BackgroundIn pediatric liver transplantation (pLT), long-term immunosuppression (IS) contributes to infection risk and chronic toxicity, yet IS minimization or withdrawal requires balancing rejection risk. Practical biomarkers for baseline risk stratification at the start of planned withdrawal remain scarce. This study investigated whether baseline neutrophil-associated proteomic signatures and histone deacetylase 1 (HDAC1) levels are associated with IS withdrawal outcomes.MethodsWithin an institutional IS withdrawal program (n = 77), 59 recipients had evaluable outcomes by the follow-up cut-off (June 30, 2025). Among these, 31 pLT recipients underwent planned IS withdrawal (primary analytic cohort), and baseline plasma from 10 patients was analyzed via liquid chromatography-mass spectrometry (LC-MS) proteomics to identify tolerance-associated proteins and pathways. HDAC1 was subsequently quantified by ELISA in 39 recipients with baseline plasma available. The diagnostic performance of HDAC1 in distinguishing immune-tolerant (IT) from non-immune-tolerant (NIT) outcomes was evaluated using receiver operating characteristic (ROC) analysis. To corroborate tissue-level consistency, HDAC1 expression was assessed by immunohistochemistry (IHC) in baseline liver biopsy sections from 10 recipients (5 IT and 5 NIT) selected from the planned withdrawal cohort.ResultsProteomic profiling revealed distinct baseline differences enriched in neutrophil-related functions, including pathways linked to degranulation and neutrophil extracellular trap (NET) formation. HDAC1 was identified as a key candidate marker, with significantly lower baseline levels observed in the IT group. In the validation cohort, plasma HDAC1 demonstrated moderate discriminative performance for baseline risk stratification (AUC = 0.81). Furthermore, IHC analysis of baseline liver biopsies showed lower intrahepatic HDAC1 staining in IT recipients compared to the NIT group, consistent with the systemic plasma findings.ConclusionsBaseline neutrophil-linked proteomic signals and diminished HDAC1 expression are associated with successful IS withdrawal in pLT recipients. These findings support HDAC1 as a hypothesis-generating candidate biomarker for baseline risk stratification and provide a clinically oriented framework to refine patient selection and enhance early monitoring during IS minimization and withdrawal protocols.

BACKGROUND & AIMS: The incidence of graft fibrosis elevated after pediatric liver transplantation (pLT) and influenced by cold ischemic time (CIT). Myosin light chain (MYL9), a member of the myosin family, could act on hepatic stellate cells (HSCs) and induce a transition to active phase. hypothesized that cold ischemic injury could stimulate expression and lead to graft fibrosis. METHODS: We tested the hypothesis by analyzing multi-omics data from human protocol liver biopsy samples 2 years LT, performing rat LT with different CIT and conducting in studies in HSC cell lines with MYL9 knockdown and overexpression. RESULTS: Clinically, CIT is an independent risk factor for graft fibrosis after pLT. Omics analysis identified MYL9 as a prominent contributor in graft fibrosis. MYL9 is strongly correlated with liver fibrosis grade and the progression of fibrosis. The study of rat LT model demonstrated MYL9 expression increases with the prolongation of CIT, and its role is specific to transplant setting. Mechanistically, in vitro experiments with HSCs exposed to hypoxia/reoxygenation revealed a substantial decrease in HSCs activation after MYL9 knockdown. Conversely, overexpression of MYL9 significantly enhanced the activation of HSCs. Subsequent transcriptome sequencing of HSCs with MYL9 knockdown unveiled that MYL9 primarily functions through the TLR4/MYD88/NF-KB signaling pathway. Liver graft fibrosis was ameliorated when toll like receptor 4 signaling was inhibited in rats. CONCLUSIONS: Our findings demonstrate that prolonged CIT up- regulates the expression of MYL9 in liver graft after LT. MYL9 activates HSCs and promotes fibrosis through a TLR4/MYD88/NF-KB signaling dependent manner. (Cell Mol Gastroenterol Hepatol 2025;19:101453; https://doi.org/10.1016/j.jcmgh.2024.101453)

BACKGROUND & AIMS:The gut-liver axis plays a critical role in metabolic dysfunction-associated steatohepatitis (MASH). Osteopontin (OPN, encoded by SPP1) is implicated in chronic liver disease; however, its expression in intestinal epithelial cells (IECs) and role in MASH remain unclear. METHODS:We evaluated intestinal OPN expression during MASH progression in patients. To determine the function of IEC-derived OPN, we generated Spp1 knock-in (Spp1KI IEC) and knock-out (Spp1ΔIEC) mice and fed them a high-fat, high-fructose, high-cholesterol diet to induce MASH. RESULTS:IEC OPN expression decreased with MASH progression and was inversely associated with liver injury. Loss of Spp1 in IECs exacerbated MASH, whereas overexpression or oral OPN administration was protective. Spp1ΔIEC mice exhibited increased hepatic inflammation, disrupted IEC morphology, elevated IEC apoptosis, reduced epithelial cell turnover, and heightened intestinal permeability. They also showed hepatic 16s rRNA presence and elevated conjugated bile acids (BAs), particularly taurocholic acid and taurodeoxycholic acid, in portal serum. These BAs promoted hepatocyte injury and activated liver macrophages, enhancing inflammation both in vitro and in vivo. Fecal microbiome analysis revealed reduced abundance of bile salt hydrolase-expressing bacteria. Fecal microbiota transplantation from Spp1ΔIEC mice or treatment with a bile salt hydrolase inhibitor further worsened MASH. CONCLUSIONS:IEC-derived OPN protects against MASH by modulating BA composition and shaping the gut microbiome.

The effectiveness of bone marrow mesenchymal stem cells (BMSCs) in post-transplantation liver fibrosis has not been studied. The aim of this study was to investigate the effect of BMSCs on liver fibrosis and their role in the Janus-activated kinase (JAK) 1/ signal transducer and activator of transcription (STAT) 5 pathway after liver transplantation (LT). A rat model of post-LT liver fibrosis induced by cold ischemia injury was successfully established. BMSCs were injected into the rats through the portal vein. Hepatic stellate cell (HSC)-T6 were co-cultured with BMSCs in vitro after hypoxia–reoxygenation. JAK1 inhibitor Abrocitinib and JAK1 agonist RO8191 were used to study the JAK1/STAT5 signaling pathway. BMSCs significantly alleviated liver fibrosis caused by cold ischemia–reperfusion injury after rat LT in vivo. After BMSCs transplantation, the levels of JAK1 and p-STAT5 in rat liver were significantly reduced. After using Abrocitinib, the stage of liver fibrosis and the levels of collagen type I alpha 1 chain (COL1A1) and actin alpha 2 (ACTA2) decreased. After using RO8191, the stage of liver fibrosis and the levels of COL1A1 and ACTA2 increased. BMSCs significantly reduced the activation of HSC-T6 after hypoxia–reoxygenation in vitro. After co-culturing with BMSCs after HSC-T6 hypoxia–reoxygenation, the levels of JAK1 and p-STAT5 were significantly reduced. After the addition of Abrocitinib, the levels of COL1A1 and ACTA2 decreased in HSC-T6; in contrast, after adding RO8191, the levels of COL1A1 and ACTA2 increased in HSC-T6 after hypoxia–reoxygenation. After using anti-IL7 antibody or anti-IL7Rα in vivo and in vitro, the stage of liver fibrosis and the levels of COL1A1 and ACTA2 decreased as well as the phosphorylation level of STAT5. BMSCs alleviate hepatic cell damage, reduce hepatic cell-derived IL7, downregulate IL7R/JAK1/STAT5 in HSCs, thereby reducing HSCs’ activation and ultimately alleviating liver fibrosis after liver transplantation. BMSCs can alleviate liver fibrosis after rat liver transplantation in vivo. BMSCs can reduce activation of HSCs after hepoxia-reoxygenation in vitro. BMSCs inhibit activation of HSCs after injury through the JAK1/STAT5 pathway, thereby reducing liver fibrosis after liver transplantation.

High mobility group box 1 (HMGB1) is a non-histone chromatin-associated protein involved in the pathogenesis of chronic liver disease. HMGB1 is expressed in myeloid cells, including conventional dendritic cells (cDCs), which play a major role in the tumor microenvironment. However, whether intracellular myeloid cell-derived HMGB1 is involved in hepatocellular carcinoma (HCC) is unknown. We hypothesize that intracellular HMGB1 drives cDC maturation towards LAMP3+ DCs, allowing effective cytotoxic CD8+ T cell responses to reduce HCC. We analyzed publicly available scRNA-seq datasets from human HCC for the expression of HMGB1 in all subsets of DCs in HCC tumor and non-tumor tissues and in hepatic draining lymph nodes (dLNs). We generated mice with conditional ablation or overexpression of Hmgb1 in myeloid cells (Hmgb1ΔMye and Hmgb1KI Mye). To induce HCC, 14-day-old male mice were injected i.p. with diethylnitrosamine (DEN) and were sacrificed at 5, 6, 8, and 10 months. Histopathological analysis of livers was performed using H&E staining. Immune cell populations were analyzed in tumor and non-tumor tissues using flow cytometry. cDCs were isolated from tumor and non-tumor tissues by FACS, and RNA-seq analysis was performed. Analysis of scRNA-seq datasets revealed LAMP3+DCs increased in human HCC tumor tissue and hepatic dLNs. LAMP3+DCs displayed increased activation and migration scores. Moreover, HMGB1 was highly expressed in cDCs (CLEC9A+and CD1C+), which can differentiate to LAMP3+DCs. Hmgb1KI Myemice were protected from HCC, whereas control mice developed HCC after 8 months, and Hmgb1ΔMyemice started developing HCC at 5 months. Macroscopic analysis and H&E staining of the livers from Hmgb1ΔMyemice showed more tumors and higher tumor volume than control and Hmgb1KI Myemice. Immunohistochemistry of HCC tumor sections revealed that Hmgb1ΔMye mice have fewer LAMP3+DCs in tumor tissues than control and Hmgb1KI Myemice. Flow cytometry analysis showed that Hmgb1KI Myemice had significantly increased cDCs in tumor tissues and dLNs than control and Hmgb1ΔMye mice. In addition, there was enhanced CD8+T cell apoptosis in the HCC tumor tissues from Hmgb1ΔMyemice. RNA-seq data of cDCs unveiled that pathways involved in antigen presentation were downregulated in Hmgb1ΔMye mice and upregulated in Hmgb1KI Mye mice compared to control mice. Ablation of myeloid-derived HMGB1 accelerates HCC development in mice. Enhancing HMGB1 expression in myeloid cells increased the number and the antigen presentation activity of cDCs. Therefore, increasing HMGB1 expression in cDCs could be a therapeutic approach to protect from HCC. Sai Santosh Babu Komakula, Xiaodong Ge, Wei Chen, Romain Desert, Hui Han, Zhuolun Song, Sukanta Das, Dipti Athavale, Nithyananthan Subramaniyam, Chao Wang, Daniel Lantvit, Abid Anwar, Natalia Nieto. The role of myeloid cell derived HMGB1 in the development of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7240.

Background: We previously identified that high-mobility group box-1 (HMGB1) is increased and undergoes post-translational modifications (PTMs) in response to alcohol consumption. Here, we hypothesized that specific PTMs, occurring mostly in hepatocytes and myeloid cells, could contribute to the pathogenesis of alcohol-associated liver disease (AALD). Methods: We used the Lieber-DeCarli (LD) model of early alcohol-induced liver injury, combined with engineered viral vectors and genetic approaches to regulate the expression of HMGB1, its PTMs (reduced [H], oxidized [O], acetylated [Ac], both [O + Ac]), and its receptors (RAGE, TLR4) in a cell-specific manner (hepatocytes and/or myeloid cells). Results: Hmgb1 ablation in hepatocytes or myeloid cells partially protected, while ablation in both prevented steatosis, inflammation, IL1B production, and alcohol-induced liver injury. Hepatocytes were a major source of [H], [O], and [Ac] HMGB1, whereas myeloid cells produced only [H] and [Ac] HMGB1. Neutralization of HMGB1 prevented, whereas injection of [H] HMGB1 increased AALD, which was worsened by injection of [O] HMGB1. While [O] HMGB1 induced liver injury, [Ac] HMGB1 protected and counteracted the effects of [O] HMGB1 in AALD. [O] HMGB1 stimulated macrophage (MF) migration, activation, IL1B production, and secretion. Ethanol-fed Rage ΔMye but not Tlr4 ΔMye, Rage ΔHep, or Tlr4 ΔHep mice were protected from AALD, indicating a crucial role of RAGE in myeloid cells for AALD. [O] HMGB1 recruited and activated myeloid cells through RAGE and contributed to steatosis, inflammation, and IL1B production in AALD. Conclusions: These results provide evidence for targeting [O] HMGB1 of hepatocyte origin as a ligand for RAGE signaling in myeloid cells and a driver of steatosis, inflammatory cell infiltration, and IL1B production in AALD. Importantly, we reveal that [Ac] HMGB1 offsets the noxious effects of [O] HMGB1 in AALD.

Objectives Previous studies elucidated that capecitabine (CAP) works as an anti-tumor agent with putative immunosuppressive effects. However, the intricate mechanisms underpinning these effects remain to be elucidated. In this study, we aimed to unravel the molecular pathways by which CAP exerts its immunosuppressive effects to reduce allograft rejection. Methods Hearts were transplanted from male BALB/c donors to male C57BL/6 recipients and treated with CAP for seven days. The rejection of these heart transplants was assessed using a range of techniques, including H&E staining, immunohistochemistry, RNA sequencing, LS-MS/MS, and flow cytometry. In vitro, naïve CD4+ T cells were isolated and cultured under Th1 condition medium with varying treatments, flow cytometry, LS-MS/MS were employed to delineate the role of thymidine synthase (TYMS) during Th1 differentiation. Results CAP treatment significantly mitigated acute allograft rejection and enhanced graft survival by reducing graft damage, T cell infiltration, and levels of circulating pro-inflammatory cytokines. Additionally, it curtailed CD4+ T cell proliferation and the presence of Th1 cells in the spleen. RNA-seq showed that TYMS, the target of CAP, was robustly increased post-transplantation in splenocytes. In vitro, TYMS and its metabolic product dTMP were differentially expressed in Th0 and Th1, and were required after activation of CD4+ T cell and Th1 differentiation. TYMS-specific inhibitor, raltitrexed, and the metabolite of capecitabine, 5-fluorouracil, could inhibit the proliferation and differentiation of Th1. Finally, the combined use of CAP and the commonly used immunosuppressant rapamycin can induce long-term survival of allograft. Conclusion CAP undergoes metabolism conversion to interfere pyrimidine metabolism, which targets TYMS-mediated differentiation of Th1, thereby playing a significant role in mitigating acute cardiac allograft rejection in murine models.

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. (c) 2023 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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.

Background. Optimizing the immunosuppressive regimen is essential to improve the long-term outcomes of pediatric liver transplant recipients. Methods. We conducted a prospective, randomized, open-label study to compare the safety and efficacy of 2 treatment approaches during pediatric liver transplantation: tacrolimus monotherapy following basiliximab induction (the study group) and a dual regimen of tacrolimus plus steroids (the control group). A total of 150 patients were enrolled, with 75 patients allocated to each group. Results. In both groups, recipients achieved graft and recipient overall survival rates exceeding 93%, with no statistically significant differences between them. However, the study group exhibited a significantly lower incidence of acute cellular rejection (ACR), delayed occurrence of ACR, and an improved ACR-free survival rate at 2 y compared with the control group. Notably, the study group also showed a significant reduction in the incidence of de novo donor-specific antibodies at 3-mo and 2-y posttransplant. Furthermore, 6 mo after the transplant, the study group demonstrated significant improvements in weight-for-age Z score and height-for-age Z score. No notable differences were observed in postoperative complications or the incidence of liver fibrosis between the 2 groups. Conclusions. Basiliximab induction combine with tacrolimus (TAC) monotherapy is a safe and effective immunosuppressive regimen to reduce the episodes of ACR without influencing the development of liver fibrosis and graft and recipient survival rate after pediatric liver transplantation.

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 induced 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.

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.

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.

BACKGROUND & AIMSNon-alcoholic 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 macrophage-derived OPN affects NASH progression is unknown.METHODSWe 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.RESULTSThis study demonstrated that MFs expressing high SPP1 are enriched in patients and mice with NAFLD, and show metabolic but not inflammatory properties. Spp1KI Mye or Spp1KI LvMF conferred protection, whereas OpnΔMye 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.CONCLUSIONMF-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 NAFLD patients.

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.