ABSTRACT
Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin (IL)-6 family that contributes to the progression of chronic liver disease. Here we investigated the role of OSM in the development and progression of hepatocellular carcinoma (HCC) in non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH). The role of OSM was investigated in (1) selected cohorts of NAFLD/NASH HCC patients, (2) liver cancer cells exposed to human recombinant OSM or stably transfected to overexpress human OSM, (3) murine HCC xenografts, and (4) a murine NASH-related model of hepatic carcinogenesis. OSM was found to be selectively overexpressed in HCC cells of NAFLD/NASH patients, depending on tumor grade. OSM serum levels, barely detectable in patients with simple steatosis or NASH, were increased in patients with cirrhosis and more evident in those carrying HCC. In this latter group, OSM serum levels were significantly higher in the subjects with intermediate/advanced HCCs and correlated with poor survival. Cell culture experiments indicated that OSM upregulation in hepatic cancer cells contributes to HCC progression by inducing epithelial-to-mesenchymal transition and increased invasiveness of cancer cells as well as by inducing angiogenesis, which is of critical relevance. In murine xenografts, OSM overexpression was associated with slower tumor growth but an increased rate of lung metastases. Overexpression of OSM and its positive correlation with the angiogenic switch were also confirmed in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Consistent with this, analysis of liver specimens from human NASH-related HCCs with vascular invasion showed that OSM was expressed by liver cancer cells invading hepatic vessels. In conclusion, OSM upregulation appears to be a specific feature of HCC arising on a NAFLD/NASH background, and it correlates with clinical parameters and disease outcome. Our data highlight a novel pro-carcinogenic contribution for OSM in NAFLD/NASH, suggesting a role of this factor as a prognostic marker and a putative potential target for therapy. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Non-alcoholic Fatty Liver Disease , Oncostatin M , Animals , Carcinogenesis , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Mice , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/pathologyABSTRACT
Annexin A1 (AnxA1) is an important effector in the resolution of inflammation which is involved in modulating hepatic inflammation in nonalcoholic steatohepatitis (NASH). In the present study, we have investigated the possible effects of treatment with AnxA1 for counteracting the progression of experimental NASH. NASH was induced in C57BL/6 mice by feeding methionine-choline deficient (MCD) or Western diets (WDs) and the animals were treated for 4-6 weeks with human recombinant AnxA1 (hrAnxA1; 1 µg, daily IP) or saline once NASH was established. In both experimental models, treatment with hrAnxA1 improved parenchymal injury and lobular inflammation without interfering with the extension of steatosis. Furthermore, administration of hrAnxA1 significantly attenuated the hepatic expression of α1-procollagen and TGF-ß1 and reduced collagen deposition, as evaluated by collagen Sirius Red staining. Flow cytometry and immunohistochemistry showed that hrAnxA1 did not affect the liver recruitment of macrophages, but strongly interfered with the formation of crown-like macrophage aggregates and reduced their capacity of producing pro-fibrogenic mediators like osteopontin (OPN) and galectin-3 (Gal-3). This effect was related to an interference with the acquisition of a specific macrophage phenotype characterized by the expression of the Triggering Receptor Expressed on Myeloid cells 2 (TREM-2), CD9 and CD206, previously associated with NASH evolution to cirrhosis. Collectively, these results indicate that, beside ameliorating hepatic inflammation, AnxA1 is specifically effective in preventing NASH-associated fibrosis by interfering with macrophage pro-fibrogenic features. Such a novel function of AnxA1 gives the rationale for the development of AnxA1 analogs for the therapeutic control of NASH evolution.
Subject(s)
Annexin A1 , Non-alcoholic Fatty Liver Disease , Animals , Annexin A1/metabolism , Disease Models, Animal , Fibrosis , Inflammation/pathology , Liver/metabolism , Liver Cirrhosis/metabolism , Methionine , Mice , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/metabolismABSTRACT
Mechanisms underlying progression of nonalcoholic fatty liver disease (NAFLD) are still incompletely characterized. Hypoxia and hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of chronic liver diseases, but the actual role of HIF-2α in the evolution of NAFLD has never been investigated in detail. In this study, we show that HIF-2α is selectively overexpressed in the cytosol and the nuclei of hepatocytes in a very high percentage (>90%) of liver biopsies from a cohort of NAFLD patients at different stages of the disease evolution. Similar features were also observed in mice with steatohepatitis induced by feeding a methionine/choline-deficient diet. Experiments performed in mice carrying hepatocyte-specific deletion of HIF-2α and related control littermates fed either a choline-deficient L-amino acid-defined or a methionine/choline-deficient diet showed that HIF-2α deletion ameliorated the evolution of NAFLD by decreasing parenchymal injury, fatty liver, lobular inflammation, and the development of liver fibrosis. The improvement in NAFLD progression in HIF-2α-deficient mice was related to a selective down-regulation in the hepatocyte production of histidine-rich glycoprotein (HRGP), recently proposed to sustain macrophage M1 polarization. In vitro experiments confirmed that the up-regulation of hepatocyte HRGP expression was hypoxia-dependent and HIF-2α-dependent. Finally, analyses performed on specimens from NAFLD patients indicated that HRGP was overexpressed in all patients showing hepatocyte nuclear staining for HIF-2α and revealed a significant positive correlation between HIF-2α and HRGP liver transcript levels in these patients. CONCLUSIONS: These results indicate that hepatocyte HIF-2α activation is a key feature in both human and experimental NAFLD and significantly contributes to the disease progression through the up-regulation of HRGP production. (Hepatology 2018;67:2196-2214).
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/physiology , Hepatocytes/metabolism , Non-alcoholic Fatty Liver Disease/etiology , Proteins/metabolism , Animals , Cells, Cultured , Disease Progression , Humans , Male , MiceABSTRACT
PURPOSE: Endotoxin is a component of the outer membrane of gram-negative bacteria that live in the intestine. Endotoxinemia is reported in non-alcoholic fatty liver disease and in cirrhotic patients, causing various biological and clinical effects in the host. It is not known whether endotoxinemia occurs in chronic hepatitis C patients (CHC), therefore we evaluated the occurrence of endotoxinemia and its effect on inflammation, liver damage, insulin resistance (IR) and atherosclerosis. METHODS: Consecutive CHC patients assessed by liver biopsy were enrolled. Endotoxinemia was evaluated by LAL test. IR was estimated by HOMA-IR. Serum TNF-α, IL-8, adiponectin and MCP-1 were measured with ELISA tests. Oxidative stress was estimated by circulating IgG against malondialdehyde adducts with human serum albumin (MDA-HAS). Carotid atherosclerosis was assessed by ultrasonography. RESULTS: Endotoxinemia was found in 60% of the 126 patients enrolled. A serum level-dependent association between endotoxinemia, steatosis (p < 0.001) and HOMA-IR (p < 0.006) was observed. Patients with endotoxinemia showed significant increase in TNF-α and IL8 levels. TNF-α correlated with steatosis (p < 0.001) and HOMA-IR (p < 0.03), whereas IL8 correlated with steatosis (p = <0.001), TNF-α (p < 0.04) and atherosclerosis (p < 0.01). The highest levels of endotoxinemia were associated with oxidative stress and a higher prevalence of carotid atherosclerosis. Multivariate logistic regression analysis showed that the independent factors associated with endotoxinemia were hepatic steatosis, HOMA-IR, IL8 and MDA-HAS. CONCLUSIONS: Endotoxinemia occurs with high frequency in CHC patients and contributes to the development of hepatic steatosis, IR and atherosclerosis through increased pro-inflammatory cytokines and oxidative stress. Anti-endotoxin treatment could be of clinical relevance.
Subject(s)
Atherosclerosis/microbiology , Endotoxemia/epidemiology , Fibrosis/microbiology , Hepatitis C, Chronic/complications , Inflammation/microbiology , Insulin Resistance , Oxidative Stress , Adolescent , Adult , Aged , Chemokines/metabolism , Cytokines/metabolism , Endotoxemia/complications , Endotoxemia/microbiology , Fatty Liver/microbiology , Female , Humans , Male , Middle Aged , Prevalence , Young AdultABSTRACT
The chemokine fractalkine (CX3CL1) and its receptor CX3CR1 are known to mediate leukocyte chemotaxis, adhesion and survival. In the liver, CX3CR1 is expressed on multiple cell types including monocytes and dendritic cells. However, the function of CX3CR1 on hepatic dendritic cells (HDCs) is still poorly understood. In this study, we investigated the role of CX3CR1 on mouse HDCs during homeostasis and following acute liver injury. At homeostasis, CX3CR1-expression was detected among CD11b+/CD103- type 2 myeloid HDCs (mHDCs) and these cells were characterized by the production of IL-10. Mice treatment with the hepatotoxic agent CCl4 up-regulated liver IL-10 expression and stimulated the expansion of CX3CR1+ mHDCs which also showed a more mature phenotype. The absence of CX3CR1 in naïve CX3CR1gfp/gfp mice specifically reduced the CD11b+/IL-10+ mHDCs as compared to CX3CR1-proficient animals (CX3CR1+/gfp). Following CCl4 poisoning, the liver recruitment and maturation of CD11b+ mHDCs was significantly attenuated in CX3CR1gfp/gfp mice. Furthermore, these mice suffered more severe hepatic injury and inflammation than CX3CR1+/gfp mice and showed a delated recovery from liver damage. Such a worsening of liver injury in CX3CR1gfp/gfp mice was associated with an impaired up-regulation of hepatic IL-10 expression and a lower number of IL-10 producing CD11b+ mHDCs. Consistently, IL-10 inactivation enhanced hepatic injury and inflammation in CX3CR1+/gfp mice receiving CCl4 Altogether, these data indicate a novel role of the CX3CL1/CX3CR1 axis in liver type 2 mHDC functions, pointing out the importance of CX3CR1 in promoting IL-10-mediated anti-inflammatory actions of HDCs.
ABSTRACT
To test the significance of lipid peroxidation in the development of alcoholic liver injury, an ethanol (EtOH) liquid diet was fed to male 129/SvJ mice (wild-type, WT) and glutathione S-transferase A4-4-null (GSTA4-/-) mice for 40 days. GSTA4-/- mice were crossed with peroxisome proliferator-activated receptor-α-null mice (PPAR-α-/-), and the effects of EtOH in the resulting double knockout (dKO) mice were compared with the other strains. EtOH increased lipid peroxidation in all except WT mice (P < 0.05). Increased steatosis and mRNA expression of the inflammatory markers CXCL2, tumor necrosis factor-α (TNF-α), and α-smooth muscle actin (α-SMA) were observed in EtOH GSTA4-/- compared with EtOH WT mice (P < 0.05). EtOH PPAR-α-/- mice had increased steatosis, serum alanine aminotransferase (ALT), and hepatic CD3+ T cell populations and elevated mRNA encoding CD14, CXCL2, TNF-α, IL-6, CD138, transforming growth factor-ß, platelet-derived growth factor receptor-ß (PDGFR-ß), matrix metalloproteinase (MMP)-9, MMP-13, α-SMA, and collagen type 1 compared with EtOH WT mice. EtOH-fed dKO mice displayed elevation of periportal hepatic 4-hydroxynonenal adducts and serum antibodies against malondialdehyde adducts compared with EtOH feeding of GSTA4-/-, PPAR-α-/-, and WT mice (P < 0.05). ALT was higher in EtOH dKO mice compared with all other groups (P < 0.001). EtOH-fed dKO mice displayed elevated mRNAs for TNF-α and CD14, histological evidence of fibrosis, and increased PDGFR, MMP-9, and MMP-13 mRNAs compared with the EtOH GSTA4-/- or EtOH PPAR-α-/- genotype (P < 0.05). These findings demonstrate the central role lipid peroxidation plays in mediating progression of alcohol-induced necroinflammatory liver injury, stellate cell activation, matrix remodeling, and fibrosis.
Subject(s)
Aldehydes/metabolism , Glutathione Transferase/metabolism , Lipid Peroxidation , Liver Diseases, Alcoholic/metabolism , PPAR alpha/metabolism , Actins/genetics , Actins/metabolism , Alanine Transaminase/blood , Aldehydes/immunology , Animals , Antibodies/blood , Chemokine CXCL2/genetics , Chemokine CXCL2/metabolism , Cytokines/genetics , Cytokines/metabolism , Fibrosis/metabolism , Gene Deletion , Glutathione Transferase/genetics , Lipopolysaccharide Receptors/genetics , Lipopolysaccharide Receptors/metabolism , Liver/metabolism , Liver/pathology , Liver Diseases, Alcoholic/immunology , Male , Matrix Metalloproteinases/genetics , Matrix Metalloproteinases/metabolism , Mice , PPAR alpha/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Platelet-Derived Growth Factor beta/genetics , Receptor, Platelet-Derived Growth Factor beta/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
UNLABELLED: Previous studies have shown that human nonalcoholic steatohepatitis (NASH) is often associated with the presence of circulating antibodies against protein adducted by lipid peroxidation products. Here we used the methionine-choline deficient (MCD) model of NASH to characterize the possible involvement of adaptive immunity in NASH. In mice fed up to 8 weeks with the MCD diet the extension of liver injury and lobular inflammation paralleled the development of immunoglobulin G (IgG) against malonyldialdehyde (MDA) and 4-hydroxynonenal (4-HNE)-derived antigens as well as with the hepatic recruitment of CD4(+) and CD8(+) T-lymphocytes responsive to the same antigens. Moreover, in these animals the individual IgG reactivity against MDA-adducts positively correlated with transaminase release and hepatic tumor necrosis factor alpha (TNF-α) expression. To substantiate the role of immune responses triggered by oxidative stress in the progression of NASH, mice were immunized with MDA-adducted bovine serum albumin (MDA-BSA) before feeding the MCD diet. MDA-BSA immunization did not affect control mice livers, but further stimulated transaminase release, lobular inflammation, and the hepatic expression of proinflammatory cytokine in MCD-fed mice. The increased severity of NASH in immunized MCD-fed mice involved liver recruitment and the T helper (Th)-1 activation of CD4(+) T cells that, in turn, further stimulated macrophage M1 responses. Moreover, hepatic fibrosis was also evident in these animals in relation with an IL-15-mediated increase of natural killer T-cells (NKT) and the up-regulation in liver production of osteopontin by NKT cells and hepatic macrophages. CONCLUSION: These results indicate that oxidative stress can contribute to the progression of NASH by stimulating both humoral and cellular immune responses, pointing to the possible role of adaptive immunity in the pathogenesis of the disease.
Subject(s)
Adaptive Immunity/immunology , Fatty Liver/immunology , Fatty Liver/metabolism , Oxidative Stress/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , Disease Models, Animal , Disease Progression , Humans , Killer Cells, Natural/immunology , Lipid Peroxidation/immunology , Liver/immunology , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Non-alcoholic Fatty Liver DiseaseABSTRACT
UNLABELLED: Annexin A1 (AnxA1) is an effector of the resolution of inflammation and is highly effective in terminating acute inflammatory responses. However, its role in chronic settings is less investigated. Because changes in AnxA1 expression within adipose tissue characterize obesity in mice and humans, we queried a possible role for AnxA1 in the pathogenesis of nonalcoholic steatohepatitis (NASH), a disease commonly associated with obesity. NASH was induced in wild-type (WT) and AnxA1 knockout (AnxA1 KO) C57BL/6 mice by feeding a methionine-choline deficient (MCD) diet up to 8 weeks. In MCD-fed WT mice, hepatic AnxA1 increased in parallel with progression of liver injury. This mediator was also detected in liver biopsies from patients with NASH and its degree of expression inversely correlated with the extent of fibrosis. In both humans and rodents, AnxA1 production was selectively localized in liver macrophages. NASH in AnxA1 KO mice was characterized by enhanced lobular inflammation resulting from increased macrophage recruitment and exacerbation of the M1 phenotype. Consistently, in vitro addition of recombinant AnxA1 to macrophages isolated from NASH livers down-modulated M1 polarization through stimulation of interleukin-10 production. Furthermore, the degree of hepatic fibrosis was enhanced in MCD-fed AnxA1 KO mice, an effect associated with augmented liver production of the profibrotic lectin, galectin-3. Accordingly, AnxA1 addition to isolated hepatic macrophages reduced galectin-3 expression. CONCLUSIONS: Macrophage-derived AnxA1 plays a functional role in modulating hepatic inflammation and fibrogenesis during NASH progression, suggesting the possible use of AnxA1 analogs for therapeutic control of this disease.
Subject(s)
Annexin A1/immunology , Fatty Liver/immunology , Hepatitis/immunology , Macrophages/immunology , Animals , Annexin A1/genetics , Choline Deficiency/genetics , Choline Deficiency/immunology , Disease Models, Animal , Disease Progression , Fatty Liver/genetics , Hepatitis/genetics , Humans , Liver Cirrhosis/genetics , Liver Cirrhosis/immunology , Male , Methionine/deficiency , Mice , Mice, Inbred C57BL , Mice, Knockout , Non-alcoholic Fatty Liver Disease , Obesity/genetics , Obesity/immunologyABSTRACT
Liver monocytes play a major role in the development of NASH (non-alcoholic steatohepatitis). In inflamed tissues, monocytes can differentiate in both macrophages and dendritic cells. In the present study, we investigated the role of moDCs (monocyte-derived inflammatory dendritic cells) in experimental steatohepatitis induced in C57BL/6 mice by feeding on a MCD (methionine/choline-deficient) diet. The evolution of steatohepatitis was characterized by an increase in hepatic CD45+ / CD11b+ myeloid cells displaying the monocyte/macrophage marker F4-80(+). In the early phases (4 weeks of treatment), Ly6C(high)/CD11b(+)/F4-80(+) inflammatory macrophages predominated. However, their frequency did not grow further with the disease progression (8 weeks of treatment), when a 4-fold expansion of CD11b(+)/F4-80(+) cells featuring the fractalkine receptor (CX3CR1) was evident. These CX3CR1+ cells were also characterized by the combined expression of inflammatory monocyte (Ly6C, CD11b) and dendritic cell (CD11c, MHCII) markers as well as by a sustained TNFα (tumour necrosis factor α) production, suggesting monocyte differentiation into inflammatory moDCs. The expansion of TNFα-producing CX3CR1+ moDCs was associated with an elevation in hepatic and circulating TNFα level and with the worsening of parenchymal injury. Hydrogen sulfide (H2S) has been shown to interfere with CX3CR1 up-regulation in monocyte-derived cells exposed to pro-inflammatory stimuli. Treating 4-week-MCD-fed mice with the H2S donor NaHS while continuing on the same diet prevented the accumulation of TNFα-producing CX3CR1+ moDCs without interfering with hepatic macrophage functions. Furthermore, NaHS reduced hepatic and circulating TNFα levels and ameliorated transaminase release and parenchymal injury. Altogether, these results show that inflammatory CX3CR1+ moDCs contributed in sustaining inflammation and liver injury during steatohepatitis progression.
Subject(s)
Dendritic Cells/immunology , Inflammation/immunology , Non-alcoholic Fatty Liver Disease/immunology , Receptors, Chemokine/immunology , Actins/genetics , Actins/immunology , Actins/metabolism , Animals , Antigens, Differentiation/genetics , Antigens, Differentiation/immunology , Antigens, Differentiation/metabolism , Antigens, Ly/genetics , CD11b Antigen/genetics , CD11b Antigen/immunology , CD11b Antigen/metabolism , CX3C Chemokine Receptor 1 , Choline Deficiency , Dendritic Cells/metabolism , Diet/adverse effects , Disease Progression , Flow Cytometry , Gene Expression/immunology , Immunohistochemistry , Inflammation/genetics , Liver/drug effects , Liver/immunology , Liver/metabolism , Methionine/deficiency , Mice, Inbred C57BL , Monocytes/immunology , Monocytes/metabolism , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/genetics , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sulfides/metabolism , Sulfides/pharmacology , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/immunology , Transforming Growth Factor beta1/metabolism , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Nonalcoholic steatohepatitis (NASH) is characterized by extensive hepatic monocyte infiltration and monocyte-derived macrophages have an important role in regulating the disease evolution. However, little is known about the functional changes occurring in liver macrophages during NASH progression. In this study, we investigated phenotypic and functional modifications of hepatic macrophages in experimental NASH induced by feeding C57BL/6 mice with a methionine-choline deficient (MCD) diet up to 8weeks. In mice with steatohepatitis liver F4/80-positive macrophages increased in parallel with the disease progression and formed small clusters of enlarged and vacuolated cells. At immunofluorescence these cells contained lipid vesicles positive for the apoptotic cell marker Annexin V suggesting the phagocytosis of apoptotic bodies derived from dead fat-laden hepatocytes. Flow cytometry revealed that these enlarged macrophages expressed inflammatory monocyte (CD11b, Ly6C, TNF-α) markers. However, as compared to regular size macrophages the enlarged sub-set was characterized by an enhanced production of arginase-1 and of the anti-inflammatory mediators IL-10 and annexin A1. Similar vacuolated macrophages producing annexin A1 were also evident in liver biopsies of NASH patients. In mice with NASH, the accumulation of enlarged F4/80(+) cells paralleled with a decline in the expression of the macrophage M1 activation markers iNOS, IL-12 and CXCL10, while the levels of M2 polarization markers arginase-1 and MGL-1 were unchanged. Interestingly, the lowering of IL-12 expression mainly involved the macrophage sub-set with regular size. We conclude that during the progression of NASH fat accumulation within liver macrophages promotes the production of anti-inflammatory mediators that influence hepatic inflammatory responses.
Subject(s)
Inflammation/pathology , Macrophages/cytology , Non-alcoholic Fatty Liver Disease/immunology , Alanine Transaminase/blood , Animals , Annexin A1/metabolism , Antigens, Ly/metabolism , Arginase/metabolism , CD11b Antigen/metabolism , Chemokine CXCL10/metabolism , Cytoskeletal Proteins , Diet , Disease Progression , Fibrosis , Homeodomain Proteins/metabolism , Humans , Inflammation/complications , Interleukin-10/metabolism , Interleukin-12/metabolism , Liver/cytology , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Monocytes/cytology , Nitric Oxide Synthase Type II/metabolism , Triglycerides/blood , Tumor Necrosis Factor-alpha/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
Background: Metabolic dysfunction-associated steatotic liver disease (MASLD), previously non-alcoholic fatty liver disease (NAFLD), is a leading cause of chronic liver disease worldwide. In 20%-30% of MASLD patients, the disease progresses to metabolic dysfunction-associated steatohepatitis (MASH, previously NASH) which can lead to fibrosis/cirrhosis, liver failure as well as hepatocellular carcinoma (HCC). Here we investigated the role of histidine-rich glycoprotein (HRG), a plasma protein produced by hepatocytes, in MASLD/MASH progression and HCC development. Methods: The role of HRG was investigated by morphological, cellular, and molecular biology approaches in (a) HRG knock-out mice (HRG-/- mice) fed on a CDAA dietary protocol or a MASH related diethyl-nitrosamine/CDAA protocol of hepatocarcinogenesis, (b) THP1 monocytic cells treated with purified HRG, and (c) well-characterized cohorts of MASLD patients with or without HCC. Results: In non-neoplastic settings, murine and clinical data indicate that HRG increases significantly in parallel with disease progression. In particular, in MASLD/MASH patients, higher levels of HRG plasma levels were detected in subjects with extensive fibrosis/cirrhosis. When submitted to the pro-carcinogenic protocol, HRG-/- mice showed a significant decrease in the volume and number of HCC nodules in relation to decreased infiltration of macrophages producing pro-inflammatory mediators, including IL-1ß, IL-6, IL-12, IL-10, and VEGF as well as impaired angiogenesis. The histopathological analysis (H-score) of MASH-related HCC indicate that the higher HRG positivity in peritumoral tissue significantly correlates with a lower overall patient survival and an increased recurrence. Moreover, a significant increase in HRG plasma levels was detected in cirrhotic (F4) patients and in patients carrying HCC vs. F0/F1 patients. Conclusion: Murine and clinical data indicate that HRG plays a significant role in MASLD/MASH progression to HCC by supporting a specific population of tumor-associated macrophages with pro-inflammatory response and pro-angiogenetic capabilities which critically support cancer cell survival. Furthermore, our data suggest HRG as a possible prognostic predictor in HCC patients with MASLD/MASH-related HCCs.
Subject(s)
Acetamides , Carcinoma, Hepatocellular , Liver Neoplasms , Metabolic Diseases , Non-alcoholic Fatty Liver Disease , Proteins , Humans , Animals , Mice , Carcinoma, Hepatocellular/etiology , Liver Neoplasms/etiology , Carcinogenesis , Liver Cirrhosis/etiology , Disease ProgressionABSTRACT
Growing evidence indicates that NF-κB (nuclear factor κB) activation contributes to the pathogenesis of NASH (non-alcoholic steatohepatisis). Among the NF-κB subunits, p50/NF-κB1 has regulatory activities down-modulating NF-κB-mediated responses. In the present study, we investigated the effects of NF-κB1 deficiency on the progression of NASH induced by feeding mice on an MCD (methionine/choline-deficient) diet. Following 4 weeks on the MCD diet, steatosis, ALT (alanine aminotransferase) release, hepatocyte apoptosis, lobular inflammation and TNFα (tumour necrosis factor α) production were higher in NF-κB1(-/-) (NF-κB1-knockout) mice than in WT (wild-type) mice. NF-κB1(-/-) mice also showed appreciable centrilobular collagen deposition, an increased number of activated hepatic stellate cells and higher type-I procollagen-α and TIMP-1 (tissue inhibitor of metalloproteases-1) mRNA expression. Although NF-κB p50 homodimers regulate macrophage activation, the number of hepatic macrophages and liver mRNAs for iNOS (inducible NO synthase), IL (interleukin)-12p40, CCL2 (CC chemokine ligand 2) and CXCL10 (CXC chemokine ligand 10) were comparable in the two strains. NASH was associated with an increase in liver infiltrating T-cells that was more evident in MCD-fed NF-κB1(-/-) than in similarly treated WT mice. Flow cytorimetry showed that T-cell recruitment involved effector CD8+ T-cells without changes in the helper CD4+ T-cell fraction. Furthermore, although NASH lowered hepatic NKT cells [NK (natural killer) T-cells] in WT mice, the NKT cell pool was selectively increased in the livers of MCD-fed NF-κB1(-/-) mice. Such NKT cell recruitment was associated with an early overexpression of IL-15, a cytokine controlling NKT cell survival and maturation. In the livers of MCD-fed NF-κB1(-/-) mice, but not in those of WT littermates, we also observed an up-regulation in the production of NKT-related cytokines IFN (interferon)-γ and osteopontin. Taken together, these results indicate that NF-κB1 down-modulation enhanced NASH progression to fibrosis by favouring NKT cell recruitment, stressing the contribution of NKT cells in the pathogenesis of NASH.
Subject(s)
Fatty Liver/etiology , NF-kappa B p50 Subunit/deficiency , Natural Killer T-Cells/metabolism , Animals , Biomarkers/metabolism , Diet , Fatty Liver/immunology , Fatty Liver/metabolism , Fatty Liver/pathology , Flow Cytometry , Liver Cirrhosis/etiology , Liver Cirrhosis/immunology , Liver Cirrhosis/metabolism , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Non-alcoholic Fatty Liver Disease , Real-Time Polymerase Chain ReactionABSTRACT
Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death worldwide. In the recent years nonalcoholic fatty liver disease (NAFLD) is becoming a growing cause of HCCs and the incidence of NAFLD-related HCCs is expected to further dramatically increase by the next decade. Chronic inflammation is regarded as the driving force of NAFLD progression and a key factor in hepatic carcinogenesis. Hepatic inflammation in NAFLD results from the persistent stimulation of innate immunity in response to hepatocellular injury and gut dysbiosis as well as by the activation of adaptive immunity. However, the relative roles of innate and adaptive immunity in the processes leading to HCC are still incompletely characterized. This is due to the complex interplay between different liver cell populations, which is also strongly influenced by gut-derived bacterial products, metabolic/nutritional signals. Furthermore, carcinogenic mechanisms in NAFLD/NASH appear to involve the activation of signals mediated by hypoxia inducible factors. This review discusses recent data regarding the contribution of different inflammatory cells to NAFLD-related HCC and their possible impact on patient response to current treatments.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Non-alcoholic Fatty Liver Disease , Humans , Carcinoma, Hepatocellular/pathology , Non-alcoholic Fatty Liver Disease/pathology , Liver Neoplasms/pathology , InflammationABSTRACT
Background and aims: Inducible T-cell Co-Stimulator (ICOS) present on T-lymphocytes and its ligand ICOSL expressed by myeloid cells play multiple roles in regulating T-cell functions. However, recent evidence indicates that reverse signalling involving ICOSL is also important in directing the differentiation of monocyte-derived cells. In this study, we investigated the involvement of ICOS/ICOSL dyad in modulating macrophage functions during the evolution of metabolic dysfunction-associated steatohepatitis (MASH). Results: In animal models of MASH, ICOS was selectively up-regulated on CD8+ T-cells in parallel with an expansion of ICOSL-expressing macrophages. An increase in circulating soluble ICOSL was also evident in patients with MASH as compared to healthy individuals. ICOSL knockout (ICOSL-/-) mice receiving choline/methionine deficient (MCD) diet for 6 weeks had milder steatohepatitis than wild type mice. MASH improvement was confirmed in mice fed with cholesterol-enriched Western diet for 24 weeks in which ICOSL deficiency greatly reduced liver fibrosis along with the formation of crown-like macrophage aggregates producing the pro-fibrogenic mediators osteopontin (OPN) and galectin-3 (Gal-3). These effects associated with a selective shewing of F4-80+/CD11bhigh monocyte-derived macrophages (MoMFs) expressing the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) to CD11blow/F4-80+ cells positive for the Kupffer cell marker C-type lectin-like type 2 receptor (CLEC-2), thus indicating an increased MoMF maturation toward monocyte-derived Kupffer cells. Conclusions: These results suggest that CD8+ T-cells interaction with monocyte-derived macrophages through ICOS/ICOSL critically supports a specific subset of TREM2+-expressing cells contributing to the evolution of steatohepatitis. The data also point ICOS/ICOSL dyad as a possible target for therapeutic interventions in MASH.
Subject(s)
CD8-Positive T-Lymphocytes , Fatty Liver , Animals , Humans , Mice , CD8-Positive T-Lymphocytes/metabolism , Inducible T-Cell Co-Stimulator Ligand/metabolism , Inducible T-Cell Co-Stimulator Protein/genetics , Interleukin-2 , Ligands , Signal TransductionSubject(s)
Liver , Non-alcoholic Fatty Liver Disease , Animals , Dendritic Cells , Hepatocytes , MiceABSTRACT
In humans, there is large inter-individual variability in the evolution of NAFLD (non-alcoholic fatty liver disease) to NASH (non-alcoholic steatohepatitis). To investigate this issue, NASH was induced with an MCD (methionine-choline-deficient) diet in C57BL/6 and Balb/c mice that are characterized by different biases in Th1/Th2 and macrophage (M1/M2) responses. Following 4 weeks on the MCD diet, steatosis and lobular inflammation were prevalent in C57BL/6 (Th1, M1 oriented) than in Balb/c (Th2, M2 oriented) mice. Consistently, hepatic TNFα (tumour necrosis factor α) mRNA expression and circulating TNFα levels were higher in MCD-fed C57BL/6 than in MCD-fed Balb/c mice. The Th1/Th2 bias did not account for the increased NASH severity, as in both strains MCD feeding did not significantly modify the liver mRNA expression of the Th1 markers IFNγ (interferon γ) and T-bet or that of the Th2 markers IL-4 (interleukin 4) and GATA-3. Conversely, MCD-fed C57BL/6 mice displayed higher liver mRNAs for the macrophage M1 activation markers iNOS (inducible NO synthase), IL-12p40 and CXCL10 (CXC chemokine ligand 10) than similarly treated Balb/c mice, without effects on the M2 polarization markers IL-10 and MGL-1 (macrophage galactose-type C-type lectin-1). Circulating IL-12 was also higher in MCD-fed C57BL/6 than in MCD-fed Balb/c mice. The analysis of macrophages isolated from the livers of MCD-fed animals confirmed an enhanced expression of M1 markers in C57BL/6 mice. Among all of the MCD-treated mice, liver iNOS, IL-12p40 and CXCL10 mRNA levels positively correlated with the frequency of hepatic necro-inflammatory foci. We concluded that the macrophage M1 bias in C57BL/6 mice may account for the increased severity of NASH in this strain, suggesting macrophage responses as important contributors to NAFLD progression.
Subject(s)
Fatty Liver/immunology , Macrophage Activation , Animals , Disease Susceptibility , Liver/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Th1-Th2 BalanceABSTRACT
NEFA (non-esterified 'free' fatty acid)-mediated lipotoxicity plays a critical role in the pathogenesis of NASH (non-alcoholic steatohepatitis). In the light of the growing need for new therapeutic options for NASH, we investigated the action of A2aR (adenosine A(2a) receptor) stimulation against lipotoxicity. The effects of the A(2a)R agonist CGS21680 [2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxyamidoadenosine] were evaluated 'in vitro' in liver cells exposed to SA (stearic acid) and 'in vivo' in rats with NASH induced by 8 weeks of feeding with an MCD diet (methionine/choline-deficient diet). In cultured hepatocytes, SA promoted apoptosis by inducing MKK4 (mitogen-activated protein kinase kinase 4)/SEK1 (stress-activated protein kinase/extracellular-signal-regulated kinase kinase-1) and JNK-1/2 (c-Jun N-terminal kinase-1/2) activation. CGS21680 addition prevented JNK-1/2 activation and reduced apoptosis without interfering with lipid accumulation. CGS21680 action required PI3K (phosphoinositide 3-kinase)/Akt-mediated block of MKK4/SEK1. Consistently, PI3K inhibition with wortmannin abolished the cytoprotective action of CGS21680 and reverted MKK4 inhibition. SA lipotoxicity was also prevented by transfecting HTC cells with a specific MKK4/SEK1 siRNA (small interfering RNA). In rats receiving the MCD diet, the development of NASH was associated with MKK4/SEK1 and JNK-1/2 activation. CGS21680 (0.5 mg/kg of body weight, intraperitoneal) administration to MCD-fed rats prevented JNK-1/2 activation by acting on MKK4/SEK1. CGS21680 also effectively reduced NASH-associated ALT (alanine aminotransferase) release, hepatocyte apoptosis, liver inflammation and fibrosis without affecting hepatic steatosis. Taken together, these results demonstrate that, by inhibiting JNK-1/2, A(2a)R stimulation reduces lipotoxicity and ameliorates NASH, giving a rationale to investigate A(2a)R agonists as possible new therapeutic agents in preventing fatty liver progression to NASH.
Subject(s)
Adenosine A2 Receptor Agonists/therapeutic use , Adenosine/analogs & derivatives , Fatty Liver/prevention & control , Phenethylamines/therapeutic use , Adenosine/pharmacology , Adenosine/therapeutic use , Adenosine A2 Receptor Agonists/pharmacology , Animals , Apoptosis/drug effects , Biomarkers/metabolism , Cells, Cultured , Drug Administration Schedule , Fatty Liver/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Injections, Intraperitoneal , JNK Mitogen-Activated Protein Kinases/metabolism , Liver/drug effects , Liver/metabolism , Liver/pathology , Male , Non-alcoholic Fatty Liver Disease , Phenethylamines/pharmacology , Rats , Rats, Wistar , Real-Time Polymerase Chain Reaction , Receptors, Adenosine A2/metabolism , Stearic Acids/toxicityABSTRACT
Expression of CCL2 (CC chemokine ligand 2) (or monocyte chemoattractant protein-1) regulates inflammatory cell infiltration in the liver and adipose tissue, favouring steatosis. However, its role in the pathogenesis of steatohepatitis is still uncertain. In the present study, we investigated the development of non-alcoholic steatohepatitis induced by an MCD diet (methionine/choline-deficient diet) in mice lacking the CCL2 gene on two different genetic backgrounds, namely Balb/C and C57/Bl6J. WT (wild-type) and CCL2-KO (knockout) mice were fed on a lipid-enriched MCD diet or a control diet for 8 weeks. In Balb/C mice fed on the MCD diet, a lack of CCL2 was associated with lower ALT (alanine transaminase) levels and reduced infiltration of inflammatory cells, together with a lower generation of oxidative-stress-related products. Sirius Red staining demonstrated pericellular fibrosis in zone 3, and image analysis showed a significantly lower matrix accumulation in CCL2-KO mice. This was associated with reduced hepatic expression of TGF-ß (transforming growth factor-ß), type I procollagen, TIMP-1 (tissue inhibitor of metalloproteinases-1) and α-smooth muscle actin. In contrast, in mice on a C57Bl/6 background, neither ALT levels nor inflammation or fibrosis were significantly different comparing WT and CCL2-KO animals fed on an MCD diet. In agreement, genes related to fibrogenesis were expressed to comparable levels in the two groups of animals. Comparison of the expression of several genes involved in inflammation and repair demonstrated that IL (interleukin)-4 and the M2 marker MGL-1 (macrophage galactose-type C-type lectin 1) were differentially expressed in Balb/C and C57Bl/6 mice. No significant differences in the degree of steatosis were observed in all groups of mice fed on the MCD diet. We conclude that, in experimental murine steatohepatitis, the effects of CCL2 deficiency are markedly dependent on the genetic background.
Subject(s)
Chemokine CCL2/genetics , Chemokine CCL2/immunology , Fatty Liver/genetics , Fatty Liver/immunology , Liver Cirrhosis/genetics , Liver Cirrhosis/immunology , Animals , Chemokine CCL2/metabolism , Collagen Type I/genetics , Dietary Fats/pharmacology , Disease Models, Animal , Fatty Liver/pathology , Hepatic Stellate Cells/immunology , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Liver Cirrhosis/pathology , Macrophages/metabolism , Macrophages/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Oxidative Stress/immunology , Species Specificity , Tissue Inhibitor of Metalloproteinase-1/genetics , Transforming Growth Factor beta/geneticsABSTRACT
BACKGROUND & AIMS: Hypoxia and hypoxia-inducible factors (HIFs) are involved in chronic liver disease progression. We previously showed that hepatocyte HIF-2α activation contributed significantly to nonalcoholic fatty liver disease progression in experimental animals and human patients. In this study, using an appropriate genetic murine model, we mechanistically investigated the involvement of hepatocyte HIF-2α in experimental nonalcoholic steatohepatitis (NASH)-related carcinogenesis. METHODS: The role of HIF-2α was investigated by morphologic, cellular, and molecular biology approaches in the following: (1) mice carrying hepatocyte-specific deletion of HIF-2α (HIF-2α-/- mice) undergoing a NASH-related protocol of hepatocarcinogenesis; (2) HepG2 cells stably transfected to overexpress HIF-2α; and (3) liver specimens from NASH patients with hepatocellular carcinoma. RESULTS: Mice carrying hepatocyte-specific deletion of HIF-2α (hHIF-2α-/-) showed a significant decrease in the volume and number of liver tumors compared with wild-type littermates. These effects did not involve HIF-1α changes and were associated with a decrease of cell proliferation markers proliferating cell nuclear antigen and Ki67. In both human and rodent nonalcoholic fatty liver disease-related tumors, HIF-2α levels were strictly associated with hepatocyte production of SerpinB3, a mediator previously shown to stimulate liver cancer cell proliferation through the Hippo/Yes-associated protein (YAP)/c-Myc pathway. Consistently, we observed positive correlations between the transcripts of HIF-2α, YAP, and c-Myc in individual hepatocellular carcinoma tumor masses, while HIF-2α deletion down-modulated c-Myc and YAP expression without affecting extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, and AKT-dependent signaling. In vitro data confirmed that HIF-2α overexpression induced HepG2 cell proliferation through YAP-mediated mechanisms. CONCLUSIONS: These results indicate that the activation of HIF-2α in hepatocytes has a critical role in liver carcinogenesis during NASH progression, suggesting that HIF-2α-blocking agents may serve as novel putative therapeutic tools.