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The opportunistic fungal pathogenCryptococcus neoformanscauses lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear howC. neoformansis recognized and phagocytosed by macrophages. Here we investigate the role of TLR4 in the nonopsonic phagocytosis ofC. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of nonopsonized cryptococci. The increased phagocytosis observed inTlr4-/-cells was dampened by pre-treatment of macrophages with either a TLR3 inhibitor or oxidised-LDL, a known ligand of scavenger receptors. The scavenger receptor, macrophage scavenger receptor 1 (MSR1) (also known as SR-A1 or CD204) was upregulated inTlr4-/-macrophages and there was a 75% decrease in phagocytosis of nonopsonized cryptococci byMsr1-/-macrophages. Furthermore, immunofluorescence imaging revealed colocalization of MSR1 and internalised cryptococci. Together, these results identify MSR1 as a key receptor for the phagocytosis of nonopsonizedC. neoformansand demonstrate TLR4/MSR1 crosstalk in the phagocytosis ofC. neoformans.

The scavenger receptor superfamily represents a highly diverse collection of evolutionarily-conserved receptors which are known to play key roles in host homeostasis, the most prominent of which is the clearance of unwanted endogenous macromolecules, such as oxidized low-density lipoproteins, from the systemic circulation. Members of this family have also been well characterized in their binding and internalization of a vast range of exogenous antigens and, consequently, are generally considered to be pattern recognition receptors, thus contributing to innate immunity. Several studies have implicated scavenger receptors in the pathophysiology of several inflammatory diseases, such as Alzheimer's and atherosclerosis. Hepatic resident cellular populations express a diverse complement of scavenger receptors in keeping with the liver's homeostatic functions, but there is gathering interest in the contribution of these receptors to hepatic inflammation and its complications. Here, we review the expression of scavenger receptors in the liver, their functionality in liver homeostasis, and their role in inflammatory liver disease and cancer.

The arginyl-glycinyl-aspartic acid (RGD) integrin alpha-v beta-6 (αvβ6) has been identified as playing a key role in the activation of transforming growth factor-β (TGFβ) that is hypothesized to be pivotal in the development of fibrosis and other diseases. In this study, αvβ6 small molecule inhibitors were characterized in a range of in vitro systems to determine affinity, kinetics, and duration of TGFβ inhibition. High αvβ6 binding affinity was shown to be correlated with slow dissociation kinetics. Compound 1 (high αvβ6 affinity, slow dissociation) and SC-68448 (low αvβ6 affinity, fast dissociation) induced concentration- and time-dependent internalization of αvβ6 in normal human bronchial epithelial (NHBE) cells. After washout, the αvβ6 cell surface repopulation was faster for SC-68448 compared with compound 1 In addition, αvβ6-dependent release of active TGFβ from NHBE cells was inhibited by compound 1 and SC-68448. After washout of SC-68448, release of active TGFβ was restored, whereas after washout of compound 1 the inhibition of TGFβ activation was maintained and only reversible in the presence of a lysosomal inhibitor (chloroquine). However, SC-68448 was able to reduce total levels of αvβ6 in NHBE cells if present continuously. These observations suggest αvβ6 can be degraded after high affinity RGD binding that sorts the integrin for lysosomal degradation after internalization, likely due to sustained engagement as a result of slow dissociation kinetics. In addition, the αvβ6 integrin can also be downregulated after sustained engagement of the RGD binding site with low affinity ligands that do not sort the integrin for immediate lysosomal degradation. SIGNIFICANCE STATEMENT: The fate of RGD integrin after ligand binding has not been widely investigated. Using the αvβ6 integrin as a case study, we have demonstrated that RGD-induced downregulation of αvβ6 is both affinity and time dependent. High affinity ligands induced downregulation via lysosomal degradation, likely due to slow dissociation, whereas sustained low affinity ligand engagement was only able to decrease αvβ6 expression over longer periods of time. Our study provides a potential unique mechanism for obtaining duration of action for drugs targeting integrins.

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality. Activation of the pro-fibrotic cytokine transforming growth factor-β (TGFβ) via the alpha-V beta-6 (αvβ6) integrin has been identified as playing a key role in the development of fibrosis. Therefore, a drug discovery programme to identify an orally bioavailable small molecule αvβ6 arginyl-glycinyl-aspartic acid (RGD)-mimetic was initiated. As part of a medicinal chemistry programme GSK3335103 was identified and profiled in a range of pre-clinical in vitro and in vivo systems. GSK3335103 was shown to bind to the αvβ6 with high affinity and demonstrated fast binding kinetics. In primary human lung epithelial cells, GSK3335103 induced concentration- and time-dependent internalisation of αvβ6 with a rapid return of integrin to the cell surface observed after washout. Following sustained engagement of the αvβ6 integrin in vitro, lysosomal degradation was induced by GSK3335103. GSK3335103 was shown to engage with the αvβ6 integrin and inhibit the activation of TGFβ in both ex vivo IPF tissue and in a murine model of bleomycin-induced lung fibrosis, as measured by αvβ6 engagement, TGFβ signalling and collagen deposition, with a prolonged duration of action observed in vivo. In summary, GSK3335103 is a potent αvβ6 inhibitor that attenuates TGFβ signalling in vitro and in vivo with a well-defined pharmacokinetic/pharmacodynamic relationship. This translates to a significant reduction of collagen deposition in vivo and therefore GSK3335103 represents a potential novel oral therapy for fibrotic disorders.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 µL media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Summary CD4+ T cells play critical roles in directing immunity, both as T helper and as regulatory T (Treg) cells. Here, we demonstrate that hepatocytes can modulate T cell populations through engulfment of live CD4+ lymphocytes. We term this phenomenon enclysis to reflect the specific enclosure of CD4+ T cells in hepatocytes. Enclysis is selective for CD4+ but not CD8+ cells, independent of antigen-specific activation, and occurs in human hepatocytes in vitro, ex vivo, and in vivo. Intercellular adhesion molecule 1 (ICAM-1) facilitates T cell early adhesion and internalization, whereas hepatocytes form membrane lamellipodia or blebs to mediate engulfment. T cell internalization is unaffected by wortmannin and Rho kinase inhibition. Hepatocytes engulf Treg cells more efficiently than non-Treg cells, but Treg cell-containing vesicles preferentially acidify overnight. Thus, enclysis is a biological process with potential effects on immunomodulation and opens a new field for research to fully understand CD4+ T cell dynamics in liver inflammation., Graphical Abstract, Highlights • Hepatocytes engulf live CD4+ T cells, with a preference for regulatory cells • We called this process enclysis and compared it with known cell-in-cell processes • ICAM-1 and β-catenin accumulated at the point of T cell engulfment • Enclysis was seen in health, in liver cancer, and especially in autoimmune disorders, Enclysis describes the enclosure of live CD4+ T cells in intracellular vesicles. Davies et al. show that hepatocytes engulf regulatory T (Treg) cells and preferentially delete them. Enclysis is distinct from entosis, efferocytosis, and suicidal emperipolesis and may be targeted to control T cell populations in the liver during inflammation.

Compound 8 is a selective αvβ1 small molecule inhibitor that has been used in pre-clinical studies to identify and characterise the αvβ1 integrin as a potential target in fibrotic disease. In this study we further investigated the selectivity and pharmacokinetics of compound 8 to determine a link between the levels of αvβ1 engagement required to achieve in vivo pharmacodynamic efficacy. The selectivity of compound 8 for the arginyl-glycinyl-aspartic acid and β1 integrins was measured using purified integrin protein preparations in radioligand binding studies with both labelled ([3H]compound 8) and unlabelled versions. The pharmacokinetic profile of compound 8 was completed in in vitro blood protein binding assays and in in vivo studies using male C57BL/6 mouse following i.v. dosing. The high selectivity of compound 8 for αvβ1 over the other αv integrins was confirmed, however a reduced selectivity was demonstrated for the β1 integrin family, with high affinity observed for α4β1 (comparable to αvβ1), moderate affinity for α2β1, α3β1 and α8β1, and low affinity for α5β1 and α9β1. Compound 8 was shown to be cleared quickly from the blood with a short half-life of 0.5 h. In conclusion, the data in this study suggest that compound 8 has the potential to engage a number of integrins in vivo beyond αvβ1, that raises a degree of uncertainty regarding its mechanism of action in models of fibrotic disease.

The arginyl-glycinyl-aspartic acid (RGD) integrin alpha-v beta-6 (

Chronic liver diseases (CLDs) are characterised by leukocyte infiltration which drives chronic inflammation, fibrosis and cirrhosis. Hepatic sinusoidal endothelial cells (HSEC) play a critical role in liver homeostasis, regulating the immune microenvironment and maintaining tolerance. HSEC line the low shear environment of the hepatic sinusoids in which leukocyte recruitment occurs. They undergo substantial phenotypic changes during CLD, yet how this influences the immune microenvironment in liver disease remains poorly understood. Mannose receptor (MR) and plasmalemma vesicle-associated protein (PLVAP) are atypical adhesion molecules, expressed within specialised vascular beds, although their contribution to CLD remains unknown. We sought to characterise MR and PLVAP expression in normal and diseased human liver tissue and aimed to understand their regulation in primary human HSEC. Immunohistochemistry studies demonstrated a distinct and mutually exclusive expression pattern, with homogenous MR expression throughout normal liver sinusoids, and PLVAP localisation to peri-venular sinusoids. In CLD, MR expression was lower, displaying disrupted homogeneity, whilst PLVAP was significantly upregulated, correlating spatially and quantitatively with collagen deposition and fibrosis independently of aetiology. Mutual exclusivity and endothelial identity of MR+ and PLVAP+ cells were confirmed by dual immunofluorescence. PLVAP co-localised with endothelial marker, CD31, whilst MR co-localised with sinusoidal marker L-SIGN (liver/lymph node-specific intercellular adhesion molecule-3 grabbing non-integrin). Primary HSEC were isolated from human liver tissue by immunomagnetic selection and MR/PLVAP expression was confirmed by immunofluorescence, allowing regulation studies to be conducted using these cells. Notably, whilst most HSEC were MR-positive, a subset of cells expressed PLVAP, recapitulating observations in situ within human liver. A high-content imaging assay was designed to investigate MR/PLVAP expression in response to various treatment conditions in a high-throughput manner. Contradictory to previous findings, MR levels did not fluctuate following pro-inflammatory stimulation (TNF-α, tumour necrosis factor-α; IL-1β, interleukin-1β; LPS, lipopolysaccharide), suggesting a distinct MR regulation mechanism within HSEC. Contrastingly, PLVAP expression increased following TNF-α and IL-1β treatment, and was significantly upregulated in the presence of VEGF (vascular endothelial growth factor), confirming previous reports. In conclusion, these data define two sinusoidal endothelial cell subsets, characterised by reciprocal MR/PLVAP expression, which may have distinct roles in homeostasis and inflammation. Furthermore, MR and PLVAP are differentially regulated within HSEC in vitro, with PLVAP being increased by pro-inflammatory stimuli and growth factors, supporting its upregulation in CLD. Targeting HSEC, with the aim of reprogramming the balance between MR and PLVAP expression, may represent a novel therapeutic approach in CLD.

The immune microenvironment plays a key role in determining the pathogenesis and progression of hepatocellular carcinoma (HCC). Some immune cell subsets promote tumour growth whilst others drive anti-tumour responses. Elucidating the mechanisms by which distinct immune subsets are recruited to the HCC microenvironment could lead to novel immunotherapies. Immune cell recruitment follows a generalized stepwise process, termed the adhesion cascade. We have previously shown that liver endothelial cells express a number of atypical adhesion molecules, such as scavenger receptors, which contribute to immune cell selectivity within the adhesion cascade and thus shape the hepatic immune microenvironment. Scavenger receptor class F member 1 (SCARF1) is thought to play an important role in the selective recruitment of CD4+ T cells to liver sinusoidal endothelial cells during chronic liver disease; however, its contribution to the pathophysiology of hepatocellular carcinoma (HCC) is currently unknown. In this study, we investigated the expression of SCARF1 in HCC tumours and explored its potential role in the recruitment of CD4+ T cells to the tumour microenvironment. Using TGCA (The Cancer Genome Atlas) RNA-sequencing datasets, we identified the downregulation of SCARF1 expression in HCC tumours, compared to non-tumourous tissues, and validated these findings with immunohistochemical staining of HCC resection specimens. We next explored the relationship of SCARF1 expression with tumour progression and found that a loss of SCARF1 expression was associated with aggressive tumour biology. Following this, we evaluated the prognostic value of SCARF1 expression in HCC tumours and demonstrated that high SCARF1 expression in HCC tumour tissues correlates with a better overall survival, disease-free survival and progression-free survival. Next, via a combination of TGCA data analysis and dual colour immunofluorescent staining, we confirmed that SCARF1 within HCC was largely associated with tumour endothelial cells. We then undertook flow-based recruitment assays under physiological levels of shear stress with primary liver-derived endothelial cells and purified CD4+ T cell subsets. We demonstrated that SCARF1 mediated a role in the specific recruitment of effector CD4+ T cells (CD4+CD25-) across liver endothelium, rather than immunosuppressive regulatory T cell subsets. Our data suggests that endothelial SCARF1 expression in tumour biopsies may provide critical prognostic information and may potentially help in selecting HCC patients for immunotherapy trials. Regulating SCARF1 levels could itself be a novel immunotherapeutic approach that re-programmes the microenvironment of HCC tumours by promoting effector CD4+ T cell infiltration.

Scavenger receptor class F member 1 (SCARF1) is thought to play an important role in the selective recruitment of CD4+ T cells to liver sinusoidal endothelial cells during chronic liver disease. However, the contribution of SCARF1 to hepatocellular carcinoma (HCC) is currently unknown. We utilized publically-available RNA-sequencing data from The Cancer Genome Atlas (TGCA) to explore SCARF1 expression in HCC and correlated it with a number of clinicopathological features. Flow adhesion assays were used to determine the role of SCARF1 in CD4+ T cell subset recruitment. SCARF1 expression was downregulated in HCC tumor tissues, compared to non-tumoral tissues, and loss of SCARF1 expression was associated with poorly differentiated/aggressive tumors. Additionally, higher SCARF1 expression in HCC tumor tissues was highly prognostic of better overall, disease-free and progression-free survival. SCARF1 within HCC was largely associated with tumor endothelial cells and adhesion studies suggested that it played a role in the specific recruitment of proinflammatory CD4+ T cells (CD4+CD25-) to HCC tumor tissues. Endothelial SCARF1 expression in tumor biopsies may provide critical prognostic information. Additionally, SCARF1 may also be a novel endothelial target that could help re-programme the microenvironment of HCC by promoting effector T cell tumor infiltration.

Promising outcomes with recent immune checkpoint inhibitor trials in hepatocellular carcinoma (HCC) have encouraged the search for novel immunotherapies. The tumour microenvironment in hepatocellular carcinoma (HCC) is highly complex but aggressive tumours are characterised by the accumulation of immunosuppressive cell populations. The endothelium is described as the gatekeeper for immunity, however, the regulation of immune cell/endothelial interaction within HCC is poorly characterised. We aimed to increase our understanding of the biological processes taking place at the level of the tumour endothelium in HCC through RNA sequencing of the endothelium in isolation, comparing this to non-tumour endothelium. In addition, we further studied the tumour microenvironment by spatial transcriptomic analysis of whole HCC tissue sections. Methods We undertook a validated technique for endothelial isolation using magnetic beads conjugated to Ulex agglutinin I, a lectin isolated from Ulex europaeus which binds specifically to the L-fucose residues present within glycoproteins on the surface of human endothelial cells. These beads were incubated with a single cell suspension of HCC tissue or distal non-tumour tissue. RNA was extracted and mRNA sequencing performed. We next analysed paraffin sections of resected HCCs with Nanostring® Digital Spatial Profiling (DSP) to provide further information on the localisation of immune signatures within the tumour microenvironment. Results 5 paired tumour and distal non-tumour samples taken from patients who underwent surgical resection were analysed. 45 genes were identified as being significantly differentially expressed between the tumour and non-tumour endothelium (adjusted p value Conclusion Our results demonstrate that the phenotype of tumour endothelium contributes to pathways which promote immune privilege in HCC. Spatial transcriptomics can provide further insight of how endothelial profiling correlates with immune cell infiltration in HCC. We have identified several new genes which need further validation but could be novel therapeutic targets that reprogramme the tumour endothelium and boost the efficacy of current immunotherapies.

The arginyl-glycinyl-aspartic acid (RGD) integrin alpha-v beta-6 (αvβ6) has been identified as playing a key role in the activation of transforming growth factor-β (TGFβ) that is hypothesised to be pivotal in the development of fibrosis. In this study αvβ6 …