Diverse potential of secretome from natural killer cells and monocyte-derived macrophages in activating stellate cells.

Chronic liver diseases, such as non-alcoholic steatohepatitis (NASH)-induced cirrhosis, are characterized by an increasing accumulation of stressed, damaged, or dying hepatocytes. Hepatocyte damage triggers the activation of resident immune cells, such as Kupffer cells (KC), as well as the recruitment of immune cells from the circulation toward areas of inflammation. After infiltration, monocytes differentiate into monocyte-derived macrophages (MoMF) which are functionally distinct from resident KC. We herein aim to compare the in vitro signatures of polarized macrophages and activated hepatic stellate cells (HSC) with ex vivo-derived disease signatures from human NASH. Furthermore, to shed more light on HSC activation and liver fibrosis progression, we investigate the effects of the secretome from primary human monocytes, macrophages, and NK cells on HSC activation. Interleukin (IL)-4 and IL-13 treatment induced transforming growth factor beta 1 (TGF-ß1) secretion by macrophages. However, the supernatant transfer did not induce HSC activation. Interestingly, PMA-activated macrophages showed strong induction of the fibrosis response genes COL10A1 and CTGF, while the supernatant of IL-4/IL-13-treated monocytes induced the upregulation of COL3A1 in HSC. The supernatant of PMA-activated NK cells had the strongest effect on COL10A1 induction in HSC, while IL-15-stimulated NK cells reduced the expression of COL1A1 and CTGF. These data indicate that other factors, aside from the well-known cytokines and chemokines, might potentially be stronger contributors to the activation of HSCs and induction of a fibrotic response, indicating a more diverse and complex role of monocytes, macrophages, and NK cells in liver fibrosis progression.

An optimized protocol for isolation of hepatic leukocytes retrieved from murine and NASH liver biopsies.

Immune dysregulation and inflammation by hepatic-resident leukocytes is considered a key step in disease progression of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis toward cirrhosis and hepatocellular carcinoma. Here, we provide a protocol for isolation and characterization of liver-resident immune cells from fine-needle biopsies obtained from a rodent model and humans. We describe steps for isolating leukocytes, cell sorting, and RNA extraction and sequencing. We then detail procedures for low-input mRNA sequencing analyses.

Hepatic C-X-C chemokine receptor type 6-expressing innate lymphocytes limit detrimental myeloid hyperactivation in acute liver injury.

BACKGROUND: Acute liver failure (ALF) is characterized by rapid clinical deterioration and high mortality. Acetaminophen (APAP or paracetamol) overdose is a leading cause of ALF, resulting in hepatocellular necrosis with subsequent inflammation, inflicting further liver damage. Infiltrating myeloid cells are early drivers of liver inflammation. However, the role of the abundant population of liver-resident innate lymphocytes, which commonly express the chemokine receptor CXCR6, is incompletely understood in ALF. METHODS: We investigated the role of CXCR6-expressing innate lymphocytes using the model of acute APAP toxicity in mice deficient in CXCR6 (Cxcr6gfp/gfp). RESULTS: APAP-induced liver injury was strongly aggravated in Cxcr6gfp/gfp mice compared with wild-type counterparts. Immunophenotyping using flow cytometry revealed a reduction in liver CD4+T cells, natural killer (NK) cells, and most prominently, NKT cells, whereas CXCR6 was dispensable for CD8+ T-cell accumulation. CXCR6-deficient mice exhibited excessive neutrophil and inflammatory macrophage infiltration. Intravital microscopy revealed dense cellular clusters of neutrophils in necrotic liver tissue, with higher numbers of clustering neutrophils in Cxcr6gfp/gfp mice. Gene expression analysis linked hyperinflammation in CXCR6 deficiency to increased IL-17 signaling. Although reduced in overall numbers, CXCR6-deficient mice had a shift in NKT cell subsets with increased RORγt-expressing NKT17 cells as a likely source of IL-17. In patients with ALF, we found a prominent accumulation of IL-17-expressing cells. Accordingly, CXCR6-deficient mice lacking IL-17 (Cxcr6gfp/gfpx Il17-/-) had ameliorated liver damage and reduced inflammatory myeloid infiltrates. CONCLUSIONS: Our study identifies a crucial role of CXCR6-expressing liver innate lymphocytes as orchestrators in acute liver injury containing IL-17-mediated myeloid cell infiltration. Hence, strengthening the CXCR6-axis or downstream inhibition of IL-17 could yield novel therapeutics in ALF.

Corrigendum: Heparan Sulfate Induces Necroptosis in Murine Cardiomyocytes: A Medical-in-Silico Approach Combining In Vitro Experiments and Machine Learning.

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Myeloid cells in liver and bone marrow acquire a functionally distinct inflammatory phenotype during obesity-related steatohepatitis.

OBJECTIVE: Bone marrow-derived myeloid cells accumulate in the liver as monocytes and macrophages during the progression of obesity-related non-alcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH). Myeloid cells comprise heterogeneous subsets, and dietary overnutrition may affect macrophages in the liver and bone marrow. We therefore aimed at characterising in depth the functional adaptations of myeloid cells in fatty liver. DESIGN: We employed single-cell RNA sequencing to comprehensively assess the heterogeneity of myeloid cells in the liver and bone marrow during NAFLD, by analysing C57BL/6 mice fed with a high-fat, high-sugar, high-cholesterol 'Western diet' for 16 weeks. We also characterised NAFLD-driven functional adaptations of macrophages in vitro and their functional relevance during steatohepatitis in vivo. RESULTS: Single-cell RNA sequencing identified distinct myeloid cell clusters in the liver and bone marrow. In both compartments, monocyte-derived populations were largely expanded in NASH-affected mice. Importantly, the liver myeloid compartment adapted a unique inflammatory phenotype during NAFLD progression, exemplarily characterised by downregulated inflammatory calprotectin (S100A8/A9) in macrophage and dendritic cell subsets. This distinctive gene signature was also found in their bone marrow precursors. The NASH myeloid phenotype was principally recapitulated by in vitro exposure of bone marrow-derived macrophages with fatty acids, depended on toll-like receptor 4 signalling and defined a characteristic response pattern to lipopolysaccharide stimulation. This imprinted and stable NASH myeloid immune phenotype functionally determined inflammatory responses following acute liver injury (acetaminophen poisoning) in vivo. CONCLUSION: Liver myeloid leucocytes and their bone marrow precursors adapt a common and functionally relevant inflammatory signature during NAFLD progression.

CX3CR1 Mediates the Development of Monocyte-Derived Dendritic Cells during Hepatic Inflammation.

Recent evidence suggests that hepatic dendritic cells (HDCs) contribute to the evolution of chronic liver diseases. However, the HDC subsets involved and the mechanisms driving these responses are still poorly understood. In this study, we have investigated the role of the fractalkine receptor CX3CR1 in modulating monocyte-derived dendritic cell (moDC) differentiation during liver inflammation. The phenotype of HDC and functional relevance of CX3CR1 was assessed in mice following necro-inflammatory liver injury induced by the hepatotoxic agent carbon tetrachloride (CCl4) and in steatohepatitis caused by a methionine/choline-deficient (MCD) diet. In both the experimental models, hepatic inflammation was associated with a massive expansion of CD11c+/MHCIIhigh/CD11b+ myeloid HDCs. These cells also expressed the monocyte markers Ly6C, chemokine (C-C Motif) receptor 2 (CCR2), F4/80 and CD88, along with CX3CR1, allowing their tentative identification as moDCs. Mice defective in CX3CR1 showed a reduction in liver-moDC recruitment following CCl4 poisoning in parallel with a defective maturation of monocytes into moDCs. The lack of CX3CR1 also affected moDC differentiation from bone marrow myeloid cells induced by granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) in vitro. In wild-type mice, treatment with the CX3CR1 antagonist CX3-AT (150 µg, i.p.) 24 h after CCl4 administration reduced liver moDCS and significantly ameliorated hepatic injury and inflammation. Altogether, these results highlight the possible involvement of moDCs in promoting hepatic inflammation following liver injury and indicated a novel role of CX3CL1/CX3CR1 dyad in driving the differentiation of hepatic moDCs.

Single Cell RNA Sequencing Identifies Subsets of Hepatic Stellate Cells and Myofibroblasts in Liver Fibrosis.

Activation of hepatic stellate cells (HSCs) and their trans-differentiation towards collagen-secreting myofibroblasts (MFB) promote liver fibrosis progression. During chronic liver disease, resting HSCs become activated by inflammatory and injury signals. However, HSCs/MFB not only produce collagen, but also secrete cytokines, participate in metabolism, and have biomechanical properties. We herein aimed to characterize the heterogeneity of these liver mesenchymal cells by single cell RNA sequencing. In vivo resting HSCs or activated MFB were isolated from C57BL6/J mice challenged by carbon tetrachloride (CCl4) intraperitoneally for 3 weeks to induce liver fibrosis and compared to in vitro cultivated MFB. While resting HSCs formed a homogenous population characterized by high platelet derived growth factor receptor ß (PDGFRß) expression, in vivo and in vitro activated MFB split into heterogeneous populations, characterized by α-smooth muscle actin (α-SMA), collagens, or immunological markers. S100 calcium binding protein A6 (S100A6) was a universal marker of activated MFB on both the gene and protein expression level. Compared to the heterogeneity of in vivo MFB, MFB in vitro sequentially and only transiently expressed marker genes, such as chemokines, during culture activation. Taken together, our data demonstrate the heterogeneity of HSCs and MFB, indicating the existence of functionally relevant subsets in hepatic fibrosis.

Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer.

Non-alcoholic fatty liver disease ranges from steatosis to non-alcoholic steatohepatitis (NASH), potentially progressing to cirrhosis and hepatocellular carcinoma (HCC). Here, we show that platelet number, platelet activation and platelet aggregation are increased in NASH but not in steatosis or insulin resistance. Antiplatelet therapy (APT; aspirin/clopidogrel, ticagrelor) but not nonsteroidal anti-inflammatory drug (NSAID) treatment with sulindac prevented NASH and subsequent HCC development. Intravital microscopy showed that liver colonization by platelets depended primarily on Kupffer cells at early and late stages of NASH, involving hyaluronan-CD44 binding. APT reduced intrahepatic platelet accumulation and the frequency of platelet-immune cell interaction, thereby limiting hepatic immune cell trafficking. Consequently, intrahepatic cytokine and chemokine release, macrovesicular steatosis and liver damage were attenuated. Platelet cargo, platelet adhesion and platelet activation but not platelet aggregation were identified as pivotal for NASH and subsequent hepatocarcinogenesis. In particular, platelet-derived GPIbα proved critical for development of NASH and subsequent HCC, independent of its reported cognate ligands vWF, P-selectin or Mac-1, offering a potential target against NASH.

The CCR2+ Macrophage Subset Promotes Pathogenic Angiogenesis for Tumor Vascularization in Fibrotic Livers.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) typically arises in fibrotic or cirrhotic livers, which are characterized by pathogenic angiogenesis. Myeloid immune cells, specifically tumor-associated macrophages (TAMs), may represent potential novel therapeutic targets in HCC, complementing current ablative or immune therapies. However, the detailed functions of TAM subsets in hepatocarcinogenesis have remained obscure. METHODS: TAM subsets were analyzed in-depth in human HCC samples and a combined fibrosis-HCC mouse model, established by i.p. injection with diethylnitrosamine after birth and repetitive carbon tetrachloride (CCl4) treatment for 16 weeks. Based on comprehensively phenotyping TAM subsets (fluorescence-activated cell sorter, transcriptomics) in mice, the function of CCR2+ TAM was assessed by a pharmacologic chemokine inhibitor. Angiogenesis was evaluated by contrast-enhanced micro-computed tomography and histology. RESULTS: We show that human CCR2+ TAM accumulate at the highly vascularized HCC border and express the inflammatory marker S100A9, whereas CD163+ immune-suppressive TAM accrue in the HCC center. In the fibrosis-cancer mouse model, we identified 3 major hepatic myeloid cell populations with distinct messenger RNA profiles, of which CCR2+ TAM particularly showed activated inflammatory and angiogenic pathways. Inhibiting CCR2+ TAM infiltration using a pharmacologic chemokine CCL2 antagonist in the fibrosis-HCC model significantly reduced pathogenic vascularization and hepatic blood volume, alongside attenuated tumor volume. CONCLUSIONS: The HCC microenvironment in human patients and mice is characterized by functionally distinct macrophage populations, of which the CCR2+ inflammatory TAM subset has pro-angiogenic properties. Understanding the functional differentiation of myeloid cell subsets in chronically inflamed liver may provide novel opportunities for modulating hepatic macrophages to inhibit tumor-promoting pathogenic angiogenesis.

CXCR6 Inhibits Hepatocarcinogenesis by Promoting Natural Killer T- and CD4+ T-Cell-Dependent Control of Senescence.

BACKGROUND & AIMS: Inflammation in the liver provokes fibrosis, but inflammation is also important for tumor surveillance. Inhibitors of chemokine pathways, such as CXCL16 and CXCR6 regulation of lymphocyte trafficking, are being tested as antifibrotic agents, but their effects on the development of hepatocellular carcinoma (HCC) are unclear. We assessed the roles of CXCR6-dependent immune mechanisms in hepatocarcinogenesis. METHODS: C57BL/6J wild-type (WT) mice and CXCR6-deficient mice (Cxcr6eGfp/eGfp) were given injections of diethylnitrosamine (DEN) to induce liver cancer and α-galactosylceramide to activate natural killer T (NKT) cells. We also performed studies in mice with conditional, hepatocyte-specific deletion of NEMO, which develop inflammation-associated liver tumors (NemoLPC-KO and NemoLPC-KOCxcr6eGfp/eGfp mice). We collected liver tissues from patients with cirrhosis (n = 43), HCC (n = 35), and neither of these diseases (control individuals, n = 25). Human and mouse liver tissues were analyzed by histology, immunohistochemistry, flow cytometry, RNA expression arrays (from sorted hepatic lymphocytes), and matrix-assisted laser desorption/ionization imaging. Bone marrow was transferred from Cxcr6eGfp/eGfp or WT mice to irradiated C57BL/6J mice, and spleen and liver cells were analyzed by flow cytometry. CD4+ T cells or NKT cells were isolated from the spleen and liver of CD45.1+ WT mice and transferred into CXCR6-deficient mice after DEN injection. RESULTS: After DEN injection, CXCR6-deficient mice had a significantly higher tumor burden than WT mice and increased tumor progression, characterized by reduced intrahepatic numbers of invariant NKT and CD4+ T cells that express tumor necrosis factor and interferon gamma. Livers of NemoLPC-KOCxcr6eGfp/eGfp mice had significantly more senescent hepatocytes than livers of NemoLPC-KO mice. In studies of bone-marrow chimeras, adoptive cell transfer experiments, and analyses of NemoLPC-KO mice, we found that NKT and CD4 T cells promote the removal of senescent hepatocytes to prevent hepatocarcinogenesis, and that this process required CXCR6. Injection of WT with α-galactosylceramide increased removal of senescent hepatocytes by NKT cells. We observed peritumoral accumulation of CXCR6-associated lymphocytes in human HCC, which appeared reduced compared with cirrhosis tissues. CONCLUSIONS: In studies of mice with liver tumors, we found that CXCR6 mediated NKT-cell and CD4+ T-cell removal of senescent hepatocytes. Antifibrotic strategies to reduce CXCR6 activity in liver, or to reduce inflammation or modulate the immune response, should be tested for their effects on hepatocarcinogenesis.

Adapted Immune Responses of Myeloid-Derived Cells in Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is considered to be one of the most frequent chronic liver diseases worldwide and is associated with an increased risk of developing liver cirrhosis and hepatocellular carcinoma. Hepatic macrophages, mainly comprising monocyte derived macrophages and tissue resident Kupffer cells, are characterized by a high diversity and plasticity and act as key regulators during NAFLD progression, in conjunction with other infiltrating myeloid cells like neutrophils or dendritic cells. The activation and polarization of myeloid immune cells is influenced by dietary components, inflammatory signals like danger-associated molecular patterns (DAMPs) or cytokines as well as gut-derived inflammatory factors such as pathogen-associated molecular patterns (PAMPs). The functionality of myeloid leukocytes in the liver is directly linked to their inflammatory polarization, which is shaped by local and systemic inflammatory mediators such as cytokines, chemokines, PAMPs, and DAMPs. These environmental signals provoke intracellular adaptations in myeloid cells, including inflammasome and transcription factor activation, inflammatory signaling pathways, or switches in cellular metabolism. Dietary changes and obesity also promote a dysbalance in intestinal microbiota, which can facilitate intestinal permeability and bacterial translocation. The aim of this review is to highlight recent findings on the activating pathways of innate immune cells during the progression of NAFLD, dissecting local hepatic and systemic signals, dietary and metabolic factors as well as pathways of the gut-liver axis. Understanding the mechanism by which plasticity of myeloid-derived leukocytes is related to metabolic changes and NAFLD progression may provide options for new therapeutic approaches.

Heparan Sulfate Induces Necroptosis in Murine Cardiomyocytes: A Medical-In silico Approach Combining In vitro Experiments and Machine Learning.

Life-threatening cardiomyopathy is a severe, but common, complication associated with severe trauma or sepsis. Several signaling pathways involved in apoptosis and necroptosis are linked to trauma- or sepsis-associated cardiomyopathy. However, the underling causative factors are still debatable. Heparan sulfate (HS) fragments belong to the class of danger/damage-associated molecular patterns liberated from endothelial-bound proteoglycans by heparanase during tissue injury associated with trauma or sepsis. We hypothesized that HS induces apoptosis or necroptosis in murine cardiomyocytes. By using a novel Medical-In silico approach that combines conventional cell culture experiments with machine learning algorithms, we aimed to reduce a significant part of the expensive and time-consuming cell culture experiments and data generation by using computational intelligence (refinement and replacement). Cardiomyocytes exposed to HS showed an activation of the intrinsic apoptosis signal pathway via cytochrome C and the activation of caspase 3 (both p < 0.001). Notably, the exposure of HS resulted in the induction of necroptosis by tumor necrosis factor α and receptor interaction protein 3 (p < 0.05; p < 0.01) and, hence, an increased level of necrotic cardiomyocytes. In conclusion, using this novel Medical-In silico approach, our data suggest (i) that HS induces necroptosis in cardiomyocytes by phosphorylation (activation) of receptor-interacting protein 3, (ii) that HS is a therapeutic target in trauma- or sepsis-associated cardiomyopathy, and (iii) indicate that this proof-of-concept is a first step toward simulating the extent of activated components in the pro-apoptotic pathway induced by HS with only a small data set gained from the in vitro experiments by using machine learning algorithms.

Dynamic plasticity of macrophage functions in diseased liver.

Liver macrophages attract increasing interest due to their crucial roles in homeostasis and hepatic diseases. Recent findings in mice and man suggest a remarkable phenotypic and functional diversity of liver macrophages. Kupffer cells, the subset of tissue resident macrophages with sentinel functions in liver, mainly arise from embryogenic precursors, whereas in injury, liver tissue is engrafted by monocyte-derived macrophages. Both principal macrophage populations respond to local or systemic signals and have substantial effects on reduction as well as aggravation of hepatic diseases. Despite contrasting functions of heterogeneous macrophage subsets in disease progression and regression, they may provide promising targets for novel therapeutic interventions in hepatology. Areas of intense research include their multifaceted roles in metabolic diseases (non-alcoholic steatohepatitis, NASH), fibrosis or liver cancer (hepatocellular or cholangiocellular carcinoma, HCC or CCA). We discuss recent findings on the origin, diversity and functional plasticity of liver macrophages in homeostasis and hepatic disease conditions.

Therapeutic inhibition of inflammatory monocyte recruitment reduces steatohepatitis and liver fibrosis.

Macrophages are key regulators of liver fibrosis progression and regression in nonalcoholic steatohepatitis (NASH). Liver macrophages comprise resident phagocytes, Kupffer cells, and monocyte-derived cells, which are recruited through the chemokine receptor C-C motif chemokine receptor 2 (CCR2). We aimed at elucidating the therapeutic effects of inhibiting monocyte infiltration in NASH models by using cenicriviroc (CVC), an oral dual chemokine receptor CCR2/CCR5 antagonist that is under clinical evaluation. Human liver tissues from NASH patients were analyzed for CCR2+ macrophages, and administration of CVC was tested in mouse models of steatohepatitis, liver fibrosis progression, and fibrosis regression. In human livers from 17 patients and 4 controls, CCR2+ macrophages increased parallel to NASH severity and fibrosis stage, with a concomitant inflammatory polarization of these cluster of differentiation 68+ , portal monocyte-derived macrophages (MoMF). Similar to human disease, we observed a massive increase of hepatic MoMF in experimental models of steatohepatitis and liver fibrosis. Therapeutic treatment with CVC significantly reduced the recruitment of hepatic Ly-6C+ MoMF in all models. In experimental steatohepatitis with obesity, therapeutic CVC application significantly improved insulin resistance and hepatic triglyceride levels. In fibrotic steatohepatitis, CVC treatment ameliorated histological NASH activity and hepatic fibrosis. CVC inhibited the infiltration of Ly-6C+ monocytes, without direct effects on macrophage polarization, hepatocyte fatty acid metabolism, or stellate cell activation. Importantly, CVC did not delay fibrosis resolution after injury cessation. RNA sequencing analysis revealed that MoMF, but not Kupffer cells, specifically up-regulate multiple growth factors and cytokines associated with fibrosis progression, while Kupffer cells activated pathways related to inflammation initiation and lipid metabolism. CONCLUSION: Pharmacological inhibition of CCR2+ monocyte recruitment efficiently ameliorates insulin resistance, hepatic inflammation, and fibrosis, corroborating the therapeutic potential of CVC in patients with NASH. (Hepatology 2018;67:1270-1283).

Differential impact of the dual CCR2/CCR5 inhibitor cenicriviroc on migration of monocyte and lymphocyte subsets in acute liver injury.

A hallmark of acute hepatic injury is the recruitment of neutrophils, monocytes and lymphocytes, including natural killer (NK) or T cells, towards areas of inflammation. The recruitment of leukocytes from their reservoirs bone marrow or spleen into the liver is directed by chemokines such as CCL2 (for monocytes) and CCL5 (for lymphocytes). We herein elucidated the impact of chemokine receptor inhibition by the dual CCR2 and CCR5 inhibitor cenicriviroc (CVC) on the composition of myeloid and lymphoid immune cell populations in acute liver injury. CVC treatment effectively inhibited the migration of bone marrow monocytes and splenic lymphocytes (NK, CD4 T-cells) towards CCL2 or CCL5 in vitro. When liver injury was induced by an intraperitoneal injection of carbon tetrachloride (CCl4) in mice, followed by repetitive oral application of CVC, flow cytometric and unbiased t-SNE analysis of intrahepatic leukocytes demonstrated that dual CCR2/CCR5 inhibition in vivo significantly decreased numbers of monocyte derived macrophages in acutely injured livers. CVC also reduced numbers of Kupffer cells (KC) or monocyte derived macrophages with a KC-like phenotype, respectively, after injury. In contrast to the inhibitory effects in vitro, CVC had no impact on the composition of hepatic lymphoid cell populations in vivo. Effective inhibition of monocyte recruitment was associated with reduced inflammatory macrophage markers and moderately ameliorated hepatic necroses at 36h after CCl4. In conclusion, dual CCR2/CCR5 inhibition primarily translates into reduced monocyte recruitment in acute liver injury in vivo, suggesting that this strategy will be effective in reducing inflammatory macrophages in conditions of liver disease.

Liver macrophages in tissue homeostasis and disease.

Macrophages represent a key cellular component of the liver, and are essential for maintaining tissue homeostasis and ensuring rapid responses to hepatic injury. Our understanding of liver macrophages has been revolutionized by the delineation of heterogeneous subsets of these cells. Kupffer cells are a self-sustaining, liver-resident population of macrophages and can be distinguished from the monocyte-derived macrophages that rapidly accumulate in the injured liver. Specific environmental signals further determine the polarization and function of hepatic macrophages. These cells promote the restoration of tissue integrity following liver injury or infection, but they can also contribute to the progression of liver diseases, including hepatitis, fibrosis and cancer. In this Review, we highlight novel findings regarding the origin, classification and function of hepatic macrophages, and we discuss their divergent roles in the healthy and diseased liver.

Chemokine (C-C motif) receptor 2-positive monocytes aggravate the early phase of acetaminophen-induced acute liver injury.

Acetaminophen (APAP, paracetamol) poisoning is a leading cause of acute liver failure (ALF) in humans and induces hepatocyte necrosis, followed by activation of the innate immune system, further aggravating liver injury. The role of infiltrating monocytes during the early phase of ALF is still ambiguous. Upon experimental APAP overdose in mice, monocyte-derived macrophages (MoMFs) massively accumulated in injured liver within 12-24 hours, whereas the number of tissue-resident macrophages (Kupffer cells) decreased. Influx of MoMFs is dependent on the chemokine receptor, chemokine (C-C motif) receptor 2 (CCR2), given that Ccr2-/- mice display reduced infiltration of monocytes and attenuated liver injury post-APAP overdose at early time points. As evidenced by intravital multiphoton microscopy of Ccr2 reporter mice, CCR2+ monocytes infiltrate liver as early as 8-12 hours post-APAP overdose and form dense cellular clusters around necrotic areas. CCR2+ MoMFs express a distinct pattern of inflammatory, but also repair-associated, genes in injured livers. Adoptive transfer experiments revealed that MoMFs primarily exert proinflammatory functions early post-APAP, thereby aggravating liver injury. Consequently, early pharmacological inhibition of either chemokine (C-C motif) ligand (CCL2; by the inhibitor, mNOX-E36) or CCR2 (by the orally available dual CCR2/CCR5 inhibitor, cenicriviroc) reduces monocyte infiltration and APAP-induced liver injury (AILI) in mice. Importantly, neither the early nor continuous inhibition of CCR2 hinder repair processes during resolution from injury. In line with this, human livers of ALF patients requiring liver transplantation reveal increased CD68+ hepatic macrophage numbers with massive infiltrates of periportal CCR2+ macrophages that display a proinflammatory polarization. CONCLUSION: Infiltrating monocyte-derived macrophages aggravate APAP hepatotoxicity, and the pharmacological inhibition of either CCL2 or CCR2 might bear therapeutic potential by reducing the inflammatory reaction during the early phase of AILI. (Hepatology 2016;64:1667-1682).

Immune mechanisms in acetaminophen-induced acute liver failure.

An overdose of acetaminophen (N-acetyl-p-aminophenol, APAP), also termed paracetamol, can cause severe liver damage, ultimately leading to acute liver failure (ALF) with the need of liver transplantation. APAP is rapidly taken up from the intestine and metabolized in hepatocytes. A small fraction of the metabolized APAP forms cytotoxic mitochondrial protein adducts, leading to hepatocyte necrosis. The course of disease is not only critically influenced by dose of APAP and the initial hepatocyte damage, but also by the inflammatory response following acetaminophen-induced liver injury (AILI). As revealed by mouse models of AILI and corresponding translational studies in ALF patients, necrotic hepatocytes release danger-associated-molecular patterns (DAMPs), which are recognized by resident hepatic macrophages, Kupffer cell (KC), and neutrophils, leading to the activation of these cells. Activated hepatic macrophages release various proinflammatory cytokines, such as TNF-α or IL-1ß, as well as chemokines (e.g., CCL2) thereby further enhancing inflammation and increasing the influx of immune cells, like bone-marrow derived monocytes and neutrophils. Monocytes are mainly recruited via their receptor CCR2 and aggravate inflammation. Infiltrating monocytes, however, can mature into monocyte-derived macrophages (MoMF), which are, in cooperation with neutrophils, also involved in the resolution of inflammation. Besides macrophages and neutrophils, distinct lymphocyte populations, especially γδ T cells, are also linked to the inflammatory response following an APAP overdose. Natural killer (NK), natural killer T (NKT) and T cells possibly further perpetuate inflammation in AILI. Understanding the complex interplay of immune cell subsets in experimental models and defining their functional involvement in disease progression is essential to identify novel therapeutic targets for human disease.