Iterative liver injury results in progressive fibrosis disrupting hepatic architecture, regeneration potential, and liver function. Hepatic stellate cells (HSCs) are a major source of pathological ...matrix during fibrosis and are thought to be a functionally homogeneous population. Here, we use single-cell RNA sequencing to deconvolve the hepatic mesenchyme in healthy and fibrotic mouse liver, revealing spatial zonation of HSCs across the hepatic lobule. Furthermore, we show that HSCs partition into topographically diametric lobule regions, designated portal vein-associated HSCs (PaHSCs) and central vein-associated HSCs (CaHSCs). Importantly we uncover functional zonation, identifying CaHSCs as the dominant pathogenic collagen-producing cells in a mouse model of centrilobular fibrosis. Finally, we identify LPAR1 as a therapeutic target on collagen-producing CaHSCs, demonstrating that blockade of LPAR1 inhibits liver fibrosis in a rodent NASH model. Taken together, our work illustrates the power of single-cell transcriptomics to resolve the key collagen-producing cells driving liver fibrosis with high precision.
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•scRNA-seq reveals spatial zonation of hepatic stellate cells (HSCs)•HSCs partition into topographically diametric lobule regions•Functional zonation of HSCs during centrilobular injury-induced fibrosis is uncovered•LPAR1 is a therapeutic target on pathological central vein-associated HSC
Dobie et al. use scRNA-seq to reveal spatial and functional zonation of hepatic stellate cells (HSCs) across the hepatic lobule, identifying central vein-associated HSCs as the dominant pathogenic collagen-producing cells during centrilobular injury-induced fibrosis. This illustrates the power of scRNA-seq to resolve the key collagen-producing cells driving liver fibrosis.
The epidermal growth factor receptor ligand Amphiregulin has a well-documented role in the restoration of tissue homeostasis after injury; however, the mechanism by which Amphiregulin contributes to ...wound repair remains unknown. Here we show that Amphiregulin functioned by releasing bioactive transforming growth factor beta (TGF-β) from latent complexes via integrin-αV activation. Using acute injury models in two different tissues, we found that by inducing TGF-β activation on mesenchymal stromal cells (pericytes), Amphiregulin induced their differentiation into myofibroblasts, thereby selectively contributing to the restoration of vascular barrier function within injured tissue. Furthermore, we identified macrophages as a critical source of Amphiregulin, revealing a direct effector mechanism by which these cells contribute to tissue restoration after acute injury. Combined, these observations expose a so far under-appreciated mechanism of how cells of the immune system selectively control the differentiation of tissue progenitor cells during tissue repair and inflammation.
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•Macrophages express Amphiregulin upon tissue damage•Amphiregulin activates integrin-αV complexes on pericytes•Integrin-αV-activated TGF-β induces pericyte into myofibroblast differentiation•Myofibroblast-derived collagen contributes to wound healing
How the immune system uses evolutionarily conserved signaling pathways involved in tissue development to orchestrate wound repair following injury has remained largely unresolved. Here, Minutti et al. show that the macrophage-derived EGFR ligand Amphiregulin induces local TGF-β activation and thereby the differentiation of tissue-resident pericytes.
Fibrosis due to extracellular matrix (ECM) secretion from myofibroblasts complicates many chronic liver diseases causing scarring and organ failure. Integrin-dependent interaction with scar ECM ...promotes pro-fibrotic features. However, the pathological intracellular mechanism in liver myofibroblasts is not completely understood, and further insight could enable therapeutic efforts to reverse fibrosis. Here, we show that integrin beta-1, capable of binding integrin alpha-11, regulates the pro-fibrotic phenotype of myofibroblasts. Integrin beta-1 expression is upregulated in pro-fibrotic myofibroblasts in vivo and is required in vitro for production of fibrotic ECM components, myofibroblast proliferation, migration and contraction. Serine/threonine-protein kinase proteins, also known as P21-activated kinase (PAK), and the mechanosensitive factor, Yes-associated protein 1 (YAP-1) are core mediators of pro-fibrotic integrin beta-1 signalling, with YAP-1 capable of perpetuating integrin beta-1 expression. Pharmacological inhibition of either pathway in vivo attenuates liver fibrosis. PAK protein inhibition, in particular, markedly inactivates the pro-fibrotic myofibroblast phenotype, limits scarring from different hepatic insults and represents a new tractable therapeutic target for treating liver fibrosis.
The omentum is a visceral adipose tissue rich in fat-associated lymphoid clusters (FALCs) that collects peritoneal contaminants and provides a first layer of immunological defense within the abdomen. ...Here, we investigated the mechanisms that mediate the capture of peritoneal contaminants during peritonitis. Single-cell RNA sequencing and spatial analysis of omental stromal cells revealed that the surface of FALCs were covered by CXCL1+ mesothelial cells, which we termed FALC cover cells. Blockade of CXCL1 inhibited the recruitment and aggregation of neutrophils at FALCs during zymosan-induced peritonitis. Inhibition of protein arginine deiminase 4, an enzyme important for the release of neutrophil extracellular traps, abolished neutrophil aggregation and the capture of peritoneal contaminants by omental FALCs. Analysis of omental samples from patients with acute appendicitis confirmed neutrophil recruitment and bacterial capture at FALCs. Thus, specialized omental mesothelial cells coordinate the recruitment and aggregation of neutrophils to capture peritoneal contaminants.
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•Specialized Cxcl13+ mesothelial cells cover the surface of FALCs and secrete CXCL1•CXCL1 mediates the recruitment of neutrophils to omental FALCs during peritonitis•PAD4-dependent neutrophil aggregates mediate the capture of zymosan by the omentum•Neutrophils form NETs on the omentum of patients with appendicitis
For decades, the omentum has been known as the policeman of the abdomen. Jackson-Jones et al. reveal the existence of specialized mesothelial cells within fat-associated lymphoid clusters of the omentum that are responsible for the capture of peritoneal contaminants via the formation of neutrophil aggregates, preventing systemic spread.
The use of single cell sequencing technologies has exploded over recent years, and is now commonly used in many non-model species. Sequencing nuclei instead of whole cells has become increasingly ...popular, as it does not require the processing of samples immediately after collection. Here we present a highly effective nucleus isolation protocol that outperforms previously available method in challenging samples in a non-model specie. This protocol can be successfully applied to extract nuclei from a variety of tissues and species.
Abstract
Aims
Pluripotent stem cell-derived endothelial cell products possess therapeutic potential in ischaemic vascular disease. However, the factors that drive endothelial differentiation from ...pluripotency and cellular specification are largely unknown. The aims of this study were to use single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape and cellular dynamics of directed differentiation of human embryonic stem cell-derived endothelial cells (hESC-EC) and to compare these cells to mature endothelial cells from diverse vascular beds.
Methods and results
A highly efficient directed 8-day differentiation protocol was used to generate a hESC-derived endothelial cell product (hESC-ECP), in which 66% of cells co-expressed CD31 and CD144. We observed largely homogeneous hESC and mesodermal populations at Days 0 and 4, respectively, followed by a rapid emergence of distinct endothelial and mesenchymal populations. Pseudotime trajectory identified transcriptional signatures of endothelial commitment and maturation during the differentiation process. Concordance in transcriptional signatures was verified by scRNA-seq analysis using both a second hESC line RC11, and an alternative hESC-EC differentiation protocol. In total, 105 727 cells were subjected to scRNA-seq analysis. Global transcriptional comparison revealed a transcriptional architecture of hESC-EC that differs from freshly isolated and cultured human endothelial cells and from organ-specific endothelial cells.
Conclusion
A transcriptional bifurcation into endothelial and mesenchymal lineages was identified, as well as novel transcriptional signatures underpinning commitment and maturation. The transcriptional architecture of hESC-ECP was distinct from mature and foetal human EC.
Rationale:
Galectin-3 (Gal-3) is an immune regulator and an important driver of fibrosis in chronic lung injury, however, its role in acute lung injury (ALI) remains unknown. Previous work has shown ...that global deletion of galectin-3 reduces collagen deposition in a bleomycin-induced pulmonary fibrosis model (MacKinnon et al., Am. J. Respir. Crit. Care Med., 2012, 185, 537–46). An inhaled Gal-3 inhibitor, GB0139, is undergoing Phase II clinical development for idiopathic pulmonary fibrosis (IPF). This work aims to elucidate the role of Gal-3 in the myeloid and mesenchymal compartment on the development of acute and chronic lung injury.
Methods:
LgalS3
fl/fl
mice were generated and crossed with mice expressing the myeloid (
LysM
) and mesenchymal (
Pdgfrb
) cre drivers to yield
LysM-cre
+/-
/LgalS3
fl/fl
and
Pdgfrb-cre
+/-
/LgalS3
fl/fl
mice. The response to acute (bleomycin or LPS) or chronic (bleomycin) lung injury was compared to globally deficient
Gal-3
−/−
mice.
Results:
Myeloid depletion of Gal-3 led to a significant reduction in Gal-3 expression in alveolar macrophages and neutrophils and a reduction in neutrophil recruitment into the interstitium but not into the alveolar space. The reduction in interstitial neutrophils corelated with decreased levels of pulmonary inflammation following acute bleomycin and LPS administration. In addition, myeloid deletion decreased Gal-3 levels in bronchoalveolar lavage (BAL) and reduced lung fibrosis induced by chronic bleomycin. In contrast, no differences in BAL Gal-3 levels or fibrosis were observed in
Pdgfrb-cre
+/-
/LgalS3
fl/fl
mice.
Conclusions:
Myeloid cell derived Galectin-3 drives acute and chronic lung inflammation and supports direct targeting of galectin-3 as an attractive new therapy for lung inflammation.
BackgroundEMT is the process whereby epithelial cells acquire mesenchymal characteristics. This process is highly regulated and required in development and tissue regeneration. However, in cancer the ...process becomes dysregulated leading to increased invasion, metastasis and tumor progression. Macrophages are one of the major infiltrating immune cells in the tumor microenvironment and have been proposed as a primary driver of pathological EMT through interactions with tumour cells. EMT and macrophage infiltration have been correlated to enhance tumor progression in multiple cancer types, making it a potential therapeutic target. In this study we generate and characterise a series of human macrophage subtypes via cytokine polarisation and show that multiple macrophage polarised states enhance the EMT phenotype.MethodsHuman macrophages were differentiated using GM-CSF or M-CSF prior to polarisation with individual cytokines (M-CSF + IFN-g; M-CSF + IL-4; M-CSF + IL-1b; M-CSF + IL-10; M-CSF + IL-6) to generate different putative macrophage phenotypes (M1, M2a, M2b, M2c, M2d) as evidenced by differences in cytokine secretion, gene transcription and protein expression. Epithelial cells were either stimulated with macrophage conditioned medium or in co-culture with macrophages in a scratch wound assay (Incucyte) and analysed by immunocytochemistry (Vimentin, SNAIL, E-cadherin). Supernatants of the stimulated macrophages were assessed by Luminex bead array and the macrophages subtypes were also assessed using bulk RNA sequencing for differences in transcriptome.ResultsSupernatants derived from macrophages and macrophage co-culture were shown to drive EMT, both in migratory capacity and EMT associated protein expression. Our results demonstrate that even a single cytokine can alter the polarisation and subsequent cytokine production of the macrophage; with IL-12 and TNF-α associated with more pro-inflammatory phenotypes (M1), and IL-10 and VEGFA more anti-inflammatory or tissue remodelling phenotypes (M2). Inhibition of the TGF-βRI by galunisertib can partially reverse the induction of EMT migration and protein expression.ConclusionsThe data shows that macrophages are plastic and the cytokines they produce can directly drive the EMT process in vitro via multiple mechanisms. This supports the conclusion that individual cytokine inhibition such as TGF-βRI alone is likely to be ineffective as macrophages produce multiple factors that can induce EMT. Using this powerful model, researchers can gain better understanding of complex interactions occurring in the tumor microenvironment, identify molecular targets with potential to block cancer cell metastasis and evaluate candidate drug efficacy.
The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In ...developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here, we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single-cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors’ competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6’s main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side effects of morphogens engaged contemporaneously in other essential functions.
The human endometrium experiences repetitive cycles of tissue wounding characterised by piecemeal shedding of the surface epithelium and rapid restoration of tissue homeostasis. In this study, we ...used a mouse model of endometrial repair and three transgenic lines of mice to investigate whether epithelial cells that become incorporated into the newly formed luminal epithelium have their origins in one or more of the mesenchymal cell types present in the stromal compartment of the endometrium. Using scRNAseq, we identified a novel population of PDGFRb + mesenchymal stromal cells that developed a unique transcriptomic signature in response to endometrial breakdown/repair. These cells expressed genes usually considered specific to epithelial cells and in silico trajectory analysis suggested they were stromal fibroblasts in transition to becoming epithelial cells. To confirm our hypothesis we used a lineage tracing strategy to compare the fate of stromal fibroblasts (PDGFRa+) and stromal perivascular cells (NG2/CSPG4+). We demonstrated that stromal fibroblasts can undergo a mesenchyme to epithelial transformation and become incorporated into the re-epithelialised luminal surface of the repaired tissue. This study is the first to discover a novel population of wound-responsive, plastic endometrial stromal fibroblasts that contribute to the rapid restoration of an intact luminal epithelium during endometrial repair. These findings form a platform for comparisons both to endometrial pathologies which involve a fibrotic response (Asherman's syndrome, endometriosis) as well as other mucosal tissues which have a variable response to wounding.
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