Christer Baeck

Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury

Objective Monocyte chemoattractant protein-1 (MCP-1, CCL2), the primary ligand for chemokine receptor C–C chemokine receptor 2 (CCR2), is increased in livers of patients with non-alcoholic steatohepatitis (NASH) and murine models of steatohepatitis and fibrosis. It was recently shown that monocyte/macrophage infiltration into the liver upon injury is critically regulated by the CCL2/CCR2 axis and is functionally important for perpetuating hepatic inflammation and fibrogenesis. The structured L-enantiomeric RNA oligonucleotide mNOX-E36 (a so-called Spiegelmer) potently binds and inhibits murine MCP-1. Pharmacological inhibition of MCP-1 with mNOX-E36 was investigated in two murine models of chronic liver diseases. Methods Pharmacological inhibition of MCP-1 by thrice-weekly mNOX-E36 subcutaneously was tested in murine models of acute or chronic carbon tetrachloride (CCl4)- and methionine–choline-deficient (MCD) diet-induced chronic hepatic injury in vivo. Results Antagonising MCP-1 by mNOX-E36 efficiently inhibited murine monocyte chemotaxis in vitro as well as migration of Gr1+ (Ly6C+) blood monocytes into the liver upon acute toxic injury in vivo. In murine models of CCl4- and MCD diet-induced hepatic injury, the infiltration of macrophages into the liver was significantly decreased in anti-MCP-1-treated mice as found by fluorescence-activated cell sorting (FACS) analysis and immunohistochemistry. In line with lower levels of intrahepatic macrophages, proinflammatory cytokines (tumour necrosis factor α, interferon γ and interleukin 6) were significantly reduced in liver tissue. Overall fibrosis progression over 6 (CCl4) or 8 weeks (MCD diet) was not significantly altered by anti-MCP-1 treatment. However, upon MCD diet challenge a lower level of fatty liver degeneration (histology score, Oil red O staining, hepatic triglyceride content, lipogenesis genes) was detected in mNOX-E36-treated animals. mNOX-E36 also ameliorated hepatic steatosis upon therapeutic administration. Conclusions These results demonstrate the successful pharmacological inhibition of hepatic monocyte/macrophage infiltration by blocking MCP-1 during chronic liver damage in two in vivo models. The associated ameliorated steatosis development suggests that inhibition of MCP-1 is an interesting novel approach for pharmacological treatment in liver inflammation and steatohepatitis.

CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis

Objectives In chronic liver injury, angiogenesis, the formation of new blood vessels from pre-existing ones, may contribute to progressive hepatic fibrosis and to development of hepatocellular carcinoma. Although hypoxia-induced expression of vascular endothelial growth factor (VEGF) occurs in advanced fibrosis, we hypothesised that inflammation may endorse hepatic angiogenesis already at early stages of fibrosis. Design Angiogenesis in livers of c57BL/6 mice upon carbon tetrachloride- or bile duct ligation-induced chronic hepatic injury was non-invasively monitored using in vivo contrast-enhanced micro computed tomography (µCT) and ex vivo anatomical µCT after hepatic Microfil perfusion. Functional contributions of monocyte-derived macrophage subsets for angiogenesis were explored by pharmacological inhibition of CCL2 using the Spiegelmer mNOX-E36. Results Contrast-enhanced in vivo µCT imaging allowed non-invasive monitoring of the close correlation of angiogenesis, reflected by functional hepatic blood vessel expansion, with experimental fibrosis progression. On a cellular level, inflammatory monocyte-derived macrophages massively accumulated in injured livers, colocalised with newly formed vessels in portal tracts and exhibited pro-angiogenic gene profiles including upregulated VEGF and MMP9. Functional in vivo and anatomical ex vivo µCT analyses demonstrated that inhibition of monocyte infiltration by targeting the chemokine CCL2 prevented fibrosis-associated angiogenesis, but not fibrosis progression. Monocyte-derived macrophages primarily fostered sprouting angiogenesis within the portal vein tract. Portal vein diameter as a measure of portal hypertension depended on fibrosis, but not on angiogenesis. Conclusions Inflammation-associated angiogenesis is promoted by CCL2-dependent monocytes during fibrosis progression. Innovative in vivo µCT methodology can accurately monitor angiogenesis and antiangiogenic therapy effects in experimental liver fibrosis.

Pharmacological inhibition of the chemokine C‐C motif chemokine ligand 2 (monocyte chemoattractant protein 1) accelerates liver fibrosis regression by suppressing Ly‐6C+ macrophage infiltration in mice

Macrophages constitute a major proinflammatory component during chronic liver diseases and are considered a key factor in promoting hepatic fibrosis. However, there is increasing evidence that distinct monocyte and macrophage subsets exert critical functions in regression from organ fibrosis as well. Experimental mouse models of fibrosis regression have identified “restorative” macrophages as Ly‐6C (Ly6C, Gr1) low‐expressing, monocyte‐derived cells. We investigated molecular pathways balancing proinflammatory and restorative macrophages during fibrosis regression as well as pharmacologically augmenting beneficial macrophage functionality in fibrosis resolution. Therefore, we employed a Spiegelmer‐based inhibitor of the chemokine, C‐C motif chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1), termed mNOX‐E36, in the regression phase of two murine models of toxic (CCl4) and metabolic (methionine‐choline–deficient diet) liver fibrosis. Although inflammation rapidly declined after cessation of injury, we observed a transient influx of Ly‐6C+ infiltrating monocytes (iMΦ), which are characterized by typical macrophage morphology, up‐regulated expression of CCR2, and the pro‐inflammatory cytokine, tumor necrosis factor (TNF), in injured liver. By inhibiting the early influx of Ly‐6C+ iMΦ by the CCL2 inhibitor, mNOX‐E36, the intrahepatic macrophage equilibration shifted toward the “restorative” Ly‐6C‐ subset of iMΦ. Consequently, fibrosis resolution was significantly accelerated upon mNOX‐E36 administration in both models. Blocking transient recruitment of infiltrating Ly‐6C+ monocytes, but not direct effects of the inhibitor on the remaining macrophages, resulted in reduced intrahepatic levels of proinflammatory cytokines. Conclusion: Transient CCL2‐dependent recruitment of infiltrating Ly‐6C+ monocytes during fibrosis regression counteracts scar resolution by perpetuating inflammatory reactions through release of proinflammatory cytokines such as TNF. Pharmacological inhibition of Ly‐6C+ monocyte recruitment using the CCL2‐inhibitor, mNOX‐E36, accelerates regression from toxic and metabolic liver fibrosis in two independent experimental models. (Hepatology 2014;59:1060–1072)

Chemokine Receptor CXCR6-Dependent Hepatic NK T Cell Accumulation Promotes Inflammation and Liver Fibrosis

Chronic liver injury characteristically results in hepatic inflammation, which represents a prerequisite for organ fibrosis. Although NKT cells are abundantly present in liver and involved in hepatic inflammation, molecular mechanisms of their recruitment in liver fibrosis remained elusive. We hypothesized that chemokine receptor CXCR6 and its ligand CXCL16 control NKT cell migration and functionality in liver fibrosis. In patients with chronic liver diseases (n = 58), CXCR6 and CXCL16 expression was intrahepatically upregulated compared with controls. In murine liver, Cxcl16 was strongly expressed by endothelium and macrophages, whereas lymphocyte populations (NKT, NK, CD4 T, CD8 T cells) expressed CXCR6. Intravital two-photon microscopy imaging of Cxcr6+/gfp and Cxcr6gfp/gfp mice and chemotaxis studies in vitro revealed that CXCR6 specifically controls hepatic NKT cell accumulation during the early response upon experimental liver damage. Hepatic invariant NKT cells expressed distinct proinflammatory cytokines including IFN-γ and IL-4 upon injury. CXCR6-deficient mice were protected from liver fibrosis progression in two independent experimental models. Macrophage infiltration and protein levels of inflammatory cytokines IFN-γ, TNF-α, and IL-4 were also reduced in fibrotic livers of Cxcr6−/− mice, corroborating that hepatic NKT cells provide essential cytokine signals perpetuating hepatic inflammation and fibrogenesis. Adoptive transfer of NKT cells, but not CD4 T cells, isolated from wild type livers restored hepatic fibrosis in Cxcr6−/− mice upon experimental steatohepatitis. Our results demonstrate that hepatic NKT cells accumulate CXCR6-dependent early upon injury, thereby accentuating the inflammatory response in the liver and promoting hepatic fibrogenesis. Interfering with CXCR6/CXCL16 might therefore bear therapeutic potential in liver fibrosis.

Pharmacological inhibition of the chemokine CXCL16 diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury.

Non-alcoholic fatty liver disease (NAFLD) is a major cause of morbidity and mortality in developed countries, resulting in steatohepatitis (NASH), fibrosis and eventually cirrhosis. Modulating inflammatory mediators such as chemokines may represent a novel therapeutic strategy for NAFLD. We recently demonstrated that the chemokine receptor CXCR6 promotes hepatic NKT cell accumulation, thereby controlling inflammation in experimental NAFLD. In this study, we first investigated human biopsies (n = 20), confirming that accumulation of inflammatory cells such as macrophages is a hallmark of progressive NAFLD. Moreover, CXCR6 gene expression correlated with the inflammatory activity (ALT levels) in human NAFLD. We then tested the hypothesis that pharmacological inhibition of CXCL16 might hold therapeutic potential in NAFLD, using mouse models of acute carbon tetrachloride (CCl4)- and chronic methionine-choline-deficient (MCD) diet-induced hepatic injury. Neutralizing CXCL16 by i.p. injection of anti-CXCL16 antibody inhibited the early intrahepatic NKT cell accumulation upon acute toxic injury in vivo. Weekly therapeutic anti-CXCL16 administrations during the last 3 weeks of 6 weeks MCD diet significantly decreased the infiltration of inflammatory macrophages into the liver and intrahepatic levels of inflammatory cytokines like TNF or MCP-1. Importantly, anti-CXCL16 treatment significantly reduced fatty liver degeneration upon MCD diet, as assessed by hepatic triglyceride levels, histological steatosis scoring and quantification of lipid droplets. Moreover, injured hepatocytes up-regulated CXCL16 expression, indicating that scavenging functions of CXCL16 might be additionally involved in the pathogenesis of NAFLD. Targeting CXCL16 might therefore represent a promising novel therapeutic approach for liver inflammation and steatohepatitis.

Balance of inflammatory pathways and interplay of immune cells in the liver during homeostasis and injury

Multiple potentially harmful stimuli challenge the liver, the chief metabolic and detoxifying organ of the human body. Due to its central anatomical location, continuous blood flow from the gastrointestinal tract through the hepatic sinusoids allows the metabolically active hepatocytes, the non-parenchymal cells and the various immune cell populations residing and patrolling in the liver to interact with antigens and microbiological components coming from the intestine. Cytokines are key mediators within the complex interplay of intrahepatic immune cells and hepatocytes, because they can activate effector functions of immune cells as well as hepatocytic intracellular signaling pathways controlling cellular homeostasis. Kupffer cells and liver-infiltrating monocyte-derived macrophages are primary sources of cytokines such as tumor necrosis factor (TNF). The liver is also enriched in natural killer (NK) and natural killer T (NKT) cells, which fulfill functions in pathogen defense, T cell recruitment and modulation of fibrogenic responses. TNF can activate specific intracellular pathways in hepatocytes that influence cell fate in different manners, e.g. pro-apoptotic signals via the caspase cascade, but also survival pathways, namely the nuclear factor (NF)-kappaB pathway. NF-kappaB regulates important functions in liver physiology and pathology. The exact dissection of the contribution of recruited and resident immune cells, their soluble cytokine and chemokine mediators and the intracellular hepatocytic response in liver homeostasis and injury could potentially identify novel targets for the treatment of acute and chronic liver disease, liver fibrosis or cirrhosis.

Serum NT-proCNP concentrations are elevated in patients with chronic liver diseases and associated with complications and unfavorable prognosis of cirrhosis

OBJECTIVES C-type natriuretic peptide (CNP) might be an important regulator of vasodilatation, fluid and sodium balance in liver cirrhosis. We aimed at assessing its regulation and prognostic relevance in liver disease patients. DESIGN AND METHODS We analyzed NT-proCNP serum levels in 193 patients with chronic liver diseases and 43 healthy controls. RESULTS Serum NT-proCNP concentrations were significantly elevated in liver disease patients compared to healthy controls, with highest levels in established hepatic cirrhosis, independent of disease etiology. NT-proCNP was associated with complications of liver diseases and portal hypertension, namely ascites, esophageal varices and hepatic encephalopathy. Circulating NT-proCNP correlated inversely with renal function. Importantly, elevated NT-proCNP levels were identified as a predictor of mortality or necessity for transplantation. NT-proCNP levels >2 pmol/L indicated adverse prognosis (sensitivity 66.7%, specificity 72.8%, RR 5.4 [95%-CI 2.6-11.2]). CONCLUSIONS Serum NT-proCNP is elevated in advanced liver diseases and has prognostic value in cirrhotic patients.

Growth Differentiation Factor-15 Is a Predictor of Mortality in Critically Ill Patients with Sepsis

Growth differentiation factor-15 (GDF-15) is a member of the transforming growth factor-β superfamily related to inflammation and macrophage activation. Serum concentrations of GDF-15 can predict poor survival in chronic diseases, but its role in sepsis is obscure. Therefore, we investigated GDF-15 as a prognostic biomarker in critically ill patients. We measured GDF-15 levels in 219 critically ill patients (146 with sepsis, 73 without sepsis) upon admission to the intensive care unit (ICU), in comparison to 66 healthy controls. GDF-15 levels were significantly increased in ICU patients compared to controls. GDF-15 was further increased in sepsis and showed a strong association with organ dysfunction (kidney, liver and lactate) and disease severity (APACHE II and SOFA score). High GDF-15 concentrations at admission independently predicted ICU (HR 3.42; 95% CI 1.33–8.78) and overall mortality (HR 2.02, 95% CI 1.02–3.88) in all ICU critically ill patients as well as in a large subgroup of sepsis patients (ICU mortality: HR 3.16; 95% CI 1.10–9.07; overall mortality: HR 2.62; 95% CI 1.14–6.02). Collectively, serum GDF-15 levels are significantly increased in critically ill patients, associated with sepsis, organ failure, and disease severity. High GDF-15 levels at ICU admission predict short- and long-term mortality risk.

The PDE4 inhibitor roflumilast reduces weight gain by increasing energy expenditure and leads to improved glucose metabolism.

To investigate the metabolic effects of the phosphodiesterase‐4 (PDE4) inhibitor roflumilast, a clinically approved anti‐inflammatory drug used for the treatment of chronic obstructive pulmonary disease.