Gene Deconvolution Reveals Aberrant Liver Regeneration and Immune Cell Infiltration in Alcohol-Associated Hepatitis.

BACKGROUND AND AIMS: Acute liver damage causes hepatocyte stress and death, but in chronic liver disease impaired hepatocyte regeneration and immune cell infiltration prevents recovery. While the roles of both impaired liver regeneration and immune infiltration have been studied extensively in chronic liver diseases, the differential contribution of these factors is difficult to assess. APPROACH AND RESULTS: We combined single-cell RNA-sequencing (RNA-seq) data from healthy livers and peripheral immune cells to measure cell proportions in chronic liver diseases. Using bulk RNA-seq data from patients with early alcohol-associated hepatitis, severe AH (sAH), HCV, HCV with cirrhosis, and NAFLD, we performed gene deconvolution to predict the contribution of different cell types in each disease. Patients with sAH had the greatest change in cell composition, with increases in both periportal hepatocytes and cholangiocyte populations. Interestingly, while central vein hepatocytes were decreased, central vein endothelial cells were expanded. Endothelial cells are thought to regulate liver regeneration through WNT signaling. WNT2, important in central vein hepatocyte development, was down in sAH, while multiple other WNTs and WNT receptors were up-regulated. Immunohistochemistry revealed up-regulation of FZD6, a noncanonical WNT receptor, in hepatocytes in sAH. Immune cell populations also differed in disease. In sAH, a specific group of inflammatory macrophages was increased and distinct from the macrophage population in patients with HCV. Network and correlation analyses revealed that changes in the cell types in the liver were highly correlated with clinical liver function tests. CONCLUSIONS: These results identify distinct changes in the liver cell populations in chronic liver disease and illustrate the power of using single-cell RNA-seq data from a limited number of samples in understanding multiple different diseases.

Growth Hormone Stops Excessive Inflammation After Partial Hepatectomy, Allowing Liver Regeneration and Survival Through Induction of H2-Bl/HLA-G.

BACKGROUND AND AIMS: Growth hormone (GH) is important for liver regeneration after partial hepatectomy (PHx). We investigated this process in C57BL/6 mice that express different forms of the GH receptor (GHR) with deletions in key signaling domains. APPROACH AND RESULTS: PHx was performed on C57BL/6 mice lacking GHR (Ghr-/- ), disabled for all GH-dependent Janus kinase 2 signaling (Box1-/- ), or lacking only GH-dependent signal transducer and activator of transcription 5 (STAT5) signaling (Ghr391-/- ), and wild-type littermates. C57BL/6 Ghr-/- mice showed striking mortality within 48 hours after PHx, whereas Box1-/- or Ghr391-/- mice survived with normal liver regeneration. Ghr-/- mortality was associated with increased apoptosis and elevated natural killer/natural killer T cell and macrophage cell markers. We identified H2-Bl, a key immunotolerance protein, which is up-regulated by PHx through a GH-mediated, Janus kinase 2-independent, SRC family kinase-dependent pathway. GH treatment was confirmed to up-regulate expression of the human homolog of H2-Bl (human leukocyte antigen G [HLA-G]) in primary human hepatocytes and in the serum of GH-deficient patients. We find that injury-associated innate immune attack by natural killer/natural killer T cell and macrophage cells are instrumental in the failure of liver regeneration, and this can be overcome in Ghr-/- mice by adenoviral delivery of H2-Bl or by infusion of HLA-G protein. Further, H2-Bl knockdown in wild-type C57BL/6 mice showed elevated markers of inflammation after PHx, whereas Ghr-/- backcrossed on a strain with high endogenous H2-Bl expression showed a high rate of survival following PHx. CONCLUSIONS: GH induction of H2-Bl expression is crucial for reducing innate immune-mediated apoptosis and promoting survival after PHx in C57BL/6 mice. Treatment with HLA-G may lead to improved clinical outcomes following liver surgery or transplantation.

Alternatively activated macrophages promote resolution of necrosis following acute liver injury.

BACKGROUND & AIM: Following acetaminophen (APAP) overdose, acute liver injury (ALI) can occur in patients that present too late for N-acetylcysteine treatment, potentially leading to acute liver failure, systemic inflammation, and death. Macrophages influence the progression and resolution of ALI due to their innate immunological function and paracrine activity. Syngeneic primary bone marrow-derived macrophages (BMDMs) were tested as a cell-based therapy in a mouse model of APAP-induced ALI (APAP-ALI). METHODS: Several phenotypically distinct BMDM populations were delivered intravenously to APAP-ALI mice when hepatic necrosis was established, and then evaluated based on their effects on injury, inflammation, immunity, and regeneration. In vivo phagocytosis assays were used to interrogate the phenotype and function of alternatively activated BMDMs (AAMs) post-injection. Finally, primary human AAMs sourced from healthy volunteers were evaluated in immunocompetent APAP-ALI mice. RESULTS: BMDMs rapidly localised to the liver and spleen within 4 h of administration. Injection of AAMs specifically reduced hepatocellular necrosis, HMGB1 translocation, and infiltrating neutrophils following APAP-ALI. AAM delivery also stimulated proliferation in hepatocytes and endothelium, and reduced levels of several circulating proinflammatory cytokines within 24 h. AAMs displayed a high phagocytic activity both in vitro and in injured liver tissue post-injection. Crosstalk with the host innate immune system was demonstrated by reduced infiltrating host Ly6Chi macrophages in AAM-treated mice. Importantly, therapeutic efficacy was partially recapitulated using clinical-grade primary human AAMs in immunocompetent APAP-ALI mice, underscoring the translational potential of these findings. CONCLUSION: We identify that AAMs have value as a cell-based therapy in an experimental model of APAP-ALI. Human AAMs warrant further evaluation as a potential cell-based therapy for APAP overdose patients with established liver injury. LAY SUMMARY: After an overdose of acetaminophen (paracetamol), some patients present to hospital too late for the current antidote (N-acetylcysteine) to be effective. We tested whether macrophages, an injury-responsive leukocyte that can scavenge dead/dying cells, could serve as a cell-based therapy in an experimental model of acetaminophen overdose. Injection of alternatively activated macrophages rapidly reduced liver injury and reduced several mediators of inflammation. Macrophages show promise to serve as a potential cell-based therapy for acute liver injury.

The Role of Complement in Liver Injury, Regeneration, and Transplantation.

The liver is both an immunologically complex and a privileged organ. The innate immune system is a central player, in which the complement system emerges as a pivotal part of liver homeostasis, immune responses, and crosstalk with other effector systems in both innate and adaptive immunity. The liver produces the majority of the complement proteins and is the home of important immune cells such as Kupffer cells. Liver immune responses are delicately tuned between tolerance to many antigens flowing in from the alimentary tract, a tolerance that likely makes the liver less prone to rejection than other solid organ transplants, and reaction to local injury, systemic inflammation, and regeneration. Notably, complement is a double-edged sword as activation is detrimental by inducing inflammatory tissue damage in, for example, ischemia-reperfusion injury and transplant rejection yet is beneficial for liver tissue regeneration. Therapeutic complement inhibition is rapidly developing for routine clinical treatment of several diseases. In the liver, targeted inhibition of damaged tissue may be a rational and promising approach to avoid further tissue destruction and simultaneously preserve beneficial effects of complement in areas of proliferation. Here, we argue that complement is a key system to manipulate in the liver in several clinical settings, including liver injury and regeneration after major surgery and preservation of the organ during transplantation.

Novel Targets in the Immune Microenvironment of the Hepatic Sinusoids for Treating Liver Diseases.

Immune dysregulation and accumulation of leukocytes is a hallmark of adult chronic liver diseases. Progressive hepatic inflammation can lead to fibrosis and cirrhosis with a high risk of liver failure or hepatocellular cancer (HCC). Recent advances have been made in the treatment of liver disease including the development of highly effective antiviral therapy for hepatitis C and the potential of immunotherapy for HCC. Despite this, the majority of other chronic liver diseases including alcoholic liver disease, fatty liver disease, and cholestatic diseases do not respond to conventional anti-inflammatory therapies. Recent studies defining the organ-specific properties that contribute to resident immune activation and immune cell recruitment from the circulation in these conditions have identified novel hepatic inflammatory pathways, which are now being targeted in clinical trials. Further understanding of how the immune microenvironment is regulated within the liver and how disease-specific mechanisms alter this process will hopefully lead to combination therapies to prevent aberrant inflammation and also promote fibrosis resolution. In this review, we focus on the advances that have been made in identifying key components of the inflammatory pathway including the recognition of danger signals, the recruitment and retention of lymphocytes from the circulation, and the pathways that promote resolution.

B Cell-Mediated Maintenance of Cluster of Differentiation 169-Positive Cells Is Critical for Liver Regeneration.

The liver has an extraordinary capacity to regenerate through activation of key molecular pathways. However, central regulators controlling liver regeneration remain insufficiently studied. Here, we show that B cell-deficient animals failed to induce sufficient liver regeneration after partial hepatectomy (PHx). Consistently, adoptive transfer of B cells could rescue defective liver regeneration. B cell-mediated lymphotoxin beta production promoted recovery from PHx. Absence of B cells coincided with loss of splenic cluster of differentiation 169-positive (CD169+ ) macrophages. Moreover, depletion of CD169+ cells resulted in defective liver regeneration and decreased survival, which was associated with reduced hepatocyte proliferation. Mechanistically, CD169+ cells contributed to liver regeneration by inducing hepatic interleukin-6 (IL-6) production and signal transducer and activator of transcription 3 activation. Accordingly, treatment of CD169+ cell-depleted animals with IL-6/IL-6 receptor rescued liver regeneration and severe pathology following PHx. Conclusion: We identified CD169+ cells to be a central trigger for liver regeneration, by inducing key signaling pathways important for liver regeneration.

Intrahepatic T-Cell Receptor ß Immune Repertoire Is Essential for Liver Regeneration.

T lymphocytes synergize with the cellular immune system to promote hepatocyte regeneration. The T-cell receptor (TCR) immune repertoire is closely associated with the host immune response and regenerative proliferation. High-throughput sequencing of TCR provides deep insight into monitoring the immune microenvironment. Here, we aimed to determine the role of the TCRß immune repertoire in liver regeneration (LR). We investigated hepatic regeneration in TCRß chain-deficient (tcrb-/- ) mice by two-thirds partial hepatectomy (PHx) method. Our results demonstrated that tcrb-/- mice revealed a reduced capacity for LR, which was characterized by impaired hepatocyte proliferation and enhanced hepatocyte apoptosis. Dysregulation of inflammatory signaling activation and inflammatory factors was observed in regenerated tcrb-/- livers. Simultaneously, significantly altered immunocyte levels and aberrant cytokine levels were observed during hepatic regeneration. In addition, we first determined the profile of the TCRß immune repertoire during LR, indicating that PHx resulted in remarkably lower TCRß diversity in intrahepatic T lymphocytes. Conclusion: Taken together, our data suggest that TCRß deficiency gives a rise to aberrant intrahepatic immune microenvironment that impairs LR, and the TCRß reconstitution is required for hepatic immunocyte recruitment and activation during LR.

IL-22 in hepatocyte's survival of Pakistani patients with end stage liver disease: an insight into IL 22 mediated hepato-regenerative pathway.

Hepatitis is the principal cause of hepatocellular carcinoma (HCC) and decompensated cirrhosis. HCC is amongst the leading causes of deaths worldwide. Current therapeutic options have proven to be unsuccessful in treating this disease due to multifactorial nature of the disease. The present study was designed to investigate the role of IL-22 mediated survival of hepatocytes during cirrhosis and HCC. Resected/explanted liver tissue samples of patients with End Stage Liver Disease were obtained from Hepato-Pancreato-Biliary Liver Transplant Unit of Sheikh Zayed Hospital, Lahore, Pakistan. Qualitative expression of IL-22, SOCS3, and IL-22 induced anti-apoptotic protein, B-cell lymphoma extra-large (Bcl-xL), were evaluated by Immunohistochemical analysis (IHC). The IHC analysis revealed significantly high expression of IL-22, SOCS3, and Bcl-xL within explanted livers of HCC patients. Overall, the expression of SOCS3 was higher than any other protein, and the expression of all proteins showed significant variation in different group of patients based on clincopathological features. The results of the current study indicated that IL-22 mediated JAK-STAT pathway i.e. liver regeneration and healing is dependent on the disease progression and type of agent responsible for causing the infection in the first place. However, quantitative analysis of these factors in future can provide further evidence of the role of this pathway in HCC for development of anti-HCC therapies.

B-Cell Activating Factor Enhances Hepatocyte-Driven Angiogenesis via B-Cell CLL/Lymphoma 10/Nuclear Factor-KappaB Signaling during Liver Regeneration.

B-cell activating factor (BAFF) is found to be associated with the histological severity of nonalcoholic steatohepatitis (NASH). BAFF was also found to have a protective role in hepatic steatosis via down regulating the expression of steatogenesis genes and enhancing steatosis in hepatocytes through BAFF-R. However, the roles of BAFF during liver regeneration are not well defined. In this study, C57/B6 mice with 70% partial hepatectomy were used as a liver regeneration model. BAFF expression was determined by enzyme immunoassay, and anti-BAFF-neutralizing antibodies were administered to confirm the effects of BAFF on liver regeneration. Western blotting, immunohistochemistry, and florescence staining determined the expression of B-cell CCL/lymphoma 10 (BCL10). The angiogenesis promoting capability was evaluated after the transfection of cells with siRNA targeting BCL10 expression, and the role of NF-κB was assessed. The results revealed that the BAFF and BCL10 levels were upregulated after partial hepatectomy. Treatment with anti-BAFF-neutralizing antibodies caused death in mice that were subjected to 70% partial hepatectomy within 72 h. In vitro, recombinant BAFF protein did not enhance hepatocyte proliferation; however, transfection with BCL10 siRNA arrested hepatocytes at the G2/M phase. Interestingly, conditioned medium from BAFF-treated hepatocytes enhanced angiogenesis and endothelial cell proliferation. Moreover, Matrix metalloproteinase-9 (MMP-9), Fibroblast growth factor 4 (FGF4), and Interleukin-8 (IL-8) proteins were upregulated by BAFF through BCL10/NF-κB signaling. In mice that were treated with anti-BAFF-neutralizing antibodies, the microvessel density (MVD) of the remaining liver tissues and liver regeneration were both reduced. Taken together, our study demonstrated that an increased expression of BAFF and activation of BCL10/NF-κB signaling were involved in hepatocyte-driven angiogenesis and survival during liver regeneration.

Sex-dependent differences in inflammatory responses during liver regeneration in a murine model of acute liver injury.

A sexual dimorphism in liver inflammation and repair was previously demonstrated. Its cellular dissection in the course of acute liver injury (ALI) was explored. BALB/c mice were treated with carbon tetrachloride (CCl4) by intraperitoneal injection and killed after 3, 5, and 8 days. Histological and hepatic cell population analyses were performed. The correlation between androgen receptor (AR) expression and liver recruited inflammatory cells was investigated by treatment with the AR antagonist flutamide. Additionally, patients with a diagnosis of drug induced liver injury (DILI) were included in the study, with a particular focus on gender dimorphism in circulating monocytes. A delayed resolution of necrotic damage and a higher expression of proinflammatory cytokines were apparent in male mice along with a slower recruitment of inflammatory monocytes. F4/80+CD11b+ macrophages and CD11bhighGr-1high monocytes expressed AR and were recruited later in male compared with female livers after CCl4 treatment. Moreover, CD11bhighAR+Gr-1high recruitment was negatively modulated by flutamide in males. Analysis of DILI patients showed overall a significant reduction in circulating mature monocytes compared with healthy subjects. More interestingly, male patients had higher numbers of immature monocytes compared with female patients.A stronger cytotoxic tissue response was correlated with an impaired recruitment of CD11bhighAR+Gr-1high cells and F4/80+CD11b+ macrophages in the early inflammatory phase under AR signaling. During DILI, a dimorphic immune response was apparent, characterized by a massive recruitment of monocytes to the liver both in males and females, but only in males was this recruitment sustained by a turnover of immature monocytes.

Systems Analysis of the Complement-Induced Priming Phase of Liver Regeneration.

Liver regeneration is a well-orchestrated process in the liver that allows mature hepatocytes to reenter the cell cycle to proliferate and replace lost or damaged cells. This process is often impaired in fatty or diseased livers, leading to cirrhosis and other deleterious phenotypes. Prior research has established the role of the complement system and its effector proteins in the progression of liver regeneration; however, a detailed mechanistic understanding of the involvement of complement in regeneration is yet to be established. In this study, we have examined the role of the complement system during the priming phase of liver regeneration through a systems level analysis using a combination of transcriptomic and metabolomic measurements. More specifically, we have performed partial hepatectomy on mice with genetic deficiency in C3, the major component of the complement cascade, and collected their livers at various time points. Based on our analysis, we show that the C3 cascade activates c-fos and promotes the TNF-α signaling pathway, which then activates acute-phase genes such as serum amyloid proteins and orosomucoids. The complement activation also regulates the efflux and the metabolism of cholesterol, an important metabolite for cell cycle and proliferation. Based on our systems level analysis, we provide an integrated model for the complement-induced priming phase of liver regeneration.

[CMV associated acute liver failure in a patient receiving tocilizumab for systemic lupus erythematosus]. / CMV-assoziiertes Leberversagen bei einer Patientin mit systemischem Lupus erythematodes unter Immunsuppression mit Tocilizumab.

A 41-year-old female patient was admitted because of febrile jaundice and acute liver failure. The quick and the bilirubin were 21 % and 258 µmol/l, and there was hepatic encephalopathy I°. AST and AP had a maximum of 612 and 215 U/l. Despite a strong left shift in the differential, the CRP had a maximum of 15 mg/l. Because of an atypically presenting systemic lupus erythematosus, she had been treated with Azathioprine, steroids and Tocilizumab until 12 days before admission. The diagnostic workup revealed CMV hepatitis and necrotizing hepatopathy, which was interpreted as toxic hepatitis. At the time of liver biopsy, on day 3 after admission, staining for Ki-67 indicated strong regenerative activity in the liver. Treatment with Valgancyclovir, antibiotics and steroids led to early recovery from liver failure. The case differs from the few described cases of severe acute liver injury related to Tocilizumab. Apparently, the combined immunosuppression (steroid, Azathioprine and Tocilizumab) led to acute liver failure secondary to CMV hepatitis and acute toxic hepatitis, which may have been aggravated by transiently impaired liver regeneration. On the other hand, stimulated liver regeneration was proven by histology despite previous IL6 blockage by Tocilizumab.

Oral ampicillin inhibits liver regeneration by breaking hepatic innate immune tolerance normally maintained by gut commensal bacteria.

UNLABELLED: Commensal bacteria have been proposed to play a role in liver repair after partial (67%) hepatectomy. However, the underlying immune mechanisms remain elusive. Here, we show that liver regeneration was impaired in antibiotic (Atb) water-treated mice and this impairment strongly correlated with commensal bacterial load. Among the various Atbs used in our cocktail, ampicillin-sensitive commensal bacterial was associated with normal liver regeneration. The number of CD1d-dependent natural killer T (NKT) cells in Atb-treated hepatectomized mice was markedly increased, and these NKT cells were functionally overactivated to produce higher interferon-γ. Deficiency of NKT cells or antibody blockade of the CD1d-NKT interaction increased hepatocyte proliferation, which improved liver regeneration. Importantly, an increased number of Kupffer cells were observed in Atb-treated mice, and these Kupffer cells produced higher interleukin-12, which then functioned to activate hepatic NKT cells. Interleukin-12p40 deficiency or treatment with an anti-interleukin-12 antibody significantly inhibited NKT cell overactivation and recovered liver regeneration in Atb-treated mice. CONCLUSION: Commensal bacteria play a critical role in maintaining Kupffer cells in a tolerant state, preventing subsequent NKT cell overactivation during liver regeneration. Moreover, our data suggest that long-term Atb use, which can impair the gut microbiota, may influence liver function by retarding liver regeneration.

IL-1ß siRNA adenovirus benefits liver regeneration by improving mesenchymal stem cells survival after acute liver failure.

UNLABELLED:   BACKGROUND: Uncontrolled hapatic inflammatory response is regarded as the primary pathological mechanism of acute liver failure and impairs the regeneration of hepatocytes and stem cell grafts. Interleukin-1 plays a key role for activating immune and inflammatory response. Recently, siRNA has made quite a few progresses in treating inflammatory response. AIM: To assess the effect of IL-1? siRNA adenovirus on MSC and the therapeutic effect of MSC combined with IL-1? siRNA adenovirus in ALF. MATERIAL AND METHODS: We implanted MSC or/and IL-1? siRNA adenovirus via the tail vein, using CCl4-induced ALF in a mice model. Mice were sacrificed at different time points. Blood samples and liver tissues were collected. Hepatic injury, liver regeneration, cytokines (CXCL1, IL-1?, IL-10, IL-6, VEGF and HGF), animal survival and vital MSC were assessed after cell transplantation. RESULTS: MSC combined with IL-1? siRNA reduced the inflammatory levels and prevented liver failure. These animals administrated with MSC and IL-1? siRNA also exhibited improved liver regeneration and increased survival rates. Immunohistochemistry and fluorescence microscopy revealed the number of vital MSC in ALF + MSC + IL-1? siRNA group were significantly more than that in ALF + MSC group. CONCLUSION: IL-1? siRNA adenovirus could enhance MSC ability of tissue regeneration through increasing its survival rate. Accordingly, combination of IL-1? siRNA adenovirus and MSC had a synergistic effect on acute liver failure.

Monocyte-endothelial cell interactions in the regulation of vascular sprouting and liver regeneration in mouse.

BACKGROUND & AIMS: Regeneration of the hepatic mass is crucial to liver repair. Proliferation of hepatic parenchyma is intimately dependent on angiogenesis and resident macrophage-derived cytokines. However the role of circulating monocyte interactions in vascular and hepatic regeneration is not well-defined. We investigated the role of these interactions in regeneration in the presence and absence of intact monocyte adhesion. METHODS: Partial hepatectomy was performed in wild-type mice and those lacking the monocyte adhesion molecule CD11b. Vascular architecture, angiogenesis and macrophage location were analyzed in the whole livers using simultaneous angiography and macrophage staining with fluorescent multiphoton microscopy. Monocyte adhesion molecule expression and sprouting-related pathways were evaluated. RESULTS: Resident macrophages (Kupffer cells) did not migrate to interact with vessels whereas infiltrating monocytes were found adjacent to sprouting points. Infiltrated monocytes colocalized with Wnt5a, angiopoietin 1 and Notch-1 in contact points and commensurate with phosphorylation and disruption of VE-cadherin. Mice deficient in CD11b showed a severe reduction in angiogenesis, liver mass regeneration and survival following partial hepatectomy, and developed unstable and leaky vessels that eventually produced an aberrant hepatic vascular network and Kupffer cell distribution. CONCLUSIONS: Direct vascular interactions of infiltrating monocytes are required for an ordered vascular growth and liver regeneration. These outcomes provide insight into hepatic repair and new strategies for hepatic regeneration.

Opposing effects of prednisolone treatment on T/NKT cell- and hepatotoxin-mediated hepatitis in mice.

UNLABELLED: Prednisolone is a corticosteroid that has been used to treat inflammatory liver diseases such as autoimmune hepatitis and alcoholic hepatitis. However, the results have been controversial, and how prednisolone affects liver disease progression remains unknown. In the current study we examined the effect of prednisolone treatment on several models of liver injury, including T/NKT cell hepatitis induced by concanavalin A (ConA) and α-galactosylceramide (α-GalCer), and hepatotoxin-mediated hepatitis induced by carbon tetrachloride (CCl4 ) and/or ethanol. Prednisolone administration attenuated ConA- and α-GalCer-induced hepatitis and systemic inflammatory responses. Treating mice with prednisolone also suppressed inflammatory responses in a model of hepatotoxin (CCl4 )-induced hepatitis, but surprisingly exacerbated liver injury and delayed liver repair. In addition, administration of prednisolone also enhanced acetaminophen-, ethanol-, or ethanol plus CCl4 -induced liver injury. Immunohistochemical and flow cytometric analyses demonstrated that prednisolone treatment inhibited hepatic macrophage and neutrophil infiltration in CCl4 -induced hepatitis and suppressed their phagocytic activities in vivo and in vitro. Macrophage and/or neutrophil depletion aggravated CCl4 -induced liver injury and impeded liver regeneration. Finally, conditional disruption of glucocorticoid receptor in macrophages and neutrophils abolished prednisolone-mediated exacerbation of hepatotoxin-induced liver injury. CONCLUSION: Prednisolone treatment prevents T/NKT cell hepatitis but exacerbates hepatotoxin-induced liver injury by inhibiting macrophage- and neutrophil-mediated phagocytic and hepatic regenerative functions. These findings may not only increase our understanding of the steroid treatment mechanism but also help us to better manage steroid therapy in liver diseases.

Constitutive Activation of the Nlrc4 Inflammasome Prevents Hepatic Fibrosis and Promotes Hepatic Regeneration after Partial Hepatectomy.

TThe molecular mechanisms responsible for the development of hepatic fibrosis are not fully understood. The Nlrc4 inflammasome detects cytosolic presence of bacterial components, activating inflammatory cytokines to facilitate clearance of pathogens and infected cells. We hypothesized that low-grade constitutive activation of the Nlrc4 inflammasome may lead to induced hepatocyte proliferation and prevent the development of hepatic fibrosis. The gene of Nlrc4 contains two single nucleotide polymorphisms (SNPs), one located within the Nlrc4 promoter and one contained within exon 5. These SNPs regulate Nlrc4 gene transcription and activation as measured through gene reporter assays and IL-1ß secretion. The 17C-6 mice have increased IL-1ß in plasma after chronic carbon tetrachloride (CCl4) administration compared to B6 mice. After two-thirds partial hepatectomy (2/3PH) 17C-6 mice have earlier restoration of liver mass with greater cyclin D1 protein and BrdU incorporation compared to B6 mice at several time points. These data reveal mild constitutive activation of the Nlrc4 inflammasome as the results of two SNPs, which leads to the stimulation of hepatocyte proliferation. The increased liver regeneration induces rapid liver mass recovery after hepatectomy and may prevent the development of hepatotoxin-induced liver fibrosis.

Expression and distribution of immunoglobulin G in the normal liver, hepatocarcinoma and postpartial hepatectomy liver.

The liver has the extraordinary properties of regeneration and immune tolerance; however, the mechanisms governing these abilities are poorly understood. To address these questions, we examined the possible expression of immunoglobulins in the human and rat liver and the relationship of IgG expression to hepatocyte proliferation, metastasis, apoptosis and immune tolerance. Immunohistochemistry, in situ hybridization, laser-guided microdissection and reverse transcription-PCR were performed to examine the expression of IgG in normal human and rat liver, severe combined immunodeficient mouse (SCID) liver and human liver cancers and corresponding cell lines. Small interfering RNA (siRNA) was transfected into cultured hepatocarcinoma cells to downregulate the expression of IgG heavy chain genes. Cell proliferation and apoptosis were assayed with flow cytometry. Cell metastasis was assayed with a Transwell cell assay. Partial hepatectomy (70%) was performed in rats to examine the relationship between hepatocyte IgG and hepatocyte proliferation. IgG, together with essential enzymes for its synthesis, were expressed in the cytoplasm of hepatocytes of normal adult human and hepatoma patients and rat livers, SCID mouse liver and BRL-3A, L-02 and HepG-2 cell lines. Downregulation of IgG inhibited cell proliferation and metastasis and promoted apoptosis. Postsurgery livers expressed significantly more IgG than the livers before surgery and decreased to the original levels when hepatocytes stopped regeneration. IgA and IgM but not IgD and IgE were also positive in hepatocytes. Our findings demonstrate that normal and malignant hepatocytes are capable of synthesizing immunoglobulin, which has important roles in hepatocyte proliferation, apoptosis and cancer growth with profound clinical implications.

Augmenter of liver regeneration attenuates inflammatory response in the postischemic mouse liver in vivo.

BACKGROUND: Augmenter of Liver Regeneration (ALR), a protein synthesized in the liver is suggested to be protective against oxidative stress-induced cell death. Hepatic ischemia-reperfusion (I/R) injury is triggered by reactive oxygen species. Here, we tested the hypothesis that ALR attenuates hepatic I/R injury in vivo. METHODS: C57BL6 mice were subjected to warm hepatic ischemia for 90 min. Either recombinant ALR (100 µg/kg) or vehicle were administered to mice prior ischemia. During reperfusion, neutrophil and CD4+ T cell migration and sinusoidal perfusion were analyzed using intravital microscopy. Alanine aminotransferase-aspartate aminotransferase (plasma) and caspase-3 (tissue) activities were determined as markers of hepatocellular necrotic and apoptotic injury. RESULTS: Hepatic I/R led to dramatic enhancement of neutrophil and CD4+ T cell recruitment in hepatic microvessels, sinusoidal perfusion failure, and strong elevation of aspartate aminotransferase-alanine aminotransferase and caspase-3 activities. During early reperfusion (60 min), the pretreatment with ALR improved postischemic perfusion failure (P < 0.05) and attenuated liver enzyme activities. Recruitment of CD4+ T cells, but not of neutrophils was attenuated. After 240 min of reperfusion, the protective effect of ALR was stronger, since the liver enzyme activity, perfusion failure, and leukocyte influx were significantly attenuated. As shown by the measurement of caspase-3 activity, postischemic apoptosis was reduced in the ALR-treated group. CONCLUSIONS: Our in vivo data show that ALR has a therapeutic potential against postischemic liver injury. As a mechanism, we suggest a direct protective effect of ALR on apoptotic and necrotic death of hepatocytes and an attenuation of inflammatory cell influx into the postischemic tissue.

A complement-IL-4 regulatory circuit controls liver regeneration.

The involvement of IL-4 in liver regeneration has not yet been recognized. In this article, we show that IL-4, produced by NKT cells that accumulate in regenerating livers after partial hepatectomy, contributes to this process by regulating the activation of complement after liver resection in mice. The mechanism of this regulation was associated with the maintenance of an appropriate level of IgM in mouse blood, because IgM deposited in liver parenchyma most likely initiated complement activation during liver regeneration. By controlling complement activation, IL-4 regulated the induction of IL-6, thereby influencing a key pathway involved in regenerating liver cell proliferation and survival. Furthermore, the secretion of IL-4 was controlled by complement through the recruitment of NKT cells to regenerating livers. Our study thus reveals the existence of a regulatory feedback mechanism involving complement and IL-4 that controls liver regeneration.