Evaggelia Liaskou

Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH‐related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild‐type (WT) mice, Patched‐deficient (Ptc+/−) (overly active Hh signaling) mice, and OPN‐deficient mice before and after feeding methionine and choline–deficient (MCD) diets to induce NASH‐related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc+/− mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN‐deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up‐regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up‐regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte‐derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). Conclusion: OPN is Hh‐regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH (HEPATOLOGY 2011)

NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease

Objective Immune responses are important in dictating non-alcoholic steatohepatitis (NASH) outcome. We previously reported that upregulation of hedgehog (Hh) and osteopontin (OPN) occurs in NASH, that Hh-regulated accumulation of natural killer T (NKT) cells promotes hepatic stellate cell (HSC) activation, and that cirrhotic livers harbour large numbers of NKT cells. Design The hypothesis that activated NKT cells drive fibrogenesis during NASH was evaluated by assessing if NKT depletion protects against NASH fibrosis; identifying the NKT-associated fibrogenic factors; and correlating plasma levels of the NKT cell-associated factor OPN with fibrosis severity in mice and humans. Results When fed methionine-choline-deficient (MCD) diets for 8 weeks, wild type (WT) mice exhibited Hh pathway activation, enhanced OPN expression, and NASH-fibrosis. Ja18‒/‒ and CD1d‒/‒ mice which lack NKT cells had significantly attenuated Hh and OPN expression and dramatically less fibrosis. Liver mononuclear cells (LMNCs) from MCD diet fed WT mice contained activated NKT cells, generated Hh and OPN, and stimulated HSCs to become myofibroblasts; neutralising these factors abrogated the fibrogenic actions of WT LMNCs. LMNCs from NKT-cell-deficient mice were deficient in fibrogenic factors, failing to activate collagen gene expression in HSCs. Human NASH livers with advanced fibrosis contained more OPN and Hh protein than those with early fibrosis. Plasma levels of OPN mirrored hepatic OPN expression and correlated with fibrosis severity. Conclusion Hepatic NKT cells drive production of OPN and Hh ligands that promote fibrogenesis during NASH. Associated increases in plasma levels of OPN may provide a biomarker of NASH fibrosis.

Monocyte subsets in human liver disease show distinct phenotypic and functional characteristics

Liver fibrosis is a wound healing response to chronic liver injury and inflammation in which macrophages and infiltrating monocytes participate in both the development and resolution phase. In humans, three monocyte subsets have been identified: the classical CD14++CD16−, intermediate CD14++CD16+, and nonclassical CD14+CD16++ monocytes. We studied the phenotype and function of these monocyte subsets in peripheral blood and liver tissue from patients with chronic inflammatory and fibrotic liver diseases. The frequency of intrahepatic monocytes increased in disease compared with control liver tissue, and in both nondiseased and diseased livers there was a higher frequency of CD14++CD16+ cells with blood. Our data suggest two nonexclusive mechanisms of CD14++CD16+ accumulation in the inflamed liver: (1) recruitment from blood, because more than twice as many CD14++CD16+ monocytes underwent transendothelial migration through hepatic endothelial cells compared with CD14++CD16− cells; and (2) local differentiation from CD14++CD16− classical monocytes in response to transforming growth factor β and interleukin (IL)‐10. Intrahepatic CD14++CD16+ cells expressed both macrophage and dendritic cell markers but showed high levels of phagocytic activity, antigen presentation, and T cell proliferation and secreted proinflammatory (tumor necrosis factor α, IL‐6, IL‐8, IL‐1β) and profibrogenic cytokines (IL‐13), chemokines (CCL1, CCL2, CCL3, CCL5), and growth factors (granulocyte colony‐stimulating factor and granulocyte‐macrophage colony‐stimulating factor), consistent with a role in the wound healing response. Conclusion: Intermediate CD14++CD16+ monocytes preferentially accumulate in chronically inflamed human liver as a consequence of enhanced recruitment from blood and local differentiation from classical CD14++CD16− monocytes. Their phagocytic potential and ability to secrete inflammatory and profibrogenic cytokines suggests they play an important role in hepatic fibrogenesis. (HEPATOLOGY 2013)

Innate immune cells in liver inflammation.

Innate immune system is the first line of defence against invading pathogens that is critical for the overall survival of the host. Human liver is characterised by a dual blood supply, with 80% of blood entering through the portal vein carrying nutrients and bacterial endotoxin from the gastrointestinal tract. The liver is thus constantly exposed to antigenic loads. Therefore, pathogenic microorganism must be efficiently eliminated whilst harmless antigens derived from the gastrointestinal tract need to be tolerized in the liver. In order to achieve this, the liver innate immune system is equipped with multiple cellular components; monocytes, macrophages, granulocytes, natural killer cells, and dendritic cells which coordinate to exert tolerogenic environment at the same time detect, respond, and eliminate invading pathogens, infected or transformed self to mount immunity. This paper will discuss the innate immune cells that take part in human liver inflammation, and their roles in both resolution of inflammation and tissue repair.

CXCR3-dependent recruitment and CCR6-mediated positioning of Th-17 cells in the inflamed liver

Background & Aims IL-17 secreting CD4 (Th17) and CD8 (Tc17) T cells have been implicated in immune-mediated liver diseases, but the molecular basis for their recruitment and positioning within the liver is unknown. Methods The phenotype and migratory behaviour of human liver-derived Th17 and Tc17 cells were investigated by flow cytometry and chemotaxis and flow-based adhesion assays. The recruitment of murine Th17 cells to the liver was studied in vivo using intra-vital microscopy. Results IL-17+ T cells comprised 1–3% of the T cell infiltrate in inflammatory liver diseases and included both CD4 (Th17) and CD8 (Tc17) cells. They expressed RORC and the IL-23 receptor and included subsets that secreted IL-22 and interferon-γ. Th17 and Tc17 cells expressed high levels of CXCR3 and CCR6, Tc17 cells also expressed CXCR6. Binding to human sinusoidal endothelium from flow was dependent on β1 and β2 integrins, CXCR3, and, in the case of Th17 cells, VAP-1. Th17 recruitment via sinusoids in mice with liver inflammation was reduced by treatment with antibodies against CXCR3 ligands, confirming the role of CXCR3 in Th17 recruitment in vivo. In human liver, IL-17+ cells were detected in portal infiltrates close to inflamed bile ducts expressing the CCR6 ligand CCL20. Cytokine-treated human cholangiocytes secreted CCL20 and induced CCR6-dependent migration of Th17 cells suggesting that local cholangiocyte chemokine secretion localises Th17 cells to bile ducts. Conclusions CXCR3 promotes recruitment of Th17 cells from the blood into the liver in both human and murine liver injury. Their subsequent positioning near bile ducts is dependent on cholangiocyte-secreted CCL20.

Regulation of mucosal addressin cell adhesion molecule 1 expression in human and mice by vascular adhesion protein 1 amine oxidase activity

Primary sclerosing cholangitis (PSC) and autoimmune hepatitis are hepatic complications associated with inflammatory bowel disease (IBD). The expression of mucosal addressin cell adhesion molecule 1 (MAdCAM‐1) on mucosal endothelium is a prerequisite for the development of IBD, and it is also detected on the hepatic vessels of patients with liver diseases associated with IBD. This aberrant hepatic expression of MAdCAM‐1 results in the recruitment of effector cells initially activated in the gut to the liver, in which they drive liver injury. However, the factors responsible for the aberrant hepatic expression of MAdCAM‐1 are not known. In this study, we show that deamination of methylamine (MA) by vascular adhesion protein 1 (VAP‐1) [a semicarbazide‐sensitive amine oxidase (SSAO) expressed in the human liver] in the presence of tumor necrosis factor α induces the expression of functional MAdCAM‐1 in hepatic endothelial cells and in intact human liver tissue ex vivo. This is associated with increased adhesion of lymphocytes from patients with PSC to hepatic vessels. Feeding mice MA, a constituent of food and cigarette smoke found in portal blood, led to VAP‐1/SSAO–dependent MAdCAM‐1 expression in mucosal vessels in vivo. Conclusion: Activation of VAP‐1/SSAO enzymatic activity by MA, a constituent of food and cigarette smoke, induces the expression of MAdCAM‐1 in hepatic vessels and results in the enhanced recruitment of mucosal effector lymphocytes to the liver. This could be an important mechanism underlying the hepatic complications of IBD. (HEPATOLOGY 2011;53:661‐672)

CX3CR1 and vascular adhesion protein‐1‐dependent recruitment of CD16+ monocytes across human liver sinusoidal endothelium

The liver contains macrophages and myeloid dendritic cells (mDCs) that are critical for the regulation of hepatic inflammation. Most hepatic macrophages and mDCs are derived from monocytes recruited from the blood through poorly understood interactions with hepatic sinusoidal endothelial cells (HSECs). Human CD16+ monocytes are thought to contain the precursor populations for tissue macrophages and mDCs. We report that CD16+ cells localize to areas of active inflammation and fibrosis in chronic inflammatory liver disease and that a unique combination of cell surface receptors promotes the transendothelial migration of CD16+ monocytes through human HSECs under physiological flow. CX3CR1 activation was the dominant pertussis‐sensitive mechanism controlling transendothelial migration under flow, and expression of the CX3CR1 ligand CX3CL1 is increased on hepatic sinusoids in chronic inflammatory liver disease. Exposure of CD16+ monocytes to immobilized purified CX3CL1 triggered β1‐integrin‐mediated adhesion to vascular cell adhesion molecule‐1 and induced the development of a migratory phenotype. Following transmigration or exposure to soluble CX3CL1, CD16+ monocytes rapidly but transiently lost expression of CX3CR1. Adhesion and transmigration across HSECs under flow was also dependent on vascular adhesion protein‐1 (VAP‐1) on the HSECs. Conclusion: Our data suggest that CD16+ monocytes are recruited by a combination of adhesive signals involving VAP‐1 and CX3CR1 mediated integrin‐activation. Thus a novel combination of surface molecules, including VAP‐1 and CX3CL1 promotes the recruitment of CD16+ monocytes to the liver, allowing them to localize at sites of chronic inflammation and fibrosis. (Hepatology 2010)

CCL25 and CCL28 promote α4β7-integrin-dependent adhesion of lymphocytes to MAdCAM-1 under shear flow

Inflammatory bowel disease is characterized by the recruitment of lymphocytes to the gut via mucosal vessels. Chemokines are believed to trigger alpha(4)beta(1)- and alpha(4)beta(7)-integrin-mediated adhesion to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on mucosal vessels, although the contribution of each pathway and the chemokines involved are not well characterized. These interactions occur under conditions of hemodynamic shear, which is critical in determining how lymphocytes integrate chemokine signals to promote transmigration. To define the role of specific chemokines in mediating lymphocyte adhesion to VCAM-1 and MAdCAM-1, we studied the ability of immobilized chemokines to activate adhesion of human lymphocytes in a flow-based adhesion assay. Adhesion to immobilized MAdCAM-1 was alpha(4)beta(7) dependent, with no contribution from alpha(4)beta(1), whereas alpha(4)beta(1) mediated rolling and static adhesion on VCAM-1. Immobilized CC-chemokine ligand (CCL) 25 and CCL28 were both able to trigger alpha(4)beta(7)-dependent lymphocyte arrest on MAdCAM-1 under shear, highlighting a potential role for these chemokines in the arrest of lymphocytes on postcapillary venules in the gut. Neither had any effect on adhesion to VCAM-1, suggesting that they selectively trigger alpha(4)beta(7)-mediated adhesion. Immobilized CCL21, CCL25, CCL28, and CXC-chemokine ligand (CXCL) 12 all converted rolling adhesion to static arrest on MAdCAM-1 by activating lymphocyte integrins, but only CCL21 and CXCL12 also triggered a motile phenotype characterized by lamelipodia and uropod formation. Thus alpha(4)beta(1)/VCAM-1 and alpha(4)beta(7)/MAdCAM-1 operate independently to support lymphocyte adhesion from flow, and chemokines may act in concert with one chemokine triggering integrin-mediated arrest and a second chemokine promoting motility and transendothelial migration.

Lymphocyte Homing and Its Role in the Pathogenesis of IBD

Inflammatory bowel disease (IBD) is an idiopathic disorder of chronic inflammation of the gastrointestinal tract. Experimental models of IBD and results from human genomewide linkage studies suggest that the primary defect that leads to IBD is an inappropriate mucosal immune response to normal intestinal microbes. Genetic alterations not only confer increased susceptibility to IBD but also appear to determine the nature and location of the intestinal inflammation, as is evident in patients with genetic alterations of NOD2 and their susceptibility for ileal Crohns disease. IBD has traditionally been classified into 2 subtypes, namely, ulcerative colitis (UC) and Crohns disease (CD), based on histological appearance and anatomical distribution. However, an increasing body of data supports the concept that IBD is an umbrella diagnosis encompassing a variety of disorders with distinct genetic, microbial, and environmental determinants that cluster either into a UC or CD phenotype. The shared common pathway is uncontrolled intestinal inflammation. A key element in the pathogenesis of intestinal inflammation in both UC and CD is increased leukocyte recruitment from the circulation, and this provides a potential target for pharmaceutical inhibition. In this article we review the current understanding of the molecules that determine leukocyte trafficking to the gut and highlight opportunities where their inhibition could be exploited to treat IBD.(Inflamm Bowel Dis 2008).

Mechanisms of tissue injury in autoimmune liver diseases

Autoimmune diseases affecting the liver are mainly represented by autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). The characteristic morphologic patterns of injury are a chronic hepatitis pattern of damage in AIH, destruction of small intrahepatic bile ducts in PBC and periductal fibrosis and inflammation involving larger bile ducts in PSC. The factors responsible for initiation and perpetuation of the injury in all the three autoimmune liver diseases are not understood completely but are likely to be environmental triggers on the background of genetic variation in immune regulation. In this review, we summarise the current understanding of the mechanisms underlying the breakdown of self-tolerance in autoimmune liver diseases.