Redox-sensitive high-mobility group box-1 isoforms contribute to liver fibrosis progression and resolution in mice.

BACKGROUND & AIMS: High-mobility group box-1 (HMGB1) significantly increases and undergoes post-translational modifications (PTMs) in response to liver injury. Since oxidative stress plays a major role in liver fibrosis and induces PTMs in proteins, we hypothesized that redox-sensitive HMGB1 isoforms contribute to liver fibrosis progression and resolution. METHODS: We used ESI-LC-MS (electrospray ionization-liquid chromatography-mass spectrometry) to study PTMs of HMGB1 during fibrosis progression and resolution. Conditional knockout mice were used for functional analyses. RESULTS: We identified that disulfide ([O]) and sulfonated ([SO3]) HMGB1 increase during carbon tetrachloride-induced liver fibrosis progression, however, while [O] HMGB1 declines, [SO3] HMGB1 drops but remains, during fibrosis resolution. Conditional knockout of Hmgb1 revealed that production of [O] and [SO3] HMGB1 occurs mostly in hepatocytes. Co-injection of [O] HMGB1 worsens carbon tetrachloride-induced liver fibrosis more than co-injection of [H] HMGB1. Conversely, ablation of [O] Hmgb1 in hepatocytes reduces liver fibrosis. Moreover, ablation of the receptor for advanced-glycation end-products (Rage) reveals that the profibrogenic effect of [O] HMGB1 is mediated by RAGE signaling in hepatic stellate cells (HSCs). Notably, injection of [SO3] HMGB1 accelerates fibrosis resolution due to RAGE-dependent stimulation of HSC apoptosis. Importantly, gene signatures activated by redox-sensitive HMGB1 isoforms in mice, classify patients with fibrosis according to fibrosis and inflammation scores. CONCLUSION: Dynamic changes in hepatocyte-derived [O] and [SO3] HMGB1 signal through RAGE-dependent mechanisms on HSCs to drive their profibrogenic phenotype and fate, contributing to progression and resolution of liver fibrosis. IMPACT AND IMPLICATIONS: Since oxidative stress plays a major role in liver fibrosis and induces post-translational modifications of proteins, we hypothesized that redox-sensitive HMGB1 isoforms contribute to liver fibrosis progression and resolution. This study is significant because a rise in [H] HMGB1 could flag 'patient at risk', the presence of [O] HMGB1 could suggest 'disease in progress or active scarring', while the appearance of [SO3] HMGB1 could point at 'resolution under way'. The latter could be used as a readout for response to pharmacological intervention with anti-fibrotic agents.

Macrophage-derived Osteopontin (SPP1) Protects From Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is characterized by steatosis, lobular inflammation, hepatocyte ballooning degeneration, and fibrosis, all of which increase the risk of progression to end-stage liver disease. Osteopontin (OPN, SPP1) plays an important role in macrophage (MF) biology, but whether MF-derived OPN affects NASH progression is unknown. METHODS: We analyzed publicly available transcriptomic datasets from patients with NASH, and used mice with conditional overexpression or ablation of Spp1 in myeloid cells and liver MFs, and fed them a high-fat, fructose, and cholesterol diet mimicking the Western diet, to induce NASH. RESULTS: This study demonstrated that MFs with high expression of SPP1 are enriched in patients and mice with nonalcoholic fatty liver disease (NAFLD), and show metabolic but not pro-inflammatory properties. Conditional knockin of Spp1 in myeloid cells (Spp1KI Mye) or in hepatic macrophages (Spp1KI LvMF) conferred protection, whereas conditional knockout of Spp1 in myeloid cells (Spp1ΔMye) worsened NASH. The protective effect was mediated by induction of arginase-2 (ARG2), which enhanced fatty acid oxidation (FAO) in hepatocytes. Induction of ARG2 stemmed from enhanced production of oncostatin-M (OSM) in MFs from Spp1KI Mye mice. OSM activated STAT3 signaling, which upregulated ARG2. In addition to hepatic effects, Spp1KI Mye also protected through sex-specific extrahepatic mechanisms. CONCLUSION: MF-derived OPN protects from NASH, by upregulating OSM, which increases ARG2 through STAT3 signaling. Further, the ARG2-mediated increase in FAO reduces steatosis. Therefore, enhancing the OPN-OSM-ARG2 crosstalk between MFs and hepatocytes may be beneficial for patients with NASH.

Deficiency of neutrophil high-mobility group box-1 in liver transplant recipients exacerbates early allograft injury in mice.

BACKGROUND AND AIMS: Early allograft dysfunction (EAD) is a severe event leading to graft failure after liver transplant (LT). Extracellular high-mobility group box-1 (HMGB1) is a damage-associated molecular pattern that contributes to hepatic ischemia-reperfusion injury (IRI). However, the contribution of intracellular HMGB1 to LT graft injury remains elusive. We hypothesized that intracellular neutrophil-derived HMGB1 from recipients protects from post-LT EAD. APPROACH AND RESULTS: We generated mice with conditional ablation or overexpression of Hmgb1 in hepatocytes, myeloid cells, or both. We performed LTs and injected lipopolysaccharide (LPS) to evaluate the effect of intracellular HMGB1 in EAD. Ablation of Hmgb1 in hepatocytes and myeloid cells of donors and recipients exacerbated early allograft injury after LT. Ablation of Hmgb1 from liver grafts did not affect graft injury; however, lack of Hmgb1 from recipient myeloid cells increased reactive oxygen species (ROS) and inflammation in liver grafts and exacerbated injury. Neutrophils lacking HMGB1 were more activated, showed enhanced pro-oxidant and pro-inflammatory signatures, and reduced biosynthesis and metabolism of inositol polyphosphates (InsPs). On LT reperfusion or LPS treatment, there was significant neutrophil mobilization and infiltration into the liver and enhanced production of ROS and pro-inflammatory cytokines when intracellular Hmgb1 was absent. Depletion of neutrophils using anti-Ly6G antibody attenuated graft injury in recipients with myeloid cell Hmgb1 ablation. CONCLUSIONS: Neutrophil HMGB1 derived from recipients is central to regulate their activation, limits the production of ROS and pro-inflammatory cytokines, and protects from early liver allograft injury.

Hepatocellular carcinomas, exhibiting intratumor fibrosis, express cancer-specific extracellular matrix remodeling and WNT/TGFB signatures, associated with poor outcome.

BACKGROUND AND AIMS: HCC, the third leading cause of cancer-related death, arises in the context of liver fibrosis. Although HCC is generally poorly fibrogenic, some tumors harbor focal intratumor extracellular matrix (ECM) deposits called "fibrous nests." To date, the molecular composition and clinical relevance of these ECM deposits have not been fully defined. APPROACH AND RESULTS: We performed quantitative matrisome analysis by tandem mass tags mass spectrometry in 20 human cancer specific matrisome (HCCs) with high or low-grade intratumor fibrosis and matched nontumor tissues, as well as in 12 livers from mice treated with vehicle, carbon tetrachloride, or diethylnitrosamine. We found 94 ECM proteins differentially abundant between high and low-grade fibrous nests, including interstitial and basement membrane components, such as several collagens, glycoproteins, proteoglycans, enzymes involved in ECM stabilization and degradation, and growth factors. Pathway analysis revealed a metabolic switch in high-grade fibrosis, with enhanced glycolysis and decreased oxidative phosphorylation. Integrating the quantitative proteomics with transcriptomics from HCCs and nontumor livers (n = 2,285 samples), we identified a subgroup of fibrous nest HCCs, characterized by cancer-specific ECM remodeling, expression of the WNT/TGFB (S1) subclass signature, and poor patient outcome. Fibrous nest HCCs abundantly expressed an 11-fibrous-nest - protein signature, associated with poor patient outcome, by multivariate Cox analysis, and validated by multiplex immunohistochemistry. CONCLUSIONS: Matrisome analysis highlighted cancer-specific ECM deposits, typical of the WNT/TGFB HCC subclass, associated with poor patient outcomes. Hence, histologic reporting of intratumor fibrosis in HCC is of clinical relevance.

Matrisome gene-based subclassification of patients with liver fibrosis identifies clinical and molecular heterogeneities.

BACKGROUND AIMS: Excessive deposition and crosslinking of extracellular matrix increases liver density and stiffness, promotes fibrogenesis, and increases resistance to fibrinolysis. An emerging therapeutic opportunity in liver fibrosis is to target the composition of the extracellular matrix or block pathogenic communication with surrounding cells. However, the type and extent of extracellular changes triggering liver fibrosis depend on the underlying etiology. Our aim was to unveil matrisome genes not dependent on etiology, which are clinically relevant to liver fibrosis. APPROACH RESULTS: We used transcriptomic profiles from liver fibrosis cases of different etiologies to identify and validate liver fibrosis-specific matrisome genes (LFMGs) and their clinical and biological relevance. Dysregulation patterns and cellular landscapes of LFMGs were further explored in mouse models of liver fibrosis progression and regression by bulk and single-cell RNA sequencing. We identified 35 LFMGs, independent of etiology, representing an LFMG signature defining liver fibrosis. Expression of the LFMG signature depended on histological severity and was reduced in regressive livers. Patients with liver fibrosis, even with identical pathological scores, could be subclassified into LFMG Low and LFMG High , with distinguishable clinical, cellular, and molecular features. Single-cell RNA sequencing revealed that microfibrillar-associated protein 4 + activated HSC increased in LFMG High patients and were primarily responsible for the LFMG signature expression and dysregulation. CONCLUSIONS: The microfibrillar-associated protein 4 + -activated HSC-derived LFMG signature classifies patients with liver fibrosis with distinct clinical and biological characteristics. Our findings unveil hidden information from liver biopsies undetectable using traditional histologic assessments.

Children with cancer at the end of life in a middle-income country: integrated pediatric palliative care improves outcomes.

BACKGROUND: In 2020, the Global Cancer Observatory reported 280,000 cases of childhood cancer worldwide, with a higher burden of disease and mortality rates in low- and middle-income countries. In 2022, the National Institute of Health reported 1708 new cases of childhood cancer in Colombia and an overall survival rate of approximately 55%. The aim of this study is to compare outcomes in children with cancer in the hospital setting during the last 72 h of life who received concurrent Pediatric Palliative Care (PPC) versus oncology care alone. METHODS: An observational descriptive study was conducted between January 2013 and June 2022 in a center for pediatric patients with oncological diagnoses. In 2017, the PPC team was created. Patients between 28 days and 17 years of age who were hospitalized at least 72 h before death were included. A retrospective review of the medical records of patients in the last 72 h of life was performed. Two cohorts were established: oncology-alone group received exclusive management by oncology, and oncology and PPC received concurrent oncology and PPC management since the diagnosis. RESULTS: We evaluated 257 medical records of deceased pediatric patients with cancer diagnoses. For the first cohort (2013-2017), 136 patients were included; for the second cohort (2018 and 2022), 121 patients were evaluated. The most frequent diagnosis was leukemia [47.1% (n = 121)]. No significant difference was found in either group between dyspnea, pain, and seizures. Dyspnea was the most frequent symptom in both groups. Agitation and anxiety were reported more frequently in children from the oncology-alone group (22.1% and 13.2%, respectively). The oncology and PPC group received more psychology and social work consultation (94.2% and 70.2% vs. 84.6 and 54.4% in the oncology alone group) and had a higher percentage of advance care planning (79.3% vs. 62.5% in the oncology alone group). CONCLUSIONS: This retrospective study highlights that PPC at the end of life (EoL) offers a holistic approach to the physical and psychosocial symptoms experienced by children with cancer; these patients received more comfort through symptom management and less aggressive treatment at the EoL. The availability of a PPC team may contribute to improvements in the quality of end-of-life care. TRIAL REGISTRATION: retrospectively registered.

Osteopontin Takes Center Stage in Chronic Liver Disease.

Osteopontin (OPN) was first identified in 1986. The prefix osteo- means bone; however, OPN is expressed in other tissues, including liver. The suffix -pontin means bridge and denotes the role of OPN as a link protein within the extracellular matrix. While OPN has well-established physiological roles, multiple "omics" analyses suggest that it is also involved in chronic liver disease. In this review, we provide a summary of the OPN gene and protein structure and regulation. We outline the current knowledge on how OPN is involved in hepatic steatosis in the context of alcoholic liver disease and non-alcoholic fatty liver disease. We describe the mechanisms whereby OPN participates in inflammation and liver fibrosis and discuss current research on its role in hepatocellular carcinoma and cholangiopathies. To conclude, we highlight important points to consider when doing research on OPN and provide direction for making progress on how OPN contributes to chronic liver disease.

Danger signals in liver injury and restoration of homeostasis.

Damage-associated molecular patterns are signalling molecules involved in inflammatory responses and restoration of homeostasis. Chronic release of these molecules can also promote inflammation in the context of liver disease. Herein, we provide a comprehensive summary of the role of damage-associated molecular patterns as danger signals in liver injury. We consider the role of reactive oxygen species and reactive nitrogen species as inducers of damage-associated molecular patterns, as well as how specific damage-associated molecular patterns participate in the pathogenesis of chronic liver diseases such as alcohol-related liver disease, non-alcoholic steatohepatitis, liver fibrosis and liver cancer. In addition, we discuss the role of damage-associated molecular patterns in ischaemia reperfusion injury and liver transplantation and highlight current studies in which blockade of specific damage-associated molecular patterns has proven beneficial in humans and mice.

High Mobility Group Box-1 Drives Fibrosis Progression Signaling via the Receptor for Advanced Glycation End Products in Mice.

High-mobility group box-1 (HMGB1) is a damage-associated molecular pattern (DAMP) increased in response to liver injury. Because HMGB1 is a ligand for the receptor for advanced glycation endproducts (RAGE), we hypothesized that induction of HMGB1 could participate in the pathogenesis of liver fibrosis though RAGE cell-specific signaling mechanisms. Liver HMGB1 protein expression correlated with fibrosis stage in patients with chronic hepatitis C virus (HCV) infection, primary biliary cirrhosis (PBC), or alcoholic steatohepatitis (ASH). Hepatic HMGB1 protein expression and secretion increased in five mouse models of liver fibrosis attributed to drug-induced liver injury (DILI), cholestasis, ASH, or nonalcoholic steatohepatitis (NASH). HMGB1 was up-regulated and secreted mostly by hepatocytes and Kupffer cells (KCs) following CCl4 treatment. Neutralization of HMGB1 protected, whereas injection of recombinant HMGB1 promoted liver fibrosis. Hmgb1 ablation in hepatocytes (Hmgb1ΔHep ) or in myeloid cells (Hmgb1ΔMye ) partially protected, whereas ablation in both (Hmgb1ΔHepΔMye ) prevented liver fibrosis in vivo. Coculture with hepatocytes or KCs from CCl4 -injected wild-type (WT) mice up-regulated Collagen type I production by hepatic stellate cells (HSCs); yet, coculture with hepatocytes from CCl4 -injected Hmgb1ΔHep or with KCs from CCl4 -injected Hmgb1ΔMye mice partially blunted this effect. Rage ablation in HSCs (RageΔHSC ) and RAGE neutralization prevented liver fibrosis. Last, we identified that HMGB1 stimulated HSC migration and signaled through RAGE to up-regulate Collagen type I expression by activating the phosphorylated mitogen-activated protein kinase kinase (pMEK)1/2, phosphorylated extracellular signal-regulated kinase (pERK)1/2 and pcJun signaling pathway. Conclusion: Hepatocyte and KC-derived HMGB1 participates in the pathogenesis of liver fibrosis by signaling through RAGE in HSCs to activate the pMEK1/2, pERK1/2 and pcJun pathway and increase Collagen type I deposition.

Signalling via the osteopontin and high mobility group box-1 axis drives the fibrogenic response to liver injury.

OBJECTIVE: Liver fibrosis is associated with significant collagen-I deposition largely produced by activated hepatic stellate cells (HSCs); yet, the link between hepatocyte damage and the HSC profibrogenic response remains unclear. Here we show significant induction of osteopontin (OPN) and high-mobility group box-1 (HMGB1) in liver fibrosis. Since OPN was identified as upstream of HMGB1, we hypothesised that OPN could participate in the pathogenesis of liver fibrosis by increasing HMGB1 to upregulate collagen-I expression. DESIGN AND RESULTS: Patients with long-term hepatitis C virus (HCV) progressing in disease stage displayed enhanced hepatic OPN and HMGB1 immunostaining, which correlated with fibrosis stage, whereas it remained similar in non-progressors. Hepatocyte cytoplasmic OPN and HMGB1 expression was significant while loss of nuclear HMGB1 occurred in patients with HCV-induced fibrosis compared with healthy explants. Well-established liver fibrosis along with marked induction of HMGB1 occurred in CCl4-injected OpnHep transgenic yet it was less in wild type and almost absent in Opn-/- mice. Hmgb1 ablation in hepatocytes (Hmgb1ΔHep) protected mice from CCl4-induced liver fibrosis. Coculture with hepatocytes that secrete OPN plus HMGB1 and challenge with recombinant OPN (rOPN) or HMGB1 (rHMGB1) enhanced collagen-I expression in HSCs, which was blunted by neutralising antibodies (Abs) and by Opn or Hmgb1 ablation. rOPN induced acetylation of HMGB1 in HSCs due to increased NADPH oxidase activity and the associated decrease in histone deacetylases 1/2 leading to upregulation of collagen-I. Last, rHMGB1 signalled via receptor for advanced glycation end-products and activated the PI3K-pAkt1/2/3 pathway to upregulate collagen-I. CONCLUSIONS: During liver fibrosis, the increase in OPN induces HMGB1, which acts as a downstream alarmin driving collagen-I synthesis in HSCs.

Cartilage oligomeric matrix protein participates in the pathogenesis of liver fibrosis.

BACKGROUND & AIMS: Liver fibrosis is characterized by significant accumulation of extracellular matrix (ECM) proteins, mainly fibrillar collagen-I, as a result of persistent liver injury. Cartilage oligomeric matrix protein (COMP) is largely found in the ECM of skeletal tissue. Increased COMP expression has been associated with fibrogenesis in systemic sclerosis, lung fibrosis, chronic pancreatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that COMP could induce fibrillar collagen-I deposition and participate in matrix remodeling thus contributing to the pathophysiology of liver fibrosis. METHODS: Thioacetamide (TAA) and carbon tetrachloride (CCl4) were used to induce liver fibrosis in wild-type (WT) and Comp-/- mice. In vitro experiments were performed with primary hepatic stellate cells (HSCs). RESULTS: COMP expression was detected in livers from control WT mice and was upregulated in response to either TAA or CCl4-induced liver fibrosis. TAA-treated or CCl4-injected Comp-/- mice showed less liver injury, inflammation and fibrosis compared to their corresponding control WT mice. Challenge of HSCs with recombinant COMP (rCOMP) induced intra- plus extracellular collagen-I deposition and increased matrix metalloproteinases (MMPs) 2, 9 and 13, albeit similar expression of transforming growth factor beta (TGFß) protein, in addition to Tgfß, tumour necrosis factor alpha (Tnfα) and tissue inhibitor of metalloproteinases-1 (Timp1) mRNAs. We demonstrated that COMP binds collagen-I; yet, it does not prevent collagen-I cleavage by MMP1. Last, rCOMP induced collagen-I expression in HSCs via CD36 receptor signaling and activation of the MEK1/2-pERK1/2 pathway. CONCLUSION: These results suggest that COMP contributes to liver fibrosis by regulating collagen-I deposition. LAY SUMMARY: Cartilage oligomeric matrix protein (COMP) induces fibrillar collagen-I deposition via the CD36 receptor signaling and activation of the MEK1/2-pERK1/2 pathway, and participates in extracellular matrix remodeling contributing to the pathophysiology of liver fibrosis.

High mobility group box-1 (HMGB1) participates in the pathogenesis of alcoholic liver disease (ALD).

Growing clinical and experimental evidence suggests that sterile inflammation contributes to alcoholic liver disease (ALD). High mobility group box-1 (HMGB1) is highly induced during liver injury; however, a link between this alarmin and ALD has not been established. Thus, the aim of this work was to determine whether HMGB1 contributes to the pathogenesis of ALD. Liver biopsies from patients with ALD showed a robust increase in HMGB1 expression and translocation, which correlated with disease stage, compared with healthy explants. Similar findings were observed in chronic ethanol-fed wild-type (WT) mice. Using primary cell culture, we validated the ability of hepatocytes from ethanol-fed mice to secrete a large amount of HMGB1. Secretion was time- and dose-dependent and responsive to prooxidants and antioxidants. Selective ablation of Hmgb1 in hepatocytes protected mice from alcohol-induced liver injury due to increased carnitine palmitoyltransferase-1, phosphorylated 5'AMP-activated protein kinase-α, and phosphorylated peroxisome proliferator-activated receptor-α expression along with elevated LDL plus VLDL export. Native and post-translationally modified HMGB1 were detected in humans and mice with ALD. In liver and serum from control mice and in serum from healthy volunteers, the lysine residues within the peptides containing nuclear localization signals (NLSs) 1 and 2 were non-acetylated, and all cysteine residues were reduced. However, in livers from ethanol-fed mice, in addition to all thiol/non-acetylated isoforms of HMGB1, we observed acetylated NLS1 and NLS2, a unique phosphorylation site in serine 35, and an increase in oxidation of HMGB1 to the disulfide isoform. In serum from ethanol-fed mice and from patients with ALD, there was disulfide-bonded hyperacetylated HMGB1, disulfide-bonded non-acetylated HMGB1, and HMGB1 phosphorylated in serine 35. Hepatocytes appeared to be a major source of these HMGB1 isoforms. Thus, hepatocyte HMGB1 participates in the pathogenesis of ALD and undergoes post-translational modifications (PTMs) that could condition its toxic effects.

AT1R-CB1R heteromerization reveals a new mechanism for the pathogenic properties of angiotensin II.

The mechanism of G protein-coupled receptor (GPCR) signal integration is controversial. While GPCR assembly into hetero-oligomers facilitates signal integration of different receptor types, cross-talk between Gαi- and Gαq-coupled receptors is often thought to be oligomerization independent. In this study, we examined the mechanism of signal integration between the Gαi-coupled type I cannabinoid receptor (CB(1)R) and the Gαq-coupled AT1R. We find that these two receptors functionally interact, resulting in the potentiation of AT1R signalling and coupling of AT1R to multiple G proteins. Importantly, using several methods, that is, co-immunoprecipitation and resonance energy transfer assays, as well as receptor- and heteromer-selective antibodies, we show that AT1R and CB(1)R form receptor heteromers. We examined the physiological relevance of this interaction in hepatic stellate cells from ethanol-administered rats in which CB(1)R is upregulated. We found a significant upregulation of AT1R-CB(1)R heteromers and enhancement of angiotensin II-mediated signalling, as compared with cells from control animals. Moreover, blocking CB(1)R activity prevented angiotensin II-mediated mitogenic signalling and profibrogenic gene expression. These results provide a molecular basis for the pivotal role of heteromer-dependent signal integration in pathology.

Osteopontin binding to lipopolysaccharide lowers tumor necrosis factor-α and prevents early alcohol-induced liver injury in mice.

UNLABELLED: Although osteopontin (OPN) is induced in alcoholic patients, its role in the pathophysiology of alcoholic liver disease (ALD) remains unclear. Increased translocation of lipopolysaccharide (LPS) from the gut is key for the onset of ALD because it promotes macrophage infiltration and activation, tumor necrosis factor-α (TNFα) production, and liver injury. Since OPN is protective for the intestinal mucosa, we postulated that enhancing OPN expression in the liver and consequently in the blood and/or in the gut could protect from early alcohol-induced liver injury. Wild-type (WT), OPN knockout (Opn(-/-)), and transgenic mice overexpressing OPN in hepatocytes (Opn(HEP) Tg) were fed either the control or the ethanol Lieber-DeCarli diet. Ethanol increased hepatic, plasma, biliary, and fecal OPN more in Opn(HEP) Tg than in WT mice. Steatosis was less in ethanol-treated Opn(HEP) Tg mice as shown by decreased liver-to-body weight ratio, hepatic triglycerides, the steatosis score, oil red-O staining, and lipid peroxidation. There was also less inflammation and liver injury as demonstrated by lower alanine aminotransferase (ALT) activity, hepatocyte ballooning degeneration, LPS levels, the inflammation score, and the number of macrophages and TNFα(+) cells. To establish if OPN could limit LPS availability and its noxious effects in the liver, binding studies were performed. OPN showed binding affinity for LPS which prevented macrophage activation, reactive oxygen, and nitrogen species generation and TNFα production. Treatment with milk OPN (m-OPN) blocked LPS translocation in vivo and protected from early alcohol-induced liver injury. CONCLUSION: Natural induction plus forced overexpression of OPN in the liver or treatment with m-OPN protect from early alcohol-induced liver injury by blocking the gut-derived LPS and TNFα effects in the liver.

Osteopontin induces ductular reaction contributing to liver fibrosis.

OBJECTIVE: In human chronic liver disease, there is association between ductular reaction (DR) and fibrosis; yet, the mechanism triggering its onset and its role in scar formation remains unknown. Since we previously showed that osteopontin (OPN) is highly induced during drug-induced liver fibrosis, we hypothesised that OPN could drive oval cells (OC) expansion and DR and signal to hepatic stellate cells (HSC) to promote scarring. RESULTS: In vivo studies demonstrated increased OPN expression in biliary epithelial cells (BEC) and in OC in thioacetamide (TAA)-treated mice. OPN ablation protected mice from TAA and bile duct ligation-induced liver injury, DR and scarring. This was associated with greater hepatocyte proliferation, lower OC expansion and DR along with less fibrosis, suggesting that OPN could activate the OC compartment to differentiate into BEC, which could then signal to HSC to enhance scarring. Since TAA-treated wild-type mice and cirrhotic patients showed TGF-ß(+) BEC, which were lacking in TAA-treated Opn(-/-) mice and in healthy human explants, this suggested that OPN could regulate TGF-ß, a profibrogenic factor. In vitro experiments confirmed that recombinant OPN (rOPN) decreases hepatocyte proliferation and increases OC and BEC proliferation. To evaluate how BEC regulate collagen-I production in HSC, co-cultures were established. Co-cultured BEC upregulated OPN and TGF-ß expression and enhanced collagen-I synthesis by HSC. Lastly, recombinant TGF-ß (rTGFß) and rOPN promoted BEC proliferation and neutralisation of OPN and TGF-ß reduced collagen-I expression in co-cultured HSC. CONCLUSIONS: OPN emerges as a key matricellular protein driving DR and contributing to scarring and liver fibrosis via TGF-ß.

Ethanol plus the Jo2 Fas agonistic antibody-induced liver injury is attenuated in mice with partial ablation of argininosuccinate synthase.

BACKGROUND: Argininosuccinate synthase (ASS) is an enzyme shared by the urea cycle and the l-citrulline/nitric oxide (NO·) cycle. ASS is the rate-limiting enzyme in the urea cycle and along with nitric oxide synthase 2 (NOS2), it endows cells with the l-citrulline/NO· salvage pathway to continuously supply l-arginine from l-citrulline for sustained NO· generation. Thus, ASS conditions NO· synthesis by NOS2. Because of the relevance of NOS2 activation for liver injury, we examined the contribution of ASS to NO· generation and how it impacts liver injury. METHODS: Wild-type (WT) mice and Ass(+/-) mice (Ass(-/-) mice are lethal) were intraperitoneally injected with ethanol (EtOH) at a dose of 2.5 g/kg of body weight twice a day for 3 days. Two hours after the last dose of EtOH, mice were administered the agonistic Jo2 anti-mouse Fas monoclonal antibody (Ab) at a dose of 0.2 µg/g of body weight. Mice were sacrificed 8 hours after the Jo2 Ab injection. Markers of nitrosative and oxidative stress as well as liver damage were analyzed. RESULTS: EtOH plus Jo2 injection induced liver injury as shown by serum alanine aminotransferase and aspartate aminotransferase activity, liver pathology, TUNEL, and cleaved caspase-3 were lower in Ass(+/-) mice compared with WT mice, suggesting that ASS contributes to EtOH plus Jo2-mediated liver injury. CYP2E1 induction, glutathione depletion, and elevated thiobarbituric acid reactive substances were comparable in both groups of mice, suggesting that CYP2E1-mediated oxidative stress is not linked to ASS-induced liver injury. In contrast, NOS2 induction, 3-nitrotyrosine adducts formation and elevated nitrites, nitrates, and S-nitrosothiols were higher in livers from WT mice than from Ass(+/-) mice. CONCLUSION: Decreased nitrosative stress causes lower EtOH plus Jo2-induced liver injury in Ass(+/-) mice.

A Protein Complex of Liver Origin Activates a Pro-inflammatory Program That Drives Hepatic and Intestinal Injury in Alcohol-Associated Liver Disease.

BACKGROUND & AIMS: There is limited information on how the liver-to-gut axis contributes to alcohol-associated liver disease (AALD). We previously identified that high-mobility group box-1 (HMGB1) undergoes oxidation in hepatocytes and demonstrated elevated serum levels of oxidized HMGB1 ([O] HMGB1) in alcoholic patients. Since interleukin-1 beta (IL-1B) increases in AALD, we hypothesized hepatocyte-derived [O] HMGB1 could interact with IL-1B to activate a pro-inflammatory program that, besides being detrimental to the liver, drives intestinal barrier dysfunction. RESULTS: Alcohol-fed RageΔMye mice exhibited decreased nuclear factor kappa B signaling, a pro-inflammatory signature, and reduced total intestinal permeability, resulting in protection from AALD. In addition, [O] HMGB1 bound and signaled through the receptor for advanced-glycation end-products (RAGE) in myeloid cells, driving hepatic inflammation, intestinal permeability, and increased portal blood lipopolysaccharide in AALD. We identified that [O] HMGB1 formed a complex with IL-1B, which was found in the livers of patients with acute alcoholic hepatitis and mice with AALD. This complex originated from the liver, because it was absent in the intestine when hepatocytes did not produce [O] HMGB1. Mechanistically, the complex bound RAGE in Kupffer cells and macrophages induces a pro-inflammatory program. Moreover, it bound RAGE in intestinal macrophages and epithelial cells, leading to intestinal inflammation, altered intestinal epithelial cell tight junction protein expression, increased intestinal permeability, and elevated portal blood lipopolysaccharide, enhancing AALD pathogenesis. CONCLUSIONS: We identified a protein complex of liver origin that amplifies the pro-inflammatory feedback loop in AALD; therefore, targeting this complex could have significant therapeutic potential.

Gut-derived ammonia contributes to alcohol-related fatty liver development via facilitating ethanol metabolism and provoking ATF4-dependent de novo lipogenesis activation.

BACKGROUND & AIMS: Dysbiosis contributes to alcohol-associated liver disease (ALD); however, the precise mechanisms remain elusive. Given the critical role of the gut microbiota in ammonia production, we herein aim to investigate whether and how gut-derived ammonia contributes to ALD. METHODS: Blood samples were collected from human subjects with/without alcohol drinking. Mice were exposed to the Lieber-DeCarli isocaloric control or ethanol-containing diets with and without rifaximin (a nonabsorbable antibiotic clinically used for lowering gut ammonia production) supplementation for five weeks. Both in vitro (NH4Cl exposure of AML12 hepatocytes) and in vivo (urease administration for 5 days in mice) hyperammonemia models were employed. RNA sequencing and fecal amplicon sequencing were performed. Ammonia and triglyceride concentrations were measured. The gene and protein expression of enzymes involved in multiple pathways were measured. RESULTS: Chronic alcohol consumption causes hyperammonemia in both mice and human subjects. In healthy livers and hepatocytes, ammonia exposure upregulates the expression of urea cycle genes, elevates hepatic de novo lipogenesis (DNL), and increases fat accumulation. Intriguingly, ammonia promotes ethanol catabolism and acetyl-CoA formation, which, together with ammonia, synergistically facilitates intracellular fat accumulation in hepatocytes. Mechanistic investigations uncovered that ATF4 activation, as a result of ER stress induction and general control nonderepressible 2 activation, plays a central role in ammonia-provoked DNL elevation. Rifaximin ameliorates ALD pathologies in mice, concomitant with blunted hepatic ER stress induction, ATF4 activation, and DNL activation. CONCLUSIONS: An overproduction of ammonia by gut microbiota, synergistically interacting with ethanol, is a significant contributor to ALD pathologies.

Ablation of secreted phosphoprotein-1 in hepatocytes increases fatty acid oxidation and ameliorates alcohol-associated liver disease.

BACKGROUND: Previously, we demonstrated that Spp1-/- mice exhibit a greater susceptibility to alcohol-induced liver injury than wild-type (WT) mice. Notably, alcohol triggers the expression of osteopontin (encoded by SPP1) in hepatocytes. However, the specific role of hepatocyte-derived SPP1 in either mitigating or exacerbating alcohol-associated liver disease (AALD) has yet to be elucidated. We hypothesized that hepatocyte-derived SPP1 plays a role in AALD by modulating the regulation of steatosis. METHODS: We analyzed hepatic SPP1 expression using four publicly available datasets from patients with alcoholic hepatitis (AH). Additionally, we examined SPP1 expression in the livers of WT mice subjected to either a control or ethanol Lieber-DeCarli (LDC) diet for 6 weeks. We compared the relationship between SPP1 expression and significantly dysregulated genes in AH with controls using correlation and enrichment analyses. To investigate the specific impact of hepatocyte-derived SPP1, we generated hepatocyte-specific Spp1 knock-out (Spp1ΔHep) mice and subjected them to either a control or ethanol Lieber-DeCarli diet for 6 weeks. RESULTS: Alcohol induced hepatic SPP1 expression in both humans and mice. Our analysis, focusing on genes correlated with SPP1, revealed an enrichment of fatty acid oxidation (FAO) in three datasets, and peroxisome proliferator-activated receptor signaling in one dataset. Notably, FAO genes correlating with SPP1 were downregulated in patients with AH. Ethanol-fed WT mice exhibited higher serum-free fatty acids (FFAs), adipose tissue lipolysis, and hepatic fatty acid (FA) transporters. In contrast, ethanol-fed Spp1ΔHep mice displayed lower liver triglycerides, FFAs, and serum alanine transaminase and greater FAO gene expression than WT mice, indicating a protective effect against AALD. Primary hepatocytes from Spp1∆Hep mice exhibited heightened expression of genes encoding proteins involved in FAO. CONCLUSIONS: Alcohol induces the expression of SPP1 in hepatocytes, leading to impaired FAO and contributing to the development of AALD.