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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.

Monocytes participate critically in atherosclerosis. There are 2 major subsets expressing different chemokine receptor patterns: CCR2 + CX3CR1 + Ly-6C hi and CCR2 -CX3CR1 ++ Ly-6C lo monocytes. Both C-C motif chemokine receptor 2 (CCR2) and C-X 3 -C motif chemokine receptor 1 (CX3CR1) are linked to progression of atherosclerotic plaques. Here, we analyzed mouse monocyte subsets in apoE-deficient mice and traced their differentiation and chemokine receptor usage as they accumulated within atherosclerotic plaques. Blood monocyte counts were elevated in apoE -/-mice and skewed toward an increased frequency of CCR2 + Ly-6C hi monocytes in apoE -/-mice fed a high-fat diet. CCR2 + Ly-6C hi monocytes efficiently accumulated in plaques, whereas CCR2 -Ly-6C lo monocytes entered less frequently but were more prone to developing into plaque cells expressing the dendritic cell-associated marker CD11c, indicating that phagocyte heterogeneity in plaques is linked to distinct types of entering monocytes. CCR2 -monocytes did not rely on CX3CR1 to enter plaques. Instead, they were partially dependent upon CCR5, which they selectively upregulated in apoE -/-mice. By comparison, CCR2 + Ly-6C hi monocytes unexpectedly required CX3CR1 in addition to CCR2 and CCR5 to accumulate within plaques. In many other inflammatory settings, these monocytes utilize CCR2, but not CX3CR1, for trafficking. Thus, antagonizing CX3CR1 may be effective therapeutically in ameliorating CCR2 + monocyte recruitment to plaques without impairing their CCR2-dependent responses to inflammation overall.

Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this 'secretory phenotype' can have pro- as well as anti-tumorigenic effects. Here we show that oncogene-induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as 'senescence surveillance'), which depends on an intact CD4(+) T-cell-mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of Nras(G12V). We also found that CD4(+) T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.

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