Cell-cell communication via ligand-receptor signaling is a fundamental feature of complex organs. Despite this, the global landscape of intercellular signaling in mammalian liver has not been ...elucidated. Here we perform single-cell RNA sequencing on non-parenchymal cells isolated from healthy and NASH mouse livers. Secretome gene analysis revealed a highly connected network of intrahepatic signaling and disruption of vascular signaling in NASH. We uncovered the emergence of NASH-associated macrophages (NAMs), which are marked by high expression of triggering receptors expressed on myeloid cells 2 (Trem2), as a feature of mouse and human NASH that is linked to disease severity and highly responsive to pharmacological and dietary interventions. Finally, hepatic stellate cells (HSCs) serve as a hub of intrahepatic signaling via HSC-derived stellakines and their responsiveness to vasoactive hormones. These results provide unprecedented insights into the landscape of intercellular crosstalk and reprogramming of liver cells in health and disease.
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•Heterogeneity and plasticity of non-parenchymal cells in healthy and NASH liver•Landscape of intrahepatic ligand-receptor signaling at single-cell resolution•Emergence of Trem2+ NASH-associated macrophages (NAMs) in mouse and human NASH•Stellakine secretion and contractile response to vasoactive hormones by HSCs
This work illustrates the heterogeneity of liver non-parenchymal cells (NPCs) and their reprogramming during NASH pathogenesis. Using single-cell RNA-sequencing analysis, the authors mapped the landscape of the intrahepatic ligand-receptor signaling network and revealed two fundamental aspects of HSC biology: stellakine secretion and contractile response to vasoactive hormones. Hepatic vascular dysfunction and emergence of Trem2+ NASH-associated macrophages (NAMs) are two conserved features of mouse and human NASH.
Background and Aims
Nonalcoholic steatohepatitis (NASH) is a progressive liver disease that is characterized by liver injury, inflammation, and fibrosis. NASH pathogenesis is linked to reprogramming ...of chromatin landscape in the liver that predisposes hepatocytes to stress‐induced tissue injury. However, the molecular nature of the putative checkpoint that maintains chromatin architecture and preserves hepatocyte health remains elusive.
Approach and Results
Here we show that heterogeneous nuclear ribonucleoprotein U (hnRNPU), a nuclear matrix protein that governs chromatin architecture and gene transcription, is a critical factor that couples chromatin disruption to NASH pathogenesis. RNA‐seq and chromatin immunoprecipitation‐seq studies revealed an extensive overlap between hnRNPU occupancy and altered gene expression during NASH. Hepatocyte‐specific inactivation of hnRNPU disrupted liver chromatin accessibility, activated molecular signature of NASH, and sensitized mice to diet‐induced NASH pathogenesis. Mechanistically, hnRNPU deficiency stimulated the expression of a truncated isoform of TrkB (TRKB‐T1) that promotes inflammatory signaling in hepatocytes and stress‐induced cell death. Brain‐derived neurotrophic factor treatment reduced membrane TRKB‐T1 protein and protected mice from diet‐induced NASH.
Conclusions
These findings illustrate a mechanism through which disruptions of chromatin architecture drive the emergence of disease‐specific signaling patterns that promote liver injury and exacerbate NASH pathogenesis.
Skeletal muscle and thermogenic adipose tissue are both critical for the maintenance of body temperature in mammals. However, whether these two tissues are interconnected to modulate thermogenesis ...and metabolic homeostasis in response to thermal stress remains inconclusive. Here, we report that human and mouse obesity is associated with elevated Musclin levels in both muscle and circulation. Intriguingly, muscle expression of Musclin is markedly increased or decreased when the male mice are housed in thermoneutral or chronic cool conditions, respectively. Beige fat is then identified as the primary site of Musclin action. Muscle-transgenic or AAV-mediated overexpression of Musclin attenuates beige fat thermogenesis, thereby exacerbating diet-induced obesity and metabolic disorders in male mice. Conversely, Musclin inactivation by muscle-specific ablation or neutralizing antibody treatment promotes beige fat thermogenesis and improves metabolic homeostasis in male mice. Mechanistically, Musclin binds to transferrin receptor 1 (Tfr1) and antagonizes Tfr1-mediated cAMP/PKA-dependent thermogenic induction in beige adipocytes. This work defines the temperature-sensitive myokine Musclin as a negative regulator of adipose thermogenesis that exacerbates the deterioration of metabolic health in obese male mice and thus provides a framework for the therapeutic targeting of this endocrine pathway.
The maintenance of DNA methylation in nascent DNA is a critical event for numerous biological processes. Following DNA replication, DNMT1 is the key enzyme that strictly copies the methylation ...pattern from the parental strand to the nascent DNA. However, the mechanism underlying this highly specific event is not thoroughly understood. In this study, we identified topoisomerase IIα (TopoIIα) as a novel regulator of the maintenance DNA methylation. UHRF1, a protein important for global DNA methylation, interacts with TopoIIα and regulates its localization to hemimethylated DNA. TopoIIα decatenates the hemimethylated DNA following replication, which might facilitate the methylation of the nascent strand by DNMT1. Inhibiting this activity impairs DNA methylation at multiple genomic loci. We have uncovered a novel mechanism during the maintenance of DNA methylation.UHRF1 is required for the maintenance of DNA methylation.
UHRF1 interacts with topoisomerase II and regulates its localization to pericentric heterochromatin.
Topoisomerase II regulates the maintenance of DNA methylation.
Our study reveals a novel molecular mechanism of the maintenance of DNA methylation.
The liver contains heterogeneous cell types that exhibit distinct spatial, molecular, and functional properties. The cells in the liver communicate with each other via a network of ligands and ...receptors and undergo cell type-specific transcriptomic reprogramming in disease. The advance of single-cell genomics has provided a powerful tool for unraveling the complexity of liver cells in health and disease with unprecedented resolution. In this review, we discuss insights gained from the recent single-cell RNA sequencing studies that shed new light on the molecular nature of liver cell heterogeneity, intercellular crosstalk, and disease-associated reprogramming.
The mammalian liver harbors heterogeneous cell types that communicate via local paracrine signaling. Recent studies have delineated the transcriptomic landscape of the liver in NASH that provides ...insights into liver cell heterogeneity, intercellular crosstalk, and disease-associated reprogramming. However, the nature of intrahepatic signaling and its role in NASH progression remain obscure.
Here, we performed transcriptomic analyses and identified cardiotrophin-like cytokine factor 1 (CLCF1), a member of the IL-6 family cytokines, as a cholangiocyte-derived paracrine factor that was elevated in the liver from diet-induced NASH mice and patients with NASH. Adenovirus-associated virus-mediated overexpression of CLCF1 in the liver ameliorated NASH pathologies in two diet-induced NASH models in mice, illustrating that CLCF1 induction may serve an adaptive and protective role during NASH pathogenesis. Unexpectedly, messenger RNA and protein levels of leukemia inhibitory factor receptor (LIFR), a subunit of the receptor complex for CLCF1, were markedly downregulated in NASH liver. Hepatocyte-specific inactivation of LIFR accelerated NASH progression in mice, supporting an important role of intrahepatic cytokine signaling in maintaining tissue homeostasis under metabolic stress conditions.
Together, this study sheds light on the molecular nature of intrahepatic paracrine signaling during NASH pathogenesis and uncovers potential targets for therapeutic intervention.
The mammalian liver comprises heterogeneous cell types within its tissue microenvironment that undergo pathophysiological reprogramming in disease states, such as non-alcoholic steatohepatitis ...(NASH). Patients with NASH are at an increased risk for the development of hepatocellular carcinoma (HCC). However, the molecular and cellular nature of liver microenvironment remodeling that links NASH to liver carcinogenesis remains obscure. Here, we show that diet-induced NASH is characterized by the induction of tumor-associated macrophage (TAM)-like macrophages and exhaustion of cytotoxic CD8+ T cells in the liver. The adipocyte-derived endocrine factor Neuregulin 4 (NRG4) serves as a hormonal checkpoint that restrains this pathological reprogramming during NASH. NRG4 deficiency exacerbated the induction of tumor-prone liver immune microenvironment and NASH-related HCC, whereas transgenic NRG4 overexpression elicited protective effects in mice. In a therapeutic setting, recombinant NRG4-Fc fusion protein exhibited remarkable potency in suppressing HCC and prolonged survival in the treated mice. These findings pave the way for therapeutic intervention of liver cancer by targeting the NRG4 hormonal checkpoint.
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•NASH induces TAM-like macrophages and T cell exhaustion in the liver•The NRG4 hormonal axis restrains the tumor-prone liver immune microenvironment in NASH•NRG4 functions as a tumor suppressor for NASH-associated liver cancer•Recombinant NRG4 fusion protein is highly potent in suppressing NASH-HCC
This work illustrates an adipose-hepatic hormonal axis that shapes the liver immune microenvironment. The adipose tissue hormone NRG4 serves as a hormonal checkpoint that restrains the tumor-prone liver microenvironment and suppresses NASH-HCC development. Recombinant NRG4-Fc fusion protein elicits potent therapeutic efficacy against NASH-HCC.
5-Hydroxymethylcytosine (5hmC) is an epigenetic modification that is generated by ten-eleven translocation (TET) protein-mediated oxidation of 5-methylcytosine (5mC). 5hmC is associated with ...transcription regulation and is decreased in many cancers including melanoma. Accumulating evidence has suggested that 5hmC is functionally distinct from 5mC. Ubiquitin-like with PHD and ring finger domains 2 (UHRF2) is the first known specific 5hmC reader that has higher affinity to 5hmC than 5mC, suggesting that UHRF2 might mediate 5hmC’s function. Structural analysis has revealed the molecular mechanism of UHRF2–5hmC binding in vitro, but it is not clear how UHRF2 recognizes 5hmC in vivo. In this study, we have identified zinc figure protein 618 (ZNF618) as a novel binding partner of UHRF2. ZNF618 specifically interacts with UHRF2 but not its paralog UHRF1. Importantly, ZNF618 co-localizes with UHRF2 at genomic loci that are enriched for 5hmC. The ZNF618 chromatin localization is independent of its interaction with UHRF2 and is through its first two zinc fingers. Instead, ZNF618 regulates UHRF2 chromatin localization. Collectively, our study suggests that ZNF618 is a key protein that regulates UHRF2 function as a specific 5hmC reader in vivo.
Nonalcoholic steatohepatitis (NASH) is a progressive liver disease that is characterized by liver injury, inflammation and fibrosis. NASH pathogenesis is linked to reprogramming of chromatin ...landscape in the liver that predisposes hepatocytes to stress-induced tissue injury. However, the molecular nature of the putative checkpoint that maintains chromatin architecture and preserves hepatocyte health remains elusive. Here we show that heterogeneous nuclear ribonucleoprotein U (hnRNPU), a nuclear matrix protein that governs chromatin architecture and gene transcription, is a critical factor that couples chromatin disruption to NASH pathogenesis. RNA-seq and ChIP-seq studies revealed an extensive overlap between hnRNPU occupancy and altered gene expression during NASH. Hepatocyte-specific inactivation of hnRNPU disrupted liver chromatin accessibility, activated molecular signature of NASH, and sensitized mice to diet-induced NASH pathogenesis. Mechanistically, hnRNPU deficiency stimulated the expression of a truncated isoform of TrkB (TRKB-T1) that promotes inflammatory signaling in hepatocytes and stress-induced cell death. BDNF treatment reduced membrane TRKB-T1 protein and protected mice from diet-induced NASH. These findings illustrate a novel mechanism through which disruptions of chromatin architecture drive the emergence of disease-specific signaling patterns that promote liver injury and exacerbate NASH pathogenesis.
The liver is a heterogeneous organ comprising a diverse array of cells including parenchymal hepatocytes, and non-parenchymal cells (NPCs) including endothelial cells, hepatic stellate cells, ...cholangiocytes, and various immune cells. These different cell types function in concert to regulate hepatic metabolism and maintain tissue homeostasis. Non-alcoholic fatty liver disease (NAFLD) is a major hepatic comorbidity of metabolic syndrome that is characterized by pathogenic fat accumulation. Chronic NAFLD may progress to non-alcoholic steatohepatitis (NASH), an inflammatory condition that is associated with liver injury, immune cell infiltration, and liver fibrosis, and increases the risk for end-stage liver diseases such as cirrhosis and hepatocellular carcinoma. Despite the prominent roles of NPCs in NASH pathogenesis, the molecular nature of intercellular crosstalk among different liver cell types and their reprogramming in disease remains poorly understood. Single-cell RNA sequencing (scRNA-seq) is a powerful technique for unraveling cellular heterogeneity in complex tissue through profiling transcriptomes of individual cells. In my thesis research, I performed scRNA-seq on NPCs isolated from healthy and diet-induced NASH mouse livers to dissect the paracrine signaling network and to uncover the immune cell landscape in NASH. We found that each cell type exhibited enriched expression of a unique subset of secreted ligands and membrane receptors, in a restricted pattern conserved from mouse to human. We confirmed expression for this paracrine network through quantitative proteomics and found macrophages and stellate cells to be major hubs of ligands and receptors which were upregulated in NASH. The hepatic stellate cells (HSCs) provide a source of stellakines which are predicted to act on endothelial and immune cells. Functionally, we showed that HSCs express a class of G-protein coupled receptors that regulate cellular contractility in response to vasoactive ligands. ScRNA-seq revealed a highly disease-specific population of NASH-associated macrophages (NAMs) marked by abundant expression of marker genes including Trem2 and Gpnmb. NAMs were found at higher levels in both human and mouse NASH and decreased upon dietary and therapeutic interventions for NASH. Their transcriptional profile indicates a propensity for phagocytosis, antigen presentation, and extracellular matrix remodeling, illustrating a potentially important role for NAMs in NASH progression. Tracing hematopoietic cells through bone marrow transplantation, we demonstrated that NAMs originate from the bone marrow and not tissue-resident progenitors. We developed a Trem2 Cre knockin mouse strain to track the emergence of NAMs during NASH progression. Our mouse liver injury and fibrosis assays were unaffected by Trem2 knockout in NASH, indicating Trem2 itself may be unimportant in NASH, but the role of the NAMs they mark are not yet precluded from pathophysiology. Finally, we discovered that NASH is linked to liver CD8+ T cell exhaustion, which is characterized by high levels of PD1 and LAG3 expression and diminished IFNγ, IL2, and TNFα secretion upon T cell stimulation. NASH-associated T cell exhaustion is attenuated by the adipose hormone Neuregulin 4 (NRG4), which protects mice from diet-induced NASH and hepatocellular carcinoma. Taken together, my thesis work has revealed the transcriptomic nature of liver cell heterogeneity, the global landscape of cell-cell signaling in the liver, and NASH-associated NPC reprogramming at a single-cell resolution.
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