha-stimulated gene 6,in animal models.Thus,MSCs have significant benefits in the treatment of chemically-induced ALF by interacting with immune cells and they may be applied to DILI in humans in the near future.     

Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2

Inflammatory cell infiltration in the liver is a hallmark of nonalcoholic steatohepatitis (NASH). The chemokine-chemokine receptor interaction induces inflammatory cell recruitment. CC-chemokine receptor (CCR)2 is expressed on hepatic macrophages and hepatic stellate cells. This study aims to investigate the therapeutic potential of CCR2 to NASH. Twenty-two weeks on a choline-deficient amino acid-defined (CDAA) diet induced steatosis, inflammatory cell infiltration, and liver fibrosis with increased CCR2 and monocyte chemoattractant protein (MCP)-1 expression in the wild-type livers. The infiltrated macrophages expressed CD68, CCR2, and a marker of bone marrow-derived monocytes, Ly6C. CCR2(-/-) mice had less steatosis, inflammatory cell infiltration, and fibrosis, and hepatic macrophages expressing CD68 and Ly6C were decreased. Toll-like receptor (TLR)4(-/-), TLR9(-/-), and MyD88(-/-) mice had reduced hepatic macrophage infiltration with decreased MCP-1 and CCR2 expression because TLR signaling is a potent inducer of MCP-1. To assess the role of Kupffer cells at the onset of NASH, Kupffer cells were depleted by liposomal clodronate. The Kupffer cell depletion ameliorated steatohepatitis with a decrease in the MCP-1 expression and recruitment of Ly6C-expressing macrophages at the onset of NASH. Finally, to test the therapeutic potential of targeting CCR2, a CCR2 inhibitor was administered to mice on a CDAA diet. The pharmaceutical inhibition of CCR2 prevented infiltration of the Ly6C-positive macrophages, resulting in an inhibition of liver inflammation and fibrosis. We concluded that CCR2 and Kupffer cells contribute to the progression of NASH by recruiting bone marrow-derived monocytes.     

Roles of heterogenous hepatic macrophages in the progression of liver diseases

Hepatic macrophages are key immune cells associated with the broad ranges of liver diseases including steatosis, inflammation and fibrosis. Hepatic macrophages interact with other immune cells and orchestrate hepatic immune circumstances. Recently, the heterogenous populations of hepatic macrophages have been discovered termed residential Kupffer cells and monocyte-derived macrophages, and identified their distinct population dynamics during the progression of various liver diseases. Liver injury lead to Kupffer cells activation with induction of inflammatory cytokines and chemokines, which triggers recruitment of inflammatory monocyte-derived macrophages. To understand liver pathology, the functions of different subtypes of liver macrophages should be regarded with different perspectives. In this review, we summarize recent advances in the roles of hepatic macrophages under liver damages and suggest hepatic macrophages as promising therapeutic targets for treating liver diseases.     

A metabonomics study of Chinese miniature pigs with acute liver failure treated with transplantation of placental mesenchymal stem cells

Metabonomics has become a highly sensitive and powerful tool for screening of biomarkers and elucidation of biochemical processes. Recently, we reported beneficial therapeutic effect of human placenta mesenchymal stem cells (hPMSCs) transplantation on d-galactosamine (GalN)-induced liver injury in Chinese miniature pigs. However, the underlying mechanism remains largely unclear. Here, UPLC/TOF/MS-based metabonomics approach was employed to analyze serum from GalN-treated pigs with the goal of identifying potential biomarkers for acute liver injury. Our results showed that obvious metabolic disturbance occurred during acute liver failure (ALF), which can be ameliorated by the hPMSCs transplantation. The metabolic profiles of the hPMSCs transplantation group returned back to the original state 5 weeks after the hPMSCs transplantation. In addition, the result obtained from metabolic trajectory analysis correlated well with those from biochemical analysis and histological examination. Serum levels of several metabolites including glycoursodeoxycholic acid, glycochenodeoxycholic acid, aromatic amino acids, phosphatidylcholine, lysophosphatidylcholine and sphingomyelin, were significantly modified during the process of ALF and cell treatment. Taken together, our study has gained new insight into the molecular mechanism on how hPMSCs administration facilitates the recovery of ALF, and provided strong support for treating liver diseases with stem cell-based therapies.     

Circulating monocytes accelerate acute liver failure by IL‐6 secretion in monkey

Acute liver failure (ALF) is associated with high mortality, and a poor understanding of the underlying pathophysiology has resulted in a lack of effective treatments so far. Here, using an amatoxin‐induced rhesus monkey model of ALF, we panoramically revealed the cellular and molecular events that lead to the development of ALF. The challenged monkeys with toxins underwent a typical course of ALF including severe hepatic injury, systemic inflammation and eventual death. Adaptive immune was not noticeably disturbed throughout the progress of ALF. A systematic examination of serum factors and cytokines revealed that IL‐6 increase was the most rapid and drastic. Interestingly, we found that IL‐6 was mainly produced by circulating monocytes. Furthermore, ablation of monocyte‐derived IL‐6 in mice decreased liver injury and systemic inflammation following chemical injection. Our findings reveal a critical role of circulating monocytes in initiating and accelerating ALF, indicating a potential therapeutic target in clinical treatment for ALF.     

Exosomes derived from human umbilical cord mesenchymal stem cells alleviate acute liver failure by reducing the activity of the NLRP3 inflammasome in macrophages

Human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-EXOs) play an important role in the regulation of the immune system and inflammatory responses; however, their role in acute liver failure (ALF) and related pathological conditions is unclear. In this study, we found that hUCMSC-EXOs can reduce the expression of the NLRP3 inflammasome and downstream inflammatory factors in acute liver failure. Western blot and ELISA results showed that hUCMSC-EXOs decreased the expression of NLRP3, caspase-1, IL-1β and IL-6 in LPS-stimulated RAW 264.7 macrophages. In vivo, the hUCMSC-EXOs repaired damaged liver tissue and decreased the expression of the NLRP3 inflammasome and the levels of ALT and AST in a mouse ALF model. The results of this study provide a new strategy for the application of human umbilical cord mesenchymal stem cell-derived exosomes in the treatment of ALF.     

A monoclonal antibody to a differentiation antigen present on mature human macrophages and absent from monocytes

A monoclonal antibody is described that has been generated in the mouse against cultured human blood monocytes/macrophages. The antibody, designated 25F9, belongs to the IgG1 subclass, detects antigens of m.w. 86,000, and does not react with freshly isolated blood monocytes but reacts with monocytes after 3 days of culture. The expression of the 25F9 antigen on macrophages increases with culture time. Furthermore, the antibody is negative on platelets, granulocytes, lymphocytes, and a large number of human cell lines except the two melanoma lines MeWo and Mel 57. In cryostat sections of normal human tissue (skin, lung, liver, thymus) and of inflammatory or neoplastic tissue (cutaneous lymphoma, eczema, BCG-granuloma, and melanoma), the antibody reacts with scattered macrophages in the dermis but not with epidermal Langerhans cells, with alveolar macrophages, with liver Kupffer cells, and with scattered macrophages in the cortex and medulla of thymus. In eczema, BCG-granuloma, and cutaneous lymphoma, only a few infiltrating macrophages were stained. On the other hand, a large number of macrophages and melanophages reacted positively in melanoma. In some cases melanoma cells also stained weakly positive. Thus, the antibody detects a differentiation antigen preferentially expressed on mature, tissue-fixed macrophages and absent from blood monocytes.     

Systematic analysis on multiple Gene Expression Omnibus data sets reveals fierce immune response in hepatitis B virus‐related acute liver failure

Acute liver failure (ALF) caused by hepatitis B virus (HBV) is common type of liver failure in the world, with high morbidity and mortality rates. However, the prevalence, genetic background and factors determining the development of HBV‐related ALF are rarely studied. In this study, we examined three Gene Expression Omnibus (GEO) data sets by bioinformatics analysis to identify differentially expressed genes (DEGs), key biological processes and pathways. Immune infiltration analysis showed high immune cells infiltration in HBV‐related ALF tissue. We then confirmed natural killer cells and macrophages infiltration in clinical samples by immunohistochemistry assay, implying these cells play a significant role in HBV‐ALF. We found 1277 genes were co‐up‐regulated and that 1082 genes were co‐down‐regulated in the 3 data sets. Inflammation‐related pathways were enriched in the co‐up‐regulated genes and synthetic metabolic pathways were enriched in the co‐down‐regulated genes. WGCNA also revealed a key module enriching in immune inflammation response and identified 10 hub genes, differentially expressed in an independent data set. In conclusion, we identified fierce immune inflammatory response to elucidate the immune‐driven mechanism of HBV‐ALF and 10 hub genes based on gene expression profiles.     

Mesenchymal stem cells increase heme oxygenase 1-activated autophagy in treatment of acute liver failure

In recent years, transplantation of mesenchymal stem cells (MSCs) has attracted much attention as a potential cell-based therapy for acute liver failure (ALF). As an inducible enzyme, heme oxygenase 1 (HO-1) has been reported to have cytoprotective, anti-apoptotic and immunoregulatory effects. Autophagy, a conserved catabolic process in cells, may be an important pathway for MSCs to treat ALF. In this study, we aimed to explore whether MSCs treat ALF by regulating autophagy and whether HO-1 was involved in the same pathway. Bone marrow-derived MSCs were isolated from Sprague-Dawley rats and cultured according to an established protocol. Co-culture systems of MSCs and hepatocytes were used to assess autophagy in the treatment of ALF. Meanwhile, MSCs were transplanted into rats with d-galactosamine (Gal)-induced ALF. Autophagy inhibitor (3-methyladenine, 3-MA), HO-1 inhibitor (zinc protoporphyrin, ZnPP) and PI3K specific inhibitor (LY294002) were employed in the study. Blood samples and liver tissues were collected before euthanasia. Survival rate, liver function, inflammatory factors, histology, Ki67 and TUNEL staining were determined. MSCs transplantation alleviated ALF both in vivo and in vitro. Autophagy and autophagy-related proteins were significantly up-regulated during MSCs treatment. 3-MA attenuated the therapeutic effect of MSCs. Administration of LY294002 before ALF induction inhibited hepatocyte autophagy. During the MSCs treatment, the HO-1 expression was increased, while inhibiting HO-1 attenuated the therapeutic effect of MSCs as well as hepatocyte autophagy. These findings suggested MSCs could alleviate ALF by increasing the HO-1 expression, which played an important role in activating autophagy through PI3K/AKT signaling pathway.     

Transplantation of miR-219 overexpressed human endometrial stem cells encapsulated in fibrin hydrogel in spinal cord injury

Oligodendrocyte (OL) loss and demyelination occur after spinal cord injury (SCI). Stimulation of remyelination through transplantation of myelinating cells may be effective in improving function. For the repair strategy to be successful, the selection of a suitable cell and maintaining cell growth when cells are injected directly to the site of injury is important. In addition to selecting the type of cell, fibrin hydrogel was used as a suitable tissue engineering scaffold for this purpose. To test the relationship between myelination and functional improvement, the human endometrial stem cells (hEnSCs) were differentiated toward oligodendrocyte progenitor cells (OPCs) using overexpression of miR-219. Adult female Wistar rats were used to induce SCI by using a compression model and were randomly assigned to the following four experimental groups: SCI, Vehicle, hEnSC, and OPC. Ten days after injury, miR-219 overexpressed hEnSC-derived OPCs encapsulated in fibrin hydrogel, as an injectable scaffold, were injected to the injury site. In this study, with a focus on promoting functional recovery after SCI, the Basso-Beattie-Bresnahan test was performed to evaluate the recovery of motor function every week for 10 weeks and the histological assay was then performed. Results showed that the rate of motor function recovery was significantly higher in OPC group compared to SCI and vehicle groups but no marked differences were found between OPC and hEnSC groups, although, the rate of myelination in the OPC group was significantly higher than the other groups. These results demonstrated that remyelination was not the cause of recovery of motor function.     

Acute liver failure-induced death of rats is delayed or prevented by blocking NMDA receptors in brain

Developing procedures to delay the mechanisms of acute liver failure-induced death would increase patients' survival by allowing time for liver regeneration or to receive a liver for transplantation. Hyperammonemia is a main contributor to brain herniation and mortality in acute liver failure (ALF). Acute ammonia intoxication in rats leads to N-methyl-D-aspartate (NMDA) receptor activation in brain. Blocking these receptors prevents ammonia-induced death. Ammonia-induced activation of NMDA receptors could contribute to ALF-induced death. If this were the case, blocking NMDA receptors could prevent or delay ALF-induced death. The aim of this work was to assess 1) whether ALF leads to NMDA receptors activation in brain in vivo and 2) whether blocking NMDA receptors prevents or delays ALF-induced death of rats. It is shown, by in vivo brain microdialysis, that galactosamine-induced ALF leads to NMDA receptors activation in brain. Blocking NMDA receptors by continuous administration of MK-801 or memantine through miniosmotic pumps affords significant protection against ALF-induced death, increasing the survival time approximately twofold. Also, when liver injury is not 100% lethal (1.5 g/kg galactosamine), blocking NMDA receptors increases the survival rate from 23 to 62%. This supports that blocking NMDA receptors could have therapeutic utility to improve survival of patients with ALF.     

Common mechanisms in pediatric acute liver failure

Acute liver failure (ALF) is a rare but potentially fatal disease in children. The etiology is multifactorial, including infection, autoimmune, and genetic disorders, as well as indeterminate hepatitis, which has a higher requirement for liver transplantation. Activation of the innate and adaptive immune systems leads to hepatocyte-specific injury which is mitigated by T regulatory cell activation. Recovery of the native liver depends on activation of apoptotic and regenerative pathways, including the integrated stress response (ISR; e.g., PERK), p53, and HNF4α. Loss-of-function mutations in these pathways cause recurrent ALF in response to non-hepatotropic viruses. Deeper understanding of these mechanisms will lead to improved diagnosis, management, and outcomes for pediatric ALF.     

In situ effector mechanisms in rat kidney allograft rejection. I. Characterization of the host cellular infiltrate in rejecting allograft parenchyma.

The infiltrating inflammatory cells were recovered with collagenase and DNase from rejecting rat kidney allografts and autografts in conditions where the enzyme treatment did not affect the expression of subclass-specific surface markers. As the differential distribution of the inflammatory cells in the dispersate was similar to the distribution of inflammatory cells in tissue imprints, and as any major blood contamination was excluded, we consider the results representative of the composition of the in situ infiltrate. At the peak of rejection on Day 6 after the transplantation, approximately 30% monocytes, 17% macrophages, 31% lymphocytes, 6% (T) lymphoblasts, and 10% (B) plasmablasts and plasma cells were present in the graft. The blast cell response, pathognomonic to immune activation, was less prominent in the recipient spleen, blood, and lymph nodes. Twenty-three percent of the infiltrating lymphocytes expressed the (T-cell-specific) Pta.A.1 surface antigen(s) and 14% were surface Ig positive. The remaining lymphocytes were double-negative “null cells.” In preparative cell electrophoresis most of the allograft-infiltrating lymphocytes carried the low electrophoretic mobility, characteristic to resting B cells. Approximately 70% of allograft-infiltrating macrophages and 50% of infiltrating monocytes but only 30% of the monocytes present in the recipient spleen expressed the Fc receptor to IgG, suggesting an activation (or increase in avidity) of this receptor during the influx of mononuclear cells into the site of inflammation and during maturation of monocytes into tissue macrophages. There was a strong in situ proliferative activity, far stronger than in the central lymphatic system of the recipient rat. After 1 hr in vivo pulse labeling with [3H]thymidine 24% of the infiltrating inflammatory cells carried the label. Most of the labeled cells were blasts or lymphocytes, but a small albeit distinct number of labeled monocytes were also present in situ. In contrast to the recipient spleen, where most of the labeled lymphoid cells had a high electrophoretic mobility of resting T cells, in the infiltrate most of the labeled lymphoid cells had a slow mobility of resting B cells.     

Human hepatocyte-derived extracellular vesicles attenuate the carbon tetrachloride-induced acute liver injury in mice

Acute liver injury (ALI) induced by chemicals or viruses can progress rapidly to acute liver failure (ALF), often resulting in death of patients without liver transplantation. Since liver transplantation is limited due to a paucity of donors, expensive surgical costs, and severe immune rejection, novel therapies are required to treat liver injury. Extracellular vesicles (EVs) are used for cellular communication, carrying RNAs, proteins, and lipids and delivering them intercellularly after being endocytosed by target cells. Recently, it was reported that EVs secreted from human hepatocytes have an ability to modulate the immune responses; however, these roles of EVs secreted from human hepatocytes were studied only with in vitro experiments. In the present study, we evidenced that EVs secreted from human hepatocytes attenuated the CCL₄-induced ALI by inhibiting the recruitment of monocytes through downregulation of chemokine receptor in the bone marrow and recruitment of neutrophils through the reduction of C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2 expression levels in the liver.Subject terms: Acute inflammation, Liver diseases     

Kinetics of Kupffer cells as shown by parabiosis and combined autoradiographic/immunohistochemical analysis

Leucocytes from syngeneic rats were labeled with tritiated thymidine and donor and recipient rats were connected by a bilateral arteriovenous shunt. Based on the time-dependent label index and labeling intensity, it was concluded that Kupffer cells, the resident macrophages of the liver, have a half-life of 12.4 days and originate from monocytes undergoing one mitosis within 8.4 days after immigration into the liver. The labeled cells were easily identified as Kupffer cells by their selective immunoreactivity with the monoclonal antibody Ki-M2R which is specific for phagocytosing macrophages in the rat. The applicability of combined autoradiography/immunohistochemistry for the identification of other poorly defined macrophage subpopulations is shown.     

Verapamil inhibits early acute liver failure through suppressing the NLRP3 inflammasome pathway

Acute liver failure (ALF) is a rare disease characterized by the sudden onset of serious hepatic injury, as manifested by a profound liver dysfunction and hepatic encephalopathy in patients without prior liver disease. In this paper, we aim to investigate whether verapamil, an antagonist of TXNIP, inhibits early ALF through suppressing the NLRP3 inflammasome pathway. Firstly, an ALF mouse model was induced by lipopolysaccharide (LPS)/D-galactosamine (GalN) treatment. The optimal concentration of verapamil in treating early ALF mice was determined followed by investigation on its mechanism in LPS/GalN-induced liver injury. Western blot analysis and co-immunoprecipitation were performed to determine the activation of the TXNIP/NLRP3 inflammasome pathway. Subsequently, overexpression of NLRP3 in mouse liver was induced by transfection with AAV-NRLP3 in vivo and in vitro to identity whether verapamil inhibited early ALF through suppressing the activation of NLRP3 inflammasome. We found that ALF was induced by LPS/GalN in mice but was alleviated by verapamil through a mechanism that correlated with suppression of the NLRP3 inflammasome pathway. Oxidative stress and inflammatory response were induced by intraperitoneal injection of LPS/GalN, but alleviated with injection of verapamil. Overexpression of NLRP3 via AAV in mouse liver in vivo and in vitro reduced the therapeutic effect of verapamil on LPS/GalN-induced ALF. Taken together, the TXNIP antagonist verapamil could inhibit activation of the NLRP3 inflammasome, inflammatory responses and oxidative stress to alleviate LPS/GalN-induced ALF.     

Hyper-activated pro-inflammatory CD16 monocytes correlate with the severity of liver injury and fibrosis in patients with chronic hepatitis B

Background

Extensive mononuclear cell infiltration is strongly correlated with liver damage in patients with chronic hepatitis B virus (CHB) infection. Macrophages and infiltrating monocytes also participate in the development of liver damage and fibrosis in animal models. However, little is known regarding the immunopathogenic role of peripheral blood monocytes and intrahepatic macrophages.

Methodology/Principal Findings

The frequencies, phenotypes, and functions of peripheral blood and intrahepatic monocyte/macrophage subsets were analyzed in 110 HBeAg positive CHB patients, including 32 immune tolerant (IT) carriers and 78 immune activated (IA) patients. Liver biopsies from 20 IA patients undergoing diagnosis were collected for immunohistochemical analysis. IA patients displayed significant increases in peripheral blood monocytes and intrahepatic macrophages as well as CD16⁺ subsets, which were closely associated with serum alanine aminotransferase (ALT) levels and the liver histological activity index (HAI) scores. In addition, the increased CD16⁺ monocytes/macrophages expressed higher levels of the activation marker HLA-DR compared with CD16⁻ monocytes/macrophages. Furthermore, peripheral blood CD16⁺ monocytes preferentially released inflammatory cytokines and hold higher potency in inducing the expansion of Th17 cells. Of note, hepatic neutrophils also positively correlated with HAI scores.

Conclusions

These distinct properties of monocyte/macrophage subpopulations participate in fostering the inflammatory microenvironment and liver damage in CHB patients and further represent a collaborative scenario among different cell types contributing to the pathogenesis of HBV-induced liver disease.