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.

Elevated circulating CD14++CD16+ intermediate monocytes are independently associated with extracardiac complications after cardiac surgery.

Elective cardiac surgery has low procedural complications. However, about 40% of patients develop extracardiac complications including delirium and acute kidney injury. We hypothesized that inflammatory processes and immune cell activation might be associated with these complications. We therefore prospectively included 104 patients undergoing cardiac surgery in our study. We assessed peripheral blood leukocyte populations by flow cytometry and circulating cytokines before operation, after surgery and at days one and four post-operatively. Patients undergoing cardiac surgery showed significantly elevated leukocytes and neutrophils after surgery. On the contrary, monocytes decreased after surgery and significantly increased at days 1 and 4, particularly classical (Mon1,CD14++CD16-) and intermediate (Mon2,CD14++CD16+) monocytes. While peripheral leukocyte subsets were unaltered in patients with infectious (n = 15) or cardiac complications (n = 31), post-operative leukocytes (p = 0.0016), neutrophils (p = 0.0061) and Mon2 (p = 0.0007) were clearly raised in patients developing extracardiac complications (n = 35). Using multiple logistic regression analyses, patient's age, ICU days, number of blood transfusions and elevated post-surgery Mon2 independently predicted extracardiac complications. Our findings demonstrate that elevated Mon2 after cardiac surgery are associated with an increased risk for extracardiac complications. These findings might improve the risk estimation after cardiac operations and the role of Mon2 for inflammation in cardiac surgery.

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.

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.

CXCR6 protects from inflammation and fibrosis in NEMOLPC-KO mice.

Chronic inflammation in the liver provokes fibrosis and, on long-term, carcinogenesis. This sequence is prototypically recapitulated in mice with hepatocyte-specific knock-out of the NF-κB essential modulator (NEMO), termed NEMOLPC-KO mice, in which increased hepatocyte apoptosis and compensatory regeneration cause steatosis, inflammation and fibrosis. Natural killer T (NKT) cells carrying the chemokine receptor CXCR6 participate in liver inflammation and injury responses. Here, we investigated the role of CXCR6 in the NEMOLPC-KO mouse model. Unexpectedly, genetic deletion of CXCR6 enhanced hepatocyte death, inflammation and fibrosis in NEMOLPC-KO mice. Although CXCR6 expression is restricted to immune cells in the liver, the adoptive transfer of CXCR6+ cells did not protect NEMOLPC-KOCxcr6-/- mice from hepatic injury. Gene array analyses revealed up-regulated stress response and metabolism pathways in hepatocytes from NEMOLPC-KOCxcr6-/- mice, functionally corresponding to an increased susceptibility of these hepatocytes to TNFα-induced cell death in vitro. These data revealed a novel CXCR6-dependent mechanism of suppressing inflammatory hepatocytic responses to cellular stress.

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).

Elevated Soluble Urokinase Plasminogen Activator Receptor and Proenkephalin Serum Levels Predict the Development of Acute Kidney Injury after Cardiac Surgery.

Acute kidney injury (AKI) develops in up to 40% of patients after cardiac surgery. The soluble urokinase plasminogen activator receptor (suPAR) has been identified as a biomarker for incident chronic kidney disease (CKD). Proenkephalin (proENK) also has been shown to be a biomarker for renal dysfunction. We hypothesized that pre-surgery suPAR and proENK levels might predict AKI in patients undergoing cardiac surgery. Consecutive patients (n = 107) undergoing elective cardiac surgery were studied prospectively. Clinical data, laboratory parameters, suPAR and proENK serum levels were assessed before operation, after operation and days one and four post-operatively. A total of 21 (19.6%) patients developed AKI within the first four days after elective surgery. Serum levels of suPAR and proENK, but not of creatinine, were significantly higher before surgery in these patients compared to those patients without AKI. This difference remained significant for suPAR, if patients with or without AKI were matched for risk factors (hypertension, diabetes, CKD). If cardiac surgery patients with pre-existing CKD (n = 10) were excluded, only pre-operative suPAR but not proENK serum levels remained significantly elevated in patients with subsequent AKI. Thus, our findings indicate that suPAR may be a predictive biomarker for AKI in the context of cardiac surgery, even in patients without underlying CKD.

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).

miR-155 targets Caspase-3 mRNA in activated macrophages.

To secure the functionality of activated macrophages in the innate immune response, efficient life span control is required. Recognition of bacterial lipopolysaccharides (LPS) by toll-like receptor 4 (TLR4) induces downstream signaling pathways, which merge to induce the expression of cytokine genes and anti-apoptotic genes. MicroRNAs (miRNAs) have emerged as important inflammatory response modulators, but information about their functional impact on apoptosis is scarce. To identify miRNAs differentially expressed in response to LPS, cDNA libraries from untreated and LPS-activated murine macrophages were analyzed by deep sequencing and regulated miRNA expression was verified by Northern blotting and qPCR. Employing TargetScan(TM) we identified CASPASE-3 (CASP-3) mRNA that encodes a key player in apoptosis as potential target of LPS-induced miR-155. LPS-dependent primary macrophage activation revealed TLR4-mediated enhancement of miR-155 expression and CASP-3 mRNA reduction. Endogenous CASP-3 and cleaved CASP-3 protein declined in LPS-activated macrophages. Accumulation of miR-155 and CASP-3 mRNA in miRNA-induced silencing complexes (miRISC) was demonstrated by ARGONAUTE 2 (AGO2) immunoprecipitation. Importantly, specific antagomir transfection effectively reduced mature miR-155 and resulted in significantly elevated CASP-3 mRNA levels in activated macrophages. In vitro translation assays demonstrated that the target site in the CASP-3 mRNA 3'UTR mediates miR-155-dependent Luciferase reporter mRNA destabilization. Strikingly, Annexin V staining of macrophages transfected with antagomir-155 and stimulated with LPS prior to staurosporine (SSP) treatment implied that LPS-induced miR-155 prevents apoptosis through CASP-3 mRNA down-regulation. In conclusion, we report that miR-155-mediated CASP-3 mRNA destabilization in LPS-activated RAW 264.7 macrophages suppresses apoptosis, as a prerequisite to maintain their crucial function in inflammation.

Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring. The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange. To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4). CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Translation control of TAK1 mRNA by hnRNP K modulates LPS-induced macrophage activation.

Macrophage activation by bacterial lipopolysaccharides (LPS) is induced through Toll-like receptor 4 (TLR4). The synthesis and activity of TLR4 downstream signaling molecules modulates the expression of pro- and anti-inflammatory cytokines. To address the impact of post-transcriptional regulation on that process, we performed RIP-Chip analysis. Differential association of mRNAs with heterogeneous nuclear ribonucleoprotein K (hnRNP K), an mRNA-specific translational regulator in differentiating hematopoietic cells, was studied in noninduced and LPS-activated macrophages. Analysis of interactions affected by LPS revealed several mRNAs encoding TLR4 downstream kinases and their modulators. We focused on transforming growth factor-ß-activated kinase 1 (TAK1) a central player in TLR4 signaling. HnRNP K interacts specifically with a sequence in the TAK1 mRNA 3' UTR in vitro. Silencing of hnRNP K does not affect TAK1 mRNA synthesis or stability but enhances TAK1 mRNA translation, resulting in elevated TNF-α, IL-1ß, and IL-10 mRNA expression. Our data suggest that the hnRNP K-3' UTR complex inhibits TAK1 mRNA translation in noninduced macrophages. LPS-dependent TLR4 activation abrogates translational repression and newly synthesized TAK1 boosts macrophage inflammatory response.

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.

Role of lymphocytes in liver cancer.

Hepatocellular carcinoma (HCC) typically occurs in patients with chronic inflammatory liver diseases, such as viral hepatitis or (non-)alcoholic steatohepatitis. Inflammation appears indeed as a crucial factor in hepatocarcinogenesis. Nevertheless, sophisticated animal models and studies of human samples revealed that the HCC also elicits antitumor immune responses. Patrolling and infiltrating lymphocytes (e.g., NKT and T cells, respectively) can exert decisive functions in the transition from chronic hepatic inflammation to cancer as well as in antitumor immune responses. An improved understanding of the cellular and molecular mechanisms whereby inflammation promotes or restricts hepatocarcinogenesis will open new avenues for therapeutic approaches to liver cancer.