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Background and aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant health concern with limited treatment options. AXL, a receptor tyrosine kinase activated by the GAS6 ligand, promotes MASH through activation of hepatic stellate cells and inflammatory macrophages. This study identified cell subsets affected by MASH progression and the effect of AXL inhibition. Methods: Mice were fed chow or different fat-enriched diets to induce MASH, and small molecule AXL kinase inhibition with bemcentinib was evaluated. Gene expression was measured by qPCR. Time-of-flight mass cytometry (CyTOF) used single cells from dissociated livers, acquired on the Fluidigm Helios, and cell populations were studied using machine learning. Results: In mice fed different fat-enriched diets, liver steatosis alone was insufficient to elevate plasma soluble AXL (sAXL) levels. However, in conjunction with inflammation, sAXL increases, serving as an early indicator of steatohepatitis progression. Bemcentinib, an AXL inhibitor, effectively reduced proinflammatory responses in MASH models, even before fibrosis appearance. Utilizing CyTOF analysis, we detected a decreased population of Kupffer cells during MASH while promoting infiltration of monocytes/macrophages and CD8+ T cells. Bemcentinib partially restored Kupffer cells, reduced pDCs and GzmB-NK cells, and increased GzmB+CD8+ T cells and LSECs. Additionally, AXL inhibition enhanced a subtype of GzmB+CD8+ tissue-resident memory T cells characterized by CX3CR1 expression. Furthermore, bemcentinib altered the transcriptomic landscape associated with MASH progression, particularly in TLR signaling and inflammatory response, exhibiting differential cytokine expression in the plasma, consistent with liver repair and decreased inflammation. Conclusion: Our findings highlight sAXL as a biomarker for monitoring MASH progression and demonstrate that AXL targeting shifted liver macrophages and CD8+ T-cell subsets away from an inflammatory phenotype toward fibrotic resolution and organ healing, presenting a promising strategy for MASH treatment. [ABSTRACT FROM AUTHOR]

Background and aims: A complete understanding of disease pathophysiology in advanced liver disease is hampered by the challenges posed by clinical specimen collection. Notably, in these patients, a transjugular liver biopsy (TJB) is the only safe way to obtain liver tissue. However, it remains unclear whether successful sequencing of this extremely small and fragile tissue can be achieved for downstream characterization of the hepatic landscape. Methods: Here we leveraged in-house available single-cell RNA-sequencing (scRNA-seq) and single-nucleus (snRNA-seq) technologies and accompanying tissue processing protocols and performed an in-patient comparison on TJB's from decompensated cirrhosis patients (n = 3). Results: We confirmed a high concordance between nuclear and whole cell transcriptomes and captured 31,410 single nuclei and 6,152 single cells, respectively. The two platforms revealed similar diversity since all 8 major cell types could be identified, albeit with different cellular proportions thereof. Most importantly, hepatocytes were most abundant in snRNA-seq, while lymphocyte frequencies were elevated in scRNA-seq. We next focused our attention on hepatic myeloid cells due to their key role in injury and repair during chronic liver disease. Comparison of their transcriptional signatures indicated that these were largely overlapping between the two platforms. However, the scRNA-seq platform failed to recover sufficient Kupffer cell numbers, and other monocytes/macrophages featured elevated expression of stressrelated parameters. Conclusion: Our results indicate that single-nucleus transcriptome sequencing provides an effective means to overcome complications associated with clinical specimen collection and could sufficiently profile all major hepatic cell types including all myeloid cell subsets. [ABSTRACT FROM AUTHOR]

Acute liver failure (ALF) is a severe liver disease caused by disruptions in the body's immune microenvironment. In the early stages of ALF, Kupffer cells (KCs) become depleted and recruit monocytes derived from the bone marrow or abdomen to replace the depleted macrophages entering the liver. These monocytes differentiate into mature macrophages, which are activated in the immune microenvironment of the liver and polarized to perform various functions. Macrophage polarization can occur in two directions: proinflammatory M1 macrophages and anti-inflammatory M2 macrophages. Controlling the ratio and direction of M1 and M2 in ALF can help reduce liver injury. However, the liver damage caused by pyroptosis should not be underestimated, as it is a caspase-dependent form of cell death. Inhibiting pyroptosis has been shown to effectively reduce liver damage induced by ALF. Furthermore, macrophage polarization and pyroptosis share common binding sites, signaling pathways, and outcomes. In the review, we describe the role of macrophage polarization and pyroptosis in the pathogenesis of ALF. Additionally, we preliminarily explore the relationship between macrophage polarization and pyroptosis, as well as their effects on ALF. [ABSTRACT FROM AUTHOR]

Background: The monocyte-macrophage-dendritic cell (DC) (MMD) system exerts crucial functions that may modulate fibrogenesis in nonalcoholic steatohepatitis (NASH). In this study, we explored the cell characteristics, distribution and developmental trajectory of the liver MMD system in NASH mice with fibrosis and clarified characteristic genes of the MMD system involved in liver fibrosis progression in NASH mice and patients. Methods: Single cells in liver tissue samples from NASH and normal mice were quantified using single-cell RNA sequencing (scRNA-seq) analysis. Differentially expressed genes (DEGs) in the MMD system by pseudotime analysis were validated by tyramide signal amplification (TSA)-immunohistochemical staining (IHC) and analyzed by second harmonic generation (SHG)/two-photon excitation fluorescence (TPEF). Results: Compared with control mice, there were increased numbers of monocytes, Kupffer cells, and DCs in two NASH mouse models. From the transcriptional profiles of these single cells, we identified 8 monocyte subsets (Mono1-Mono8) with different molecular and functional properties. Furthermore, the pseudotime analysis showed that Mono5 and Mono6 were at the beginning of the trajectory path, whereas Mono2, Mono4, Kupffer cells and DCs were at a terminal state. Genes related to liver collagen production were at the late stage of this trajectory path. DEGs analysis revealed that the genes Fmnl1 and Myh9 in the MMD system were gradually upregulated during the trajectory. By TSA-IHC, the Fmnl1 and Myh9 expression levels were increased and associated with collagen production and fibrosis stage in NASH mice and patients. Conclusions: Our transcriptome data provide a novel landscape of theMMDsystem that is involved in advanced NASH disease status. Fmnl1 and Myh9 expression in the MMD system was associated with the progression of NASH fibrosis. [ABSTRACT FROM AUTHOR]

Macrophages are key regulators of inflammation and repair, but their heterogeneity and multiple roles in the liver are not fully understood. We aimed herein to map the intrahepatic macrophage populations and their function(s) during acute liver injury. We used flow cytometry, gene expression analysis, multiplex-immunofluorescence, 3D-reconstruction, and spatial image analysis to characterize the intrahepatic immune landscape in mice post-CCl4-induced acute liver injury during three distinct phases: necroinflammation, and early and late repair. We observed hepatocellular necrosis and a reduction in liver resident lymphocytes during necroinflammation accompanied by the infiltration of circulating myeloid cells and upregulation of inflammatory cytokines. These parameters returned to baseline levels during the repair phase while pro-repair chemokines were upregulated. We identified resident CLEC4F+ Kupffer cells (KCs) and infiltrating IBA1+CLEC4F- monocyte-derived macrophages (MoMFs) as the main hepatic macrophage populations during this response to injury. While occupying most of the necrotic area, KCs and MoMFs exhibited distinctive kinetics, distribution and morphology at the site of injury. The necroinflammation phase was characterized by low levels of KCs and a remarkable invasion of MoMFs suggesting their potential role in phagoctosing necrotic hepatocytes, while opposite kinetics/distribution were observed during repair. During the early repair phase, yolksac - derived KCs were restored, whereas MoMFs diminished gradually then dissipated during late repair. MoMFs interacted with hepatic stellate cells during the necroinflammatory and early repair phases, potentially modulating their activation state and influencing their fibrogenic and pro-repair functions that are critical for wound healing. Altogether, our study reveals novel and distinctspatial and temporal distribution of KCs and MoMFs and provides insights into their complementary roles during acute liver injury. [ABSTRACT FROM AUTHOR]

Alcoholic liver disease (ALD) is a leading chronic liver disease in which immune cells play a vital role. Myeloid cells have been extensively studied in ALD, including granulocytes, macrophages, monocytes, and dendritic cells, which are involved in the occurrence and progression of steatosis, inflammation, fibrosis, and eventual cirrhosis. These cells can be popularly targeted and regulated by factors from different sources, including cytokines secreted by other cells, extracellular vesicles, and substances in serum--for example, infiltration of monocytes or neutrophils, activation of Kupffer cells, and polarization of macrophages. These processes can affect and change the function and phenotype of myeloid cells. Here we mainly review the key mediators that affect the infiltration and function of mainly myeloid cells in ALD as well as their regulatory mechanisms on target cells, which may provide novel immunotherapeutic approaches. The single-cell multimodal omics of myeloid cells is also discussed to help transform them into basic research or therapeutic strategy of ALD clinically. [ABSTRACT FROM AUTHOR]

Our research explores the immunomodulatory effects of sulforaphane (SFN), a well-known nuclear factor erythroid 2-related factor 2 (Nrf2) pathway agonist, on the sterile inflammation of and ischemia-reperfusion injuries to the liver after hemorrhagic shock (HS) followed by resuscitation (R). Male C57/BL6 wild-type and transgenic ARE- luc mice were exposed to mean arterial pressure-controlled HS. Fluid resuscitation was performed after 90 min of HS, and SFN was administrated intraperitoneally after that. The animals were sacrificed at 6 h, 24 h, and 72 h after resuscitation, and their livers were extracted to perform H&E staining and myeloperoxidase (MPO) activity analysis. The Kupffer cells were isolated for cytokines profile measurements and Nrf2 immunofluorescence staining. Further, the ARE- luc mice were used to assess hepatic Nrf2 activity in vivo. We identified that SFN-activated Kupffer cells' Nrf2 pathway and modulated its cytokines expression, including TNF-α, MCP-1, KC/CXCL1, IL-6, and IL-10. Furthermore, SFN mitigated liver ischemia-reperfusion injury, as evidenced by the downregulation of the Suzuki score and the enhanced hepatic Nrf2 activity. The in vivo SFN treatment decreased neutrophils infiltration, as shown by the decreased MPO levels. Our study shows that SFN can decrease HS/R-induced hepatic ischemia-reperfusion injury and modulate the activity of Kupffer cells via an Nrf2-dependent pathway. [ABSTRACT FROM AUTHOR]

Fatty liver disease, characterized by excessive inflammation and lipid deposition, is becoming one of the most prevalent liver metabolic diseases worldwide owing to the increasing global incidence of obesity. However, the underlying mechanisms of fatty liver disease are poorly understood. Accumulating evidence suggests that hepatic macrophages, specifically Kupffer cells (KCs), act as key players in the progression of fatty liver disease. Thus, it is essential to examine the current evidence of the roles of hepatic macrophages (both KCs and monocyte-derived macrophages). In this review, we primarily address the heterogeneities and multiple patterns of hepatic macrophages participating in the pathogenesis of fatty liver disease, including Toll-like receptors (TLRs), NLRP3 inflammasome, lipotoxicity, glucotoxicity, metabolic reprogramming, interaction with surrounding cells in the liver, and iron poisoning. A better understanding of the diverse roles of hepatic macrophages in the development of fatty liver disease may provide a more specific and promising macrophage-targeting therapeutic strategy for inflammatory liver diseases. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that NODs are involved in liver diseases; however, the underlying mechanisms remain obscure. In the present study, we analyzed the effect of NOD1 agonist pretreatment on acute liver failure induced by lipopolysaccharide (LPS) in D-galactosamine (D-GalN)-sensitized mice. We found that pretreatment with the NOD1 agonist markedly reduced LPS/D-GalN-induced mortality, elevation of serum ALT levels, and hepatocyte apoptosis. The protective effect of NOD1 agonist was independent of tumor necrosis factor (TNF)-α inhibition. NOD1 agonist pretreatment also attenuated TNF-α/D-GalN-induced apoptotic liver damage. The anti-apoptotic protein A20 expression was more pronounced in NOD1 agonist pretreated mice than in controls, and knockdown of A20 abrogated the protective effect of NOD1 agonist on LPS/D-GalN-induced liver injury and hepatocyte apoptosis. Further experiments showed that NOD1 agonist-induced A20 upregulation required the presence of kupffer cells and TNF-α. Taken together, our data strongly indicate that NOD1 is involved in the regulation of liver injury and could be a potential therapeutic target for liver diseases. [ABSTRACT FROM AUTHOR]

Macrophages are pivotal in mounting liver inflammatory and tissue repair responses upon hepatic injury, showing remarkable functional plasticity. The molecular mechanisms determining macrophage transition from inflammatory to restorative phenotypes in the damaged liver remain unclear. Using mouse models of acute (APAP) and chronic (CCl4) drug-induced hepatotoxic injury we show that the immune receptor Trem-2 controls phenotypic shifts of liver macrophages and impacts endothelial cell differentiation during tissue recovery. Trem-2 gene ablation led to a delayed re-population of Kupffer cells correlating with deterred resolution of hepatic damage following acute and chronic injury. During tissue recovery, we found that macrophages transitioning to Kupffer cells expressed high levels of Trem-2. Acquisition of the transition phenotype was associated with a unique transcriptomic profile denoting strong responsiveness to oxidative stress and downmodulation of the pro-inflammatory phenotype, which was not observed in absence of Trem-2. During tissue recovery, lack of Trem-2 favored accumulation of a liver-damage associated endothelial cell population (LDECs), whose transcriptional program was compatible with endothelial de-differentiation. Accordingly, LDECs precursor potential is supported by the downregulation of surface endothelial cell markers and by striking in vitro morphological changes towards typical endothelial cells. In conclusion, we found that the dynamics of liver macrophages in response to liver injury are critically controlled by Trem-2 and this regulation is interlinked with the de-differentiation of endothelial cells and heightened liver pathology. We propose that Trem-2 promotes the transition from pro-inflammatory to tissue repair phase by driving the acquisition of restorative properties in phagocytic macrophages. [ABSTRACT FROM AUTHOR]

Kupffer cells, the resident macrophages of the liver, comprise the largest pool of tissue macrophages in the body. Within the liver sinusoids Kupffer cells perform functions common across many tissue macrophages including response to tissue damage and antigen presentation. They also engage in specialized activities including iron scavenging and the uptake of opsonized particles from the portal blood. Here, we review recent studies of the epigenetic pathways that establish Kupffer cell identity and function. We describe a model by which liver-environment specific signals induce lineage determining transcription factors necessary for differentiation of Kupffer cells from bone-marrow derived monocytes. We conclude by discussing how these lineage determining transcription factors (LDTFs) drive Kupffer cell behavior during both homeostasis and disease, with particular focus on the relevance of Kupffer cell LDTF pathways in the setting of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. [ABSTRACT FROM AUTHOR]

Human liver myeloid cells are imperfectly defined, but it is broadly agreed that cells of stellate appearance in situ , expressing the markers CD11b and CD68, are the liver's resident macrophages, classically termed Kupffer cells. Recent investigations using single cell RNA sequencing and unsupervised clustering algorithms suggest there are two populations of cells with the characteristics of tissue macrophages in human liver. We therefore analyzed dissociated human liver tissue using the markers CD11b and CD68 to define macrophage-like cells and found within this population two subsets that differ in their expression of multiple surface markers. These subsets were FACS-sorted based on CD32 expression, and gene expression analysis identified them with human liver myeloid cell subsets that were previously defined by two independent single cell RNA sequencing studies. Using qRT-PCR we found that the two subsets differed in the expression of genes associated with T cell activation and immunosuppression, suggesting distinct roles in T cell tolerance. In addition, one subset expressed two markers, CD1C and CD11c, more often seen on classical dendritic cells. Criteria used to distinguish macrophages from dendritic cells in other tissues may need to be revised in the human liver. [ABSTRACT FROM AUTHOR]

Macrophages play central roles in inflammatory reactions and initiation of immune responses during infections. More than 80% of total tissue macrophages are described to be located in the liver as liver-resident macrophages, also named Kupffer cells (KCs). While studies in mice have established a central role of liver-resident KCs in regulating liver inflammation, their phenotype and function are not well-characterized in humans. Comparing paired human liver and peripheral blood samples, we observed significant differences in the distribution of macrophage (Mφ) subsets, with lower frequencies of CD14hiCD16lo and higher frequencies of CD14int−hiCD16int Mφ in human livers. Intrahepatic Mφ consisted of diverse subsets with differential expression of CD49a, a liver-residency marker previously described for human and mice NK cells, and VSIG4 and/or MARCO, two recently described human tissue Mφ markers. Furthermore, intrahepatic CD49a+ Mφ expressed significantly higher levels of maturation and activation markers, exhibited higher baseline levels of TNF-α, IL-12, and IL-10 production, but responded less to additional in vitro TLR stimulation. In contrast, intrahepatic CD49a− Mφ were highly responsive to stimulation with TLR ligands, similar to what was observed for CD49a− monocytes (MOs) in peripheral blood. Taken together, these studies identified populations of CD49a+, VSIG4+, and/or MARCO+ Mφ in human livers, and demonstrated that intrahepatic CD49a+ Mφ differed in phenotype and function from intrahepatic CD49a− Mφ as well as from peripheral blood-derived monocytes. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide, both in adults and children. It is characterized by an aberrant lipid storage in hepatocytes, named hepatic steatosis. Simple steatosis remains a benign process in most affected patients, while some of them develop superimposed necroinflammatory activity with a non-specific inflammatory infiltrate and a progression to non-alcoholic steatohepatitis with or without fibrosis. Deep similarity and interconnections between innate immune cells and those of liver parenchyma have been highlighted and showed to play a key role in the development of chronic liver disease. The liver can be considered as an "immune organ" because it hosts non-lymphoid cells, such as macrophage Kupffer cells, stellate and dendritic cells, and lymphoid cells. Many of these cells are components of the classic innate immune system, enabling the liver to play a major role in response to pathogens. Although the liver provides a "tolerogenic" environment, aberrant activation of innate immune signaling may trigger "harmful" inflammation that contributes to tissue injury, fibrosis, and carcinogenesis. Pathogen recognition receptors, such as toll-like receptors and nucleotide oligomerization domain-like receptors, are responsible for the recognition of immunogenic signals, and represent the major conduit for sensing hepatic and non-hepatic noxious stimuli. A pivotal role in liver inflammation is also played by cytokines, which can initiate or have a part in immune response, triggering hepatic intracellular signaling pathways. The sum of inflammatory signals and deranged substrate handling induce most of the metabolic alteration traits: insulin resistance, obesity, diabetes, hyperlipidemia, and their compounded combined effects. In this review, we discuss the relevant role of innate immune cell activation in relation to NAFLD, the metabolic complications associated to this pathology, and the possible pharmacological tools. [ABSTRACT FROM AUTHOR]