Shishir Shetty

Distinct Roles for CCR4 and CXCR3 in the Recruitment and Positioning of Regulatory T Cells in the Inflamed Human Liver

Regulatory T cells (Tregs) are found at sites of chronic inflammation where they mediate bystander and Ag-specific suppression of local immune responses. However, little is known about the molecular control of Treg recruitment into inflamed human tissues. We report that up to 18% of T cells in areas of inflammation in human liver disease are forkhead family transcriptional regulator box P3 (FoxP3)+ Tregs. We isolated CD4+CD25+CD127lowFoxP3+ Tregs from chronically inflamed human liver removed at transplantation; compared with blood-derived Tregs, liver-derived Tregs express high levels of the chemokine receptors CXCR3 and CCR4. In flow-based adhesion assays using human hepatic sinusoidal endothelium, Tregs used CXCR3 and α4β1 to bind and transmigrate, whereas CCR4 played no role. The CCR4 ligands CCL17 and CCL22 were absent from healthy liver, but they were detected in chronically inflamed liver where their expression was restricted to dendritic cells (DCs) within inflammatory infiltrates. These DCs were closely associated with CD8 T cells and CCR4+ Tregs in the parenchyma and septal areas. Ex vivo, liver-derived Tregs migrated to CCR4 ligands secreted by intrahepatic DCs. We propose that CXCR3 mediates the recruitment of Tregs via hepatic sinusoidal endothelium and that CCR4 ligands secreted by DCs recruit Tregs to sites of inflammation in patients with chronic hepatitis. Thus, different chemokine receptors play distinct roles in the recruitment and positioning of Tregs at sites of hepatitis in chronic liver disease.

Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells across human hepatic sinusoidal endothelium.

The common lymphatic endothelial and vascular endothelial receptor (CLEVER-1; also known as FEEL-1 and stabilin-1) is a recycling and intracellular trafficking receptor with multifunctional properties. In this study, we demonstrate increased endothelial expression of CLEVER-1/stabilin-1 at sites of leukocyte recruitment to the inflamed human liver including sinusoids, septal vessels, and lymphoid follicles in inflammatory liver disease and tumor-associated vessels in hepatocellular carcinoma. We used primary cultures of human hepatic sinusoidal endothelial cells (HSEC) to demonstrate that CLEVER-1/stabilin-1 expression is enhanced by hepatocyte growth factor but not by classical proinflammatory cytokines. We then showed that CLEVER-1/stabilin-1 supports T cell transendothelial migration across HSEC under conditions of flow with strong preferential activity for CD4 FoxP3+ regulatory T cells (Tregs). CLEVER-1/stabilin-1 inhibition reduced Treg transendothelial migration by 40% and when combined with blockade of ICAM-1 and vascular adhesion protein-1 (VAP-1) reduced it by >80%. Confocal microscopy demonstrated that 60% of transmigrating Tregs underwent transcellular migration through HSEC via ICAM-1– and VAP-1–rich transcellular pores in close association with CLEVER-1/stabilin-1. Thus, CLEVER-1/stabilin-1 and VAP-1 may provide an organ-specific signal for Treg recruitment to the inflamed liver and to hepatocellular carcinoma.

The Role of Chemokines in the Recruitment of Lymphocytes to the Liver

Chemokines direct leukocyte trafficking and positioning within tissues, thus playing critical roles in regulating immune responses and inflammation. The chemokine system is complex, involving interactions between multiple chemokines and their receptors that operate in combinatorial cascades with adhesion molecules. The involvement of multiple chemokines and chemokine receptors in these processes brings flexibility and specificity to recruitment. The hepatic vascular bed is a unique low-flow environment through which leukocytes are recruited to the liver during homeostatic immune surveillance and in response to infection or injury. The rate of leukocyte recruitment and the nature of cells recruited through the sinusoids in response to inflammatory signals will shape the severity of disease. At one end of the spectrum, fulminant liver failure results from a rapid recruitment of leukocytes that leads to hepatocyte destruction and liver failure; at the other end, diseases such as chronic hepatitis C infection may progress over many years from hepatitis to fibrosis and cirrhosis. Chronic hepatitis is characterized by a T lymphocyte-rich infiltrate and the nature and outcome of hepatitis will depend on the T cell subsets recruited, their activation and function within the liver. Different subsets of effector T cells have been described based on their secretion of cytokines and specific functions. These include Th1 and Th2 cells, and more recently Th17 and Th9 cells, which are associated with different types of immune response and which express distinct patterns of chemokine receptors that promote their recruitment under particular conditions. The effector function of these cells is balanced by the recruitment of regulatory T cells that are able to suppress antigen-specific effectors to allow resolution of immune responses and restoration of immune homeostasis. Understanding the signals that are responsible for recruiting different lymphocyte subsets to the liver will elucidate disease pathogenesis and open up new therapeutic approaches to modulate recruitment in favor of resolution rather than injury.

Lymphocyte recruitment to the liver: molecular insights into the pathogenesis of liver injury and hepatitis.

Recirculation of blood lymphocytes through the liver occurs under normal conditions as part of the process of immune surveillance. In response to injury or infection recruitment from blood increases and the nature and distribution of the infiltrate will determine the type and outcome of the resulting hepatitis. Recruitment from blood occurs via the hepatic sinusoids and is controlled by interactions between circulating lymphocytes and the highly specialised sinusoidal endothelial cells. This is a low flow vascular bed and the molecular basis of recruitment differs from other tissues. In this review we outline the molecular basis of lymphocyte recruitment to the liver and the effect on it of the local tissue microenvironment and how dysregulation of these processes can lead to uncontrolled inflammation and liver damage.

Recruitment mechanisms of primary and malignant B cells to the human liver

B cells are present within chronically inflamed liver tissue and recent evidence implicates them in the progression of liver disease. In addition, a large proportion of hepatic lymphomas are of B‐cell origin. The molecular signals that regulate normal and malignant B‐cell recruitment into peripheral tissue from blood are poorly understood, leading us to study human B‐cell migration through hepatic sinusoidal endothelial cells in flow‐based adhesion assays. In such assays, human blood‐derived B cells were captured from shear flow without a previous rolling phase and underwent firm adhesion mediated by vascular cell adhesion molecule‐1 (VCAM‐1). Unlike T cells, which displayed vigorous crawling behavior on the endothelium, B cells remained static before a proportion underwent transendothelial migration mediated by a combination of intercellular adhesion molecule‐1 (ICAM‐1), vascular adhesion protein‐1, common lymphatic endothelial and vascular endothelial receptor‐1/stabilin‐1, and the chemokine receptors, CXCR3 and CXCR4. B‐cell lymphoma cell lines and primary malignant B cells from patients with chronic lymphocytic leukemia and marginal zone B cell lymphoma also underwent integrin‐mediated firm adhesion involving ICAM‐1 and/or VCAM‐1 and demonstrated ICAM‐1‐dependent shape‐change and crawling behavior. Unlike primary lymphocytes, the malignant cells did not undergo transendothelial migration, which could explain why lymphomas are frequently characterized by the intravascular accumulation of malignant cells in the hepatic sinusoids. Conclusion: Our findings demonstrate that distinct combinations of signals promote B‐cell recruitment to the liver, suggesting the possibility of novel targets to modulate liver inflammation in disease. Certain features of lymphocyte homing are maintained in lymphoma recruitment to the liver, suggesting that therapeutic targets for lymphocyte recruitment may also prevent hepatic lymphoma dissemination. (HEPATOLOGY 2012)

Stabilin-1 expression defines a subset of macrophages that mediate tissue homeostasis and prevent fibrosis in chronic liver injury

Significance Organ fibrosis is a major cause of global morbidity and mortality. It is driven by chronic inflammation and associated oxidative stress with depletion of cellular antioxidant defenses. We demonstrate a pathway in which the evolutionarily conserved receptor stabilin-1 on tissue-infiltrating macrophages provides a second-line defense to prevent tissue damage from oxidative stress. Stabilin-1+ monocytes take up malondialdehyde-LDL (MDA-LDL), a major product of oxidative lipid peroxidation, to form ceroid-laden macrophages. Through the uptake of MDA-LDL, stabilin-1 suppresses production of the profibrogenic chemokine CCL3 and prevents excessive collagen deposition in experimental models of liver fibrosis. We propose that macrophage stabilin-1 is a critical defense against oxidative tissue damage and thereby maintains tissue homeostasis. Macrophages are key regulators of fibrosis development and resolution. Elucidating the mechanisms by which they mediate this process is crucial for establishing their therapeutic potential. Here, we use experimental models of liver fibrosis to show that deficiency of the scavenger receptor, stabilin-1, exacerbates fibrosis and delays resolution during the recovery phase. We detected a subset of stabilin-1+ macrophages that were induced at sites of cellular injury close to the hepatic scar in mouse models of liver fibrosis and in human liver disease. Stabilin-1 deficiency abrogated malondialdehyde-LDL (MDA-LDL) uptake by hepatic macrophages and was associated with excess collagen III deposition. Mechanistically, the lack of stabilin-1 led to elevated intrahepatic levels of the profibrogenic chemokine CCL3 and an increase in GFAP+ fibrogenic cells. Stabilin-1−/− macrophages demonstrated a proinflammatory phenotype during liver injury and the normal induction of Ly6Clo monocytes during resolution was absent in stabilin-1 knockouts leading to persistence of fibrosis. Human stabilin-1+ monocytes efficiently internalized MDA-LDL and this suppressed their ability to secrete CCL3, suggesting that loss of stabilin-1 removes a brake to CCL3 secretion. Experiments with cell-lineage–specific knockouts revealed that stabilin-1 expression in myeloid cells is required for the induction of this subset of macrophages and that increased fibrosis occurs in their absence. This study demonstrates a previously unidentified regulatory pathway in fibrogenesis in which a macrophage scavenger receptor protects against organ fibrosis by removing fibrogenic products of lipid peroxidation. Thus, stabilin-1+ macrophages shape the tissue microenvironment during liver injury and healing.

Human liver sinusoidal endothelial cells promote intracellular crawling of lymphocytes during recruitment‐ a new step in migration

The recruitment of lymphocytes via the hepatic sinusoidal channels and positioning within liver tissue is a critical event in the development and persistence of chronic inflammatory liver diseases. The hepatic sinusoid is a unique vascular bed lined by hepatic sinusoidal endothelial cells (HSECs), a functionally and phenotypically distinct subpopulation of endothelial cells. Using flow‐based adhesion assays to study the migration of lymphocytes across primary human HSECs, we found that lymphocytes enter into HSECs, confirmed by electron microscopy demonstrating clear intracellular localization of lymphocytes in vitro and by studies in human liver tissues. Stimulation by interferon‐γ increased intracellular localization of lymphocytes within HSECs. Furthermore, using confocal imaging and time‐lapse recordings, we demonstrated “intracellular crawling” of lymphocytes entering into one endothelial cell from another. This required the expression of intracellular adhesion molecule‐1 and stabilin‐1 and was facilitated by the junctional complexes between HSECs. Conclusion: Lymphocyte migration is facilitated by the unique structure of HSECs. Intracellular crawling may contribute to optimal lymphocyte positioning in liver tissue during chronic hepatitis. (Hepatology 2017;65:294‐309).

Post-transplant liver biopsy and the immune response: lessons for the clinician

With the improvements in post-transplant immunosuppression, the incidence of acute cellular rejection and chronic rejection has decreased significantly over the last few years. This has led to alterations in the presentation of rejection with more patients suffering from late acute rejection, which commonly has different histological appearances compared with the early post-transplant period. There is now a shift in interest to the long-term outcome of liver allografts, with recurrent disease being the most common cause of abnormal histology. The combination of long-term immunosuppression and recurrent disease leads to complex and atypical features on biopsy specimens. Other causes of liver graft inflammation include de novo autoimmune disease and ‘idiopathic’ post-transplant hepatitis. There is gathering evidence that idiopathic hepatitis can have significant consequences in terms of tissue fibrosis and progression to cirrhosis. Future developments in genetic and immune profiling may lead to liver biopsy becoming a predictive tool to identify patients in which immunosuppression can be safely withdrawn.

Comparing clinical presentations, treatments and outcomes of hepatocellular carcinoma due to hepatitis C and non-alcoholic fatty liver disease

Introduction: Hepatocellular carcinoma (HCC) is increasing in incidence in the UK and globally. Liver cirrhosis is the common cause for developing HCC. The common reasons for liver cirrhosis are viral hepatitis C (HCV), viral hepatitis B and alcohol. However, HCC caused by non-alcoholic fatty liver disease (NAFLD)-cirrhosis is now increasingly as a result of rising worldwide obesity. Aim: To compare the clinical presentation, treatment options and outcomes of HCC due to HCV and NAFLD patients. Methods: Data were collected from two liver transplant centres in the UK (Birmingham and Newcastle upon Tyne) between 2000 and 2014. We compared 275 patients with HCV-related HCC against 212 patients with NAFLD- related HCC. Results: Patients in the NAFLD group were found to be significantly older (P < 0.001) and more likely to be Caucasian (P < 0.001). They had lower rates of cirrhosis (P < 0.001) than those in HCV-HCC group. The NAFLD group presented with significantly larger tumours (P = 0.009), whilst HCV patients had a higher alpha fetoprotein (P = 0.018). NAFLD patients were more commonly treated with TACE (P = 0.005) than the HCV patients, whilst the HCV group were significantly more likely to be transplanted (P < 0.001). In patients selected for liver transplantation, 5-year survival rates in NAFLD were not significantly different from HCV-HCC (44 and 56% respectively, P = 0.102). Conclusion: In this study, NAFLD patients presented with larger tumours that were less likely to be amenable to curative therapy, as compared with HCV patients. Despite this disadvantage, patients with NAFLD had similar overall survival compared to patients with HCV.

Role of stabilin-1 in chronic liver tissue injury and repair

Abstract Background Organ fibrosis is a major cause of global mortality. Few medical therapies are available to treat this condition. We have previously demonstrated increased expression of stabilin-1 at sites of chronic liver disease. Using models of chronic liver injury, we aimed to elucidate the role of stabilin-1 during fibrosis and repair. Methods We used full knockout and cell specific knockouts of stabilin-1 in murine models of chronic liver injury including carbon tetrachloride treatment and a methionine and choline deficient diet. A resolution phase after carbon tetrachloride treatment was used to study tissue repair. Tissue was analysed for fibrosis by confocal microscopy, collagen quantification, and immune cell infiltrate analysis. RNA sequencing and cell transfer experiments were done to elucidate underlying mechanisms. Human tissue was also analysed for stabilin-1 expression in chronic liver injury. Findings We detected a subset of stabilin-1 macrophages that were induced at sites of cellular injury close to the hepatic scar in mouse models of liver fibrosis and in human liver disease. Stabilin-1 deficiency abrogated malondialdehyde-LDL uptake by hepatic macrophages and was associated with excess collagen III deposition. Mechanistically, the lack of stabilin-1 led to elevated intrahepatic levels of the profibrogenic chemokine CCL3 and an increase in GFAP+ fibrogenic cells. Stabilin-1 knockout macrophages demonstrated a proinflammatory phenotype during liver injury and this led to delayed wound healing. Interpretation We have demonstrated a pathway in which the evolutionarily conserved receptor stabilin-1 on tissue-infiltrating macrophages promotes the uptake of products of lipid peroxidation and thus prevents excess scarring. We propose that macrophage stabilin-1 is a crucial defence against oxidative tissue damage and thereby maintains tissue homoeostasis. Funding Wellcome Trust.