SCARF-1 promotes adhesion of CD4+ T cells to human hepatic sinusoidal endothelium under conditions of shear stress

Abstract Liver-resident cells are constantly exposed to gut-derived antigens via portal blood and, as a consequence, they express a unique repertoire of scavenger receptors. Whilst there is increasing evidence that the gut contributes to chronic inflammatory liver disease, the role of scavenger receptors in regulating liver inflammation remains limited. Here, we describe for the first time the expression of scavenger receptor class F, member 1 (SCARF-1) on hepatic sinusoidal endothelial cells (HSEC). We report that SCARF-1 shows a highly localised expression pattern and co-localised with endothelial markers on sinusoidal endothelium. Analysis of chronically inflamed liver tissue demonstrated accumulation of SCARF-1 at sites of CD4+ T cell aggregation. We then studied the regulation and functional role of SCARF-1 in HSEC and showed that SCARF-1 expression by HSEC is regulated by proinflammatory cytokines and bacterial lipopolysaccharide (LPS). Furthermore, SCARF-1 expression by HSEC, induced by proinflammatory and gut-derived factors acts as a novel adhesion molecule, present in adhesive cup structures, that specifically supports CD4+ T cells under conditions of physiological shear stress. In conclusion, we show that SCARF-1 contributes to lymphocyte subset adhesion to primary human HSEC and could play an important role in regulating the inflammatory response during chronic liver disease

Multivariable association discovery in population-scale meta-omics studies.

It is challenging to associate features such as human health outcomes, diet, environmental conditions, or other metadata to microbial community measurements, due in part to their quantitative properties. Microbiome multi-omics are typically noisy, sparse (zero-inflated), high-dimensional, extremely non-normal, and often in the form of count or compositional measurements. Here we introduce an optimized combination of novel and established methodology to assess multivariable association of microbial community features with complex metadata in population-scale observational studies. Our approach, MaAsLin 2 (Microbiome Multivariable Associations with Linear Models), uses generalized linear and mixed models to accommodate a wide variety of modern epidemiological studies, including cross-sectional and longitudinal designs, as well as a variety of data types (e.g., counts and relative abundances) with or without covariates and repeated measurements. To construct this method, we conducted a large-scale evaluation of a broad range of scenarios under which straightforward identification of meta-omics associations can be challenging. These simulation studies reveal that MaAsLin 2\u27s linear model preserves statistical power in the presence of repeated measures and multiple covariates, while accounting for the nuances of meta-omics features and controlling false discovery. We also applied MaAsLin 2 to a microbial multi-omics dataset from the Integrative Human Microbiome (HMP2) project which, in addition to reproducing established results, revealed a unique, integrated landscape of inflammatory bowel diseases (IBD) across multiple time points and omics profiles

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

Vascular adhesion protein-1 is elevated in primary sclerosing cholangitis, is predictive of clinical outcome, and facilitates recruitment of gut-tropic lymphocytes to liver in a substrate-dependent manner

ObjectivePrimary sclerosing cholangitis (PSC) is the classical hepatobiliary manifestation of IBD. This clinical association is linked pathologically to the recruitment of mucosal T cells to the liver, via vascular adhesion protein (VAP)-1-dependent enzyme activity. Our aim was to examine the expression, function and enzymatic activation of the ectoenzyme VAP-1 in patients with PSC.DesignWe examined VAP-1 expression in patients with PSC, correlated levels with clinical characteristics and determined the functional consequences of enzyme activation by specific enzyme substrates on hepatic endothelium.ResultsThe intrahepatic enzyme activity of VAP-1 was elevated in PSC versus immune-mediated disease controls and non-diseased liver (p&lt;0.001). The adhesion of gut-tropic α4β7+lymphocytes to hepatic endothelial cells in vitro under flow was attenuated by 50% following administration of the VAP-1 inhibitor semicarbazide (p&lt;0.01). Of a number of natural VAP-1 substrates tested, cysteamine—which can be secreted by inflamed colonic epithelium and gut bacteria—was the most efficient (yielded the highest enzymatic rate) and efficacious in its ability to induce expression of functional mucosal addressin cell adhesion molecule-1 on hepatic endothelium. In a prospectively evaluated patient cohort with PSC, elevated serum soluble (s)VAP-1 levels predicted poorer transplant-free survival for patients, independently (HR: 3.85, p=0.003) and additively (HR: 2.02, p=0.012) of the presence of liver cirrhosis.ConclusionsVAP-1 expression is increased in PSC, facilitates adhesion of gut-tropic lymphocytes to liver endothelium in a substrate-dependent manner, and elevated levels of its circulating form predict clinical outcome in patients.</jats:sec

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

The role of amphoterin-induced gene and open reading frame (AMIGO) family in myelination and neurodegeneration and Investigating the roles of mesenchymal and hematopoietic stem cell conditioned media on neutrophil recruitment in a model of inflammation

Project 1: Myelination plays a key role in both the developing CNS and repair from neurodegenerative diseases such as multiple sclerosis (MS). Previous work has suggested a role for the amphoterin-induced gene and open reading frame (AMIGO) family of proteins in this process. Data shown here proposes a novel role for the AMIGOs in both the developmental and repair processes of myelination. Immunofluorescent staining showed AMIGO expression in the developing mouse brain in a similar, progressive pattern to myelination. AMIGO expression was also found in oligodendrocyte precursor cells (OPCs), oligodendrocytes and astrocytes in the spinal cord during experimental autoimmune encephalomyelitis (EAE). These cells are either myelinating or have myelination-regulating function. These combined results suggest a role for the AMIGOs as potential positive regulators of myelination. Project 2: Evidence suggests that both mesenchymal and hematopoietic stem cells (MSCs and HSCs) can alter an inflammatory environment in vivo by secretion of inflammation-modulating agents. This was examined by first developing an in vitro model of inflammation. Addition of MSC and HSC conditioned media increased Kc-activated neutrophil adhesion to TNF-α-activated colon and renal endothelium, respectively. This increase in adhesion was enhanced by pre-stimulation of SCs with cytokines and chemokines, particularly H2O2 and TNF-α. Similarly, treatment with different SC-conditioned media altered the adhesion of activated neutrophils to VCAM-1, ICAM-1, MAdCAM1 and hyaluronan. This effect was further manipulated by treatment of SCs with cytokines and chemokines, particularly H2O2, TNF-α and IFN-γ. This data suggest that MSCs and HSCs potentially have multiple effects on the inflammatory sit

The role of vascular adhesion protein (VAP)-1 during inflammatory liver disease

Liver disease is the fifth largest killer in the United Kingdom and with the numbers of diagnoses increasing each year there is an urgent need for novel therapeutic interventions. This thesis examines one potential target, Vascular Adhesion Protein (VAP)-1: an amine oxidase enzyme with reported adhesin functionality. The results herein confirm a primarily sinusoidal localisation of VAP-1, expression of which was upregulated in the liver during chronic inflammation correlated with diminished enzyme activity. Functional analysis of sinusoidal VAP-1 in vitro did not demonstrate any effect of inhibition on leukocyte recruitment, unlike that observed in other tissues. Furthermore, only neutrophils were capable of binding to recombinant VAP-1 under flow conditions. Further investigation highlighted the importance of this intimate relationship during neutrophil-endothelial interactions; revealing the first evidence that neutrophils also express catalytically active VAP-1. Neutrophil effector functions, such as the formation of extracellular traps, were also hindered by recombinant VAP-1. This was also observed in wild-type mice but not those expressing a catalytically inactive form of VAP-1 (SSAOKO). Following acute injury, these mice also exhibited expanded intrahepatic macrophage and NKT cell populations compared to control. In combination, these data highlight the complex role that VAP-1 plays during inflammatory liver disease

Facilitating high throughput bispecific antibody production and potential applications within biopharmaceutical discovery workflows

ABSTRACTA major driver for the recent investment surge in bispecific antibody (bsAb) platforms and products is the multitude of distinct mechanisms of action that bsAbs offer compared to a combination of two monoclonal antibodies. Four bsAb products were granted first regulatory approvals in the US or EU during 2023 and the biopharmaceutical industry pipeline is brimming with bsAb candidates across a broad range of therapeutic applications. In previously reported bsAb discovery campaigns, following a hypothesis-based choice of two specific target proteins, selections and screening activities have often been performed in mono-specific formats. The conversion to bispecific modalities has usually been positioned toward the end of the discovery process and has involved small numbers of lead molecules, largely due to challenges in expressing, purifying, and analyzing large numbers of bsAbs. In this review, we discuss emerging strategies to facilitate the production of expanded bsAb panels, focusing particularly upon combinatorial methods to generate bsAb matrices. Such technologies will enable screening in. bispecific formats at earlier stages of discovery campaigns, not only widening the accessible protein space to maximize chances of success, but also advancing empirical bi-target validation activities to assess initial target selection hypotheses