Currently there are no effective antifibrotic therapies for liver cirrhosis, a major killer worldwide. To obtain a cellular resolution of directly-relevant pathogenesis and to inform therapeutic design, we profile the transcriptomes of over 100,000 human single cells, yielding molecular definitions for non-parenchymal cell types present in healthy and cirrhotic human liver. We uncover a novel scar-associated TREM2 + CD9 + macrophage subpopulation, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define novel ACKR1 + and PLVAP + endothelial cells which expand in cirrhosis, are topographically scar-restricted and enhance leucocyte transmigration. Multi-lineage ligand-receptor modelling of interactions between the novel scar-associated macrophages, endothelial cells and PDGFRα + collagenproducing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides the conceptual framework required to discover rational therapeutic targets in liver cirrhosis. Recent estimates suggest that 844 million people worldwide have chronic liver disease, with two million deaths per year and a rising incidence 1. Iterative liver injury secondary to any cause leads to progressive fibrosis ultimately resulting in liver cirrhosis. Importantly, the degree of liver fibrosis predicts adverse patient outcomes 2. Hence, effective antifibrotic therapies for patients with chronic liver disease are urgently required 3,4. Liver fibrosis involves a complex interplay between multiple non-parenchymal cell (NPC) lineages including immune, endothelial and mesenchymal cells spatially located within areas of scarring, termed the fibrotic niche. Despite progress in our understanding of liver fibrogenesis accrued using rodent models, there remains a significant 'translational gap' Ramachandran et al.

Fat-associated lymphoid clusters (FALCs) are a recently discovered type of lymphoid tissue associated with visceral fat. Here we show that distribution of FALCs was heterogeneous with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 B cells in the peritoneal cavity through high expression of the chemokine CXCL13 and supported B cell proliferation and germinal center differentiation during peritoneal immune challenges. FALC formation was induced by inflammation, which triggered recruitment of myeloid cells that express tumor necrosis factor (TNF) necessary for TNF receptor-signaling in stromal cells. CD1d-restricted Natural killer T (NKT) cells were likewise required for inducible formation of FALCs. Thus, FALCs support and coordinate innate B and T cell activation during serosal immune responses.

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Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to the shortage of donor organs. Developing renewable sources of human liver tissue is therefore attractive. Pluripotent stem cell-derived liver tissue represents a potential alternative to cadaver derived hepatocytes and whole organ transplant. At present, two-dimensional differentiation procedures deliver tissue lacking certain functions and long-term stability. Efforts to overcome these limiting factors have led to the building of three-dimensional (3D) cellular aggregates. Although enabling for the field, their widespread application is limited due to their reliance on variable biological components. Our studies focused on the development of 3D liver tissue under defined conditions. In vitro generated 3D tissues exhibited stable phenotype for over 1 year in culture, providing an attractive resource for long-term in vitro studies. Moreover, 3D derived tissue provided critical liver support in two animal models, including immunocompetent recipients. Therefore, we believe that our study provides stable human tissue to better model liver biology ‘in the dish’, and in the future may permit the support of compromised liver function in humans.Electronic supplementary materialThe online version of this article (10.1007/s00204-018-2280-2) contains supplementary material, which is available to authorized users.

To enable a better understanding of the regulation of neutrophil migration, we investigated the kinetics of adhesion and migration over, through and under endothelial monolayers. Neutrophils were perfused over human umbilical vein endothelial cells (HUVEC) which had been treated with tumour necrosis factor-α (TNF; 2–1,000 U/ml) for 4 h. Videomicroscopy showed that transendothelial migration was complete within about 5 min of completion of perfusion of a bolus of neutrophils. Separate populations of adherent cells could then be observed, either rolling, migrating over the surface of the HUVEC or migrating underneath, at different characteristic speeds. Increasing concentration of TNF had little effect on the kinetics of migration, but shifted the balance from rolling adhesion to transendothelial migration. When individual neutrophils were followed from the moment they bound to HUVEC treated with 100 U/ml TNF, we found that ∼40% immobilised essentially immediately on contact, while ∼40% immobilised after rolling for varying periods (average 26 s) and ∼20% rolled continuously. Most of the immobilised cells went on to migrate through the monolayer after spending 20–200 s migrating on top, and took about 60 s to pass through. Overall, the time from first binding to completion of transmigration averaged 152 s (range ∼60–240 s). Interestingly, neutrophils moved relatively slowly on top of the monolayer (about 8 μm/min) but more rapidly underneath (about 16 μm/min). We suggest that the different stages during neutrophil transmigration have characteristic kinetics with separate control mechanisms, which critically influence the efficiency and rate of clearance from the vasculature.

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Neutrophils migrate through endothelium using an ordered sequence of adhesive interactions and activating signals. To investigate the consequences of disruption of this sequence, we characterized adhesion and migration of neutrophils perfused over HUVEC that had been treated with TNF-α for 4 h and evaluated changes caused by exogenously added chemotactic agents. When HUVEC were treated with 2 U/ml TNF, flowing neutrophils adhered, with the majority rolling and relatively few migrating through the monolayer. If fMLP, IL-8, zymosan-activated plasma (a source of activated complement factor C5a), epithelial cell-derived neutrophil-activating peptide (ENA-78), or growth-regulating oncogene, GRO-α, was perfused over these neutrophils, they stopped rolling and rapidly migrated over the monolayer, but did not penetrate it. When HUVEC were treated with 100 U/ml TNF, the majority of adherent neutrophils transmigrated. If neutrophils were treated with fMLP, IL-8, C5a, ENA-78, or GRO-α just before perfusion over this HUVEC, transmigration, but not adhesion, was abolished. However, when platelet-activating factor was used to activate neutrophils, migration through HUVEC treated with 100 U/ml TNF was not impaired, and migration through HUVEC treated with 2 U/ml TNF was actually increased. Transmigration required ligation of CXC chemokine receptor-2 on neutrophils, and differential desensitization of this receptor (e.g., by fMLP but not platelet-activating factor) may explain the pattern of disruption of migration. Thus, transmigration may require presentation of the correct activators in the correct sequence, and inappropriate activation (e.g., by systemic activators) could cause pathological accumulation of neutrophils in the vessel lumen.