Organ-on-a-chip for studying immune cell adhesion to liver sinusoidal endothelial cells: the potential for testing immunotherapies and cell therapy trafficking.

Immunotherapy has changed the landscape of treatment options for patients with hepatocellular cancer. Checkpoint inhibitors are now standard of care for patients with advanced tumours, yet the majority remain resistant to this therapy and urgent approaches are needed to boost the efficacy of these agents. Targeting the liver endothelial cells, as the orchestrators of immune cell recruitment, within the tumour microenvironment of this highly vascular cancer could potentially boost immune cell infiltration. We demonstrate the successful culture of primary human liver endothelial cells in organ-on-a-chip technology followed by perfusion of peripheral blood mononuclear cells. We confirm, with confocal and multiphoton imaging, the capture and adhesion of immune cells in response to pro-inflammatory cytokines in this model. This multicellular platform sets the foundation for testing the efficacy of new therapies in promoting leukocyte infiltration across liver endothelium as well as a model for testing cell therapy, such as chimeric antigen receptor (CAR)-T cell, capture and migration across human liver endothelium.

Expression of E-cadherin by CD8+ T cells promotes their invasion into biliary epithelial cells.

The presence of CD8+ T cells in the cytoplasm of biliary epithelial cells (BEC) has been correlated with biliary damage associated with primary biliary cholangitis (PBC). Here, we characterise the mechanism of CD8+ T cell invasion into BEC. CD8+ T cells observed within BEC were large, eccentric, and expressed E-cadherin, CD103 and CD69. They were also not contained within secondary vesicles. Internalisation required cytoskeletal rearrangements which facilitated contact with BEC. Internalised CD8+ T cells were observed in both non-cirrhotic and cirrhotic diseased liver tissues but enriched in PBC patients, both during active disease and at the time of transplantation. E-cadherin expression by CD8+ T cells correlated with frequency of internalisation of these cells into BEC. E-cadherin+ CD8+ T cells formed ß-catenin-associated interactions with BEC, were larger than E-cadherin- CD8+ T cells and invaded into BEC more frequently. Overall, we unveil a distinct cell-in-cell structure process in the liver detailing the invasion of E-cadherin+ CD103+ CD69+ CD8+ T cells into BEC.

The Role of B Cells in Adult and Paediatric Liver Injury.

B lymphocytes are multitasking cells that direct the immune response by producing pro- or anti-inflammatory cytokines, by presenting processed antigen for T cell activation and co-stimulation, and by turning into antibody-secreting cells. These functions are important to control infection in the liver but can also exacerbate tissue damage and fibrosis as part of persistent inflammation that can lead to end stage disease requiring a transplant. In transplantation, immunosuppression increases the incidence of lymphoma and often this is of B cell origin. In this review we bring together information on liver B cell biology from different liver diseases, including alcohol-related and metabolic fatty liver disease, autoimmune hepatitis, primary biliary and primary sclerosing cholangitis, viral hepatitis and, in infants, biliary atresia. We also discuss the impact of B cell depletion therapy in the liver setting. Taken together, our analysis shows that B cells are important in the pathogenesis of liver diseases and that further research is necessary to fully characterise the human liver B cell compartment.

Targeting Enclysis in Liver Autoimmunity, Transplantation, Viral Infection and Cancer.

Persistent liver inflammation can lead to cirrhosis, which associates with significant morbidity and mortality worldwide. There are no curative treatments beyond transplantation, followed by long-term immunosuppression. The global burden of end stage liver disease has been increasing and there is a shortage of donor organs, therefore new therapies are desperately needed. Harnessing the power of the immune system has shown promise in certain autoimmunity and cancer settings. In the context of the liver, regulatory T cell (Treg) therapies are in development. The hypothesis is that these specialized lymphocytes that dampen inflammation may reduce liver injury in patients with chronic, progressive diseases, and promote transplant tolerance. Various strategies including intrinsic and extracorporeal expansion of Treg cells, aim to increase their abundance to suppress immune responses. We recently discovered that hepatocytes engulf and delete Treg cells by enclysis. Herein, we propose that inhibition of enclysis may potentiate existing regulatory T cell therapeutic approaches in patients with autoimmune liver diseases and in patients receiving a transplant. Moreover, in settings where the abundance of Treg cells could hinder beneficial immunity, such us in chronic viral infection or liver cancer, enhancement of enclysis could result in transient, localized reduction of Treg cell numbers and tip the balance towards antiviral and anti-tumor immunity. We describe enclysis as is a natural process of liver immune regulation that lends itself to therapeutic targeting, particularly in combination with current Treg cell approaches.

Cell-in-Cell Structures in the Liver: A Tale of Four E's.

The liver is our largest internal organ and it plays major roles in drug detoxification and immunity, where the ingestion of extracellular material through phagocytosis is a critical pathway. Phagocytosis is the deliberate endocytosis of large particles, microbes, dead cells or cell debris and can lead to cell-in-cell structures. Various types of cell endocytosis have been recently described for hepatic epithelia (hepatocytes), which are non-professional phagocytes. Given that up to 80% of the liver comprises hepatocytes, the biological impact of cell-in-cell structures in the liver can have profound effects in liver regeneration, inflammation and cancer. This review brings together the latest reports on four types of endocytosis in the liver -efferocytosis, entosis, emperipolesis and enclysis, with a focus on hepatocyte biology.

Clearance of Apoptotic Cells by Tissue Epithelia: A Putative Role for Hepatocytes in Liver Efferocytosis.

Toxic substances and microbial or food-derived antigens continuously challenge the liver, which is tasked with their safe neutralization. This vital organ is also important for the removal of apoptotic immune cells during inflammation and has been previously described as a "graveyard" for dying lymphocytes. The clearance of apoptotic and necrotic cells is known as efferocytosis and is a critical liver function to maintain tissue homeostasis. Much of the research into this form of immunological control has focused on Kupffer cells, the liver-resident macrophages. However, hepatocytes (and other liver resident cells) are competent efferocytes and comprise 80% of the liver mass. Little is known regarding the mechanisms of apoptotic and necrotic cell capture by epithelia, which lack key receptors that mediate phagocytosis in macrophages. Herein, we discuss recent developments that increased our understanding of efferocytosis in tissues, with a special focus on the liver parenchyma. We discuss the impact of efferocytosis in health and in inflammation, highlighting the role of phagocytic epithelia.

MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure.

OBJECTIVE: Acute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response. DESIGN: Flow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer-/-) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice. RESULTS: We demonstrate a significant expansion of resolution-like MerTK+HLA-DRhigh cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCIIhigh macrophages during the resolution phase in ALF, APAP-treated Mer-/- mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DRhigh phenotype which promotes neutrophil apoptosis and their subsequent clearance. CONCLUSIONS: We identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury.

Commensal-derived OMVs elicit a mild proinflammatory response in intestinal epithelial cells.

Under normal physiological conditions, the intestinal immunity remains largely hyporesponsive to the commensal microbiota, yet also retains the inherent ability to rapidly respond to pathogenic antigens. However, immunomodulatory activities of extracellular products from commensal bacteria have been little studied, with previous investigations generally utilizing the live bacterium to study microbiota-epithelial interactions. In this study, we demonstrate that extracellular products of a commensal bacterium, Escherichia coli C25, elicit a moderate release of proinflammatory IL-8 and stimulate transcriptional up-regulation of Toll-like receptors (TLRs) in intestinal epithelial cell lines HT29-19A and Caco-2. Additionally, we show that removal of outer membrane vesicles (OMVs) reduces the proinflammatory effect of secreted products from E. coli C25. Furthermore, we show that isolated OMVs have a dose-dependent proinflammatory effect on intestinal epithelial cells (IECs). Interestingly, a relatively high concentration (40 µg ml-1 protein) of OMVs had no significant regulatory effects on TLR mRNA expression in both cell lines. Finally, we also demonstrate that pre-incubation with E. coli C25-derived OMVs subsequently inhibited the internalization of the bacterium itself in both cell lines. Taken together, our results suggest that commensal-derived extracellular products, in particular OMVs, could significantly contribute to intestinal homeostasis. We also demonstrate a unique interaction between commensal-derived OMVs and host cells.

In Vitro and Ex Vivo Models to Study T Cell Migration Through the Human Liver Parenchyma.

The liver is the largest internal organ and filters around 3 pints of blood per minute. This continuous flux of blood should not be confused with rapid egress of lymphocytes through the liver; this organ has intricate corridors of specialized sinusoidal spaces, ensuring that immune cells decelerate to shear flow rates, and providing ample opportunities to interact with parenchymal cells. Migration has been intricately linked to T cell function; it is therefore important to study liver T cell biology into context within the liver microenvironment. Here we discuss the highly organized architecture of liver-resident cells (sinusoidal endothelia, Kupffer cells, stellate cells/myofibroblasts, and biliary and hepatic epithelia) and showcase basic, multicellular, and complex systems to model T cell migration through the human liver microenvironment in vitro and ex vivo.