RESUMO
The liver has a unique ability to regenerate1,2; however, in the setting of acute liver failure (ALF), this regenerative capacity is often overwhelmed, leaving emergency liver transplantation as the only curative option3-5. Here, to advance understanding of human liver regeneration, we use paired single-nucleus RNA sequencing combined with spatial profiling of healthy and ALF explant human livers to generate a single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+ migratory hepatocyte subpopulation, which emerges during human liver regeneration, and a corollary subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. Four-dimensional intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 reduces hepatocyte growth factor-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation that mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut-liver barrier may advance new areas of therapeutic discovery in regenerative medicine.
Assuntos
Falência Hepática Aguda , Regeneração Hepática , Animais , Feminino , Humanos , Masculino , Camundongos , Acetaminofen/farmacologia , Linhagem da Célula , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/patologia , Falência Hepática Aguda/patologia , Falência Hepática Aguda/induzido quimicamente , Regeneração Hepática/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Necrose/induzido quimicamente , Medicina Regenerativa , Análise da Expressão Gênica de Célula Única , CicatrizaçãoRESUMO
Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing 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 a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis.
Assuntos
Células Endoteliais/patologia , Cirrose Hepática/patologia , Fígado/patologia , Macrófagos/patologia , Análise de Célula Única , Animais , Estudos de Casos e Controles , Linhagem da Célula , Sistema do Grupo Sanguíneo Duffy/metabolismo , Células Endoteliais/metabolismo , Feminino , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/citologia , Cirrose Hepática/genética , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Fenótipo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Tetraspanina 29/metabolismo , Transcriptoma , Migração Transendotelial e TransepitelialRESUMO
BACKGROUND: Regulatory T cells (Treg) are enriched in human colorectal cancer (CRC) where they suppress anti-tumour immunity. The chemokine receptor CCR5 has been implicated in the recruitment of Treg from blood into CRC and tumour growth is delayed in CCR5-/- mice, associated with reduced tumour Treg infiltration. METHODS: Tissue and blood samples were obtained from patients undergoing resection of CRC. Tumour-infiltrating lymphocytes were phenotyped for chemokine receptors using flow cytometry. The presence of tissue chemokines was assessed. Standard chemotaxis and suppression assays were performed and the effects of CCR5 blockade were tested in murine tumour models. RESULTS: Functional CCR5 was highly expressed by human CRC infiltrating Treg and CCR5(high) Treg were more suppressive than their CCR5(low) Treg counterparts. Human CRC-Treg were more proliferative and activated than other T cells suggesting that local proliferation could provide an alternative explanation for the observed tumour Treg enrichment. Pharmacological inhibition of CCR5 failed to reduce tumour Treg infiltration in murine tumour models although it did result in delayed tumour growth. CONCLUSIONS: CCR5 inhibition does not mediate anti-tumour effects as a consequence of inhibiting Treg recruitment. Other mechanisms must be found to explain this effect. This has important implications for anti-CCR5 therapy in CRC.
Assuntos
Antineoplásicos/farmacologia , Antagonistas dos Receptores CCR5/farmacologia , Neoplasias Colorretais/imunologia , Cicloexanos/farmacologia , Linfócitos T Reguladores/imunologia , Triazóis/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Quimiocina CCL4/metabolismo , Quimiotaxia de Leucócito , Neoplasias Colorretais/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Maraviroc , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Receptores CCR5/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
BACKGROUND: Alcohol and obesity synergise to increase the risk of liver-related mortality. We examined the influence of adiposity on clinical outcomes in alcoholic hepatitis (AH) and the underlying inflammatory crosstalk between adipose tissue (AT) and the liver. METHODS: A cohort of 233 patients with AH from the UK and USA provided data to analyse the effects of obesity in AH. Body mass index was corrected for the severity of ascites, termed cBMI. Inflammatory and metabolic profiling was undertaken by proteome analysis of human serum samples. The effect of alcohol on adipose tissue and CXCL11 expression was studied in 3â¯T3-derived adipocytes and in mice using the high-fat diet-plus-binge ethanol model. FINDINGS: Obesity was common amongst patients with AH, seen in 19% of individuals. Obesity (HR 2.22, 95%CI 1.1-4.3, pâ¯=â¯.022) and underweight (HR 2.38, 1.00-5.6, pâ¯=â¯.049) were independently associated with mortality at 3â¯months. Proteome analysis demonstrated multiple metabolic and inflammatory factors differentially expressed in obese AH verse lean AH, with CXCL11 being the most elevated factor in obese AH. In vitro analysis of cultured adipocytes and in vivo analysis of mouse models showed that alcohol induced CXCL11 expression in AT, but not in liver. INTERPRETATION: Obesity is common in AH and associated with a greater than two-fold increase in short-term mortality. Obese AH is associated with a different inflammatory phenotype, with the greatest elevation in CXCL11. These data confirm that adiposity is clinically important in acute alcohol-related liver disease and illustrate the adipose-liver inflammatory axis in AH. FUND: This work was supported in part by an EASL Sheila Sherlock Physician Scientist Fellowship. The funder played no role in gathering or analysing data or writing the manuscript. This paper presents independent research supported by the NIHR Birmingham Biomedical Research Centre at the University Hospitals Birmingham NHS Foundation Trust and the University of Birmingham. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.
Assuntos
Adiposidade/genética , Quimiocina CXCL11/genética , Hepatite Alcoólica/genética , Obesidade/genética , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Adiposidade/efeitos dos fármacos , Animais , Estudos de Coortes , Dieta Hiperlipídica/efeitos adversos , Feminino , Regulação da Expressão Gênica/genética , Hepatite Alcoólica/complicações , Hepatite Alcoólica/fisiopatologia , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Morbidade , Obesidade/complicações , Obesidade/patologiaRESUMO
We have expressed and purified a protein fragment from Entamoeba histolytica. It catalyses transhydrogenation between analogues of NAD(H) and NADP(H). The characteristics of this reaction resemble those of the reaction catalysed by a complex of the NAD(H)- and NADP(H)-binding subunits of proton-translocating transhydrogenases from bacteria and mammals. It is concluded that the complete En. histolytica protein, which, along with similar proteins from other protozoan parasites, has an unusual subunit organisation, is also a proton-translocating transhydrogenase. The function of the transhydrogenase, thought to be located in organelles which do not have the enzymes of oxidative phosphorylation, is not clear.
Assuntos
Entamoeba histolytica/enzimologia , NADP Trans-Hidrogenases/metabolismo , Animais , Catálise , Concentração de Íons de Hidrogênio , NAD/metabolismo , NADP/metabolismo , NADP Trans-Hidrogenases/química , Subunidades Proteicas , Bombas de Próton/química , Bombas de Próton/metabolismo , PrótonsRESUMO
AIMS: Lysyl oxidase (LOX) expression is elevated in colorectal cancer (CRC) tissue and associated with disease progression. A blood test may form a more acceptable diagnostic test for CRC although LOX has not previously been measured in the serum. We therefore sought to determine the clinical usefulness of a serum LOX test for CRC in a symptomatic population. METHODS: Adult patients referred to a hospital colorectal clinic with bowel symptoms completed a questionnaire and provided a blood sample for serum LOX measurement. Associations between presenting symptoms, serum LOX concentrations and outcomes of investigations were tested by univariate and multivariate analyses to determine if serum LOX was clinically useful in the prediction of CRC. LOX expression in CRC and adjacent colon biopsies was evaluated by ELISA and immunohistochemistry. RESULTS: Thirty-one cases of colorectal cancer and 16 high-risk polyps were identified from a total of 962 participants. There was no association between serum LOX concentration and the presence of CRC, high-risk polyps or cancers at any site. LOX expression was significantly increased in CRC tissue compared to adjacent colon. CONCLUSION: Despite overexpression of LOX in CRC tissue, elevated serum levels could not be demonstrated. Serum LOX measurement is therefore not a clinically useful test for CRC.
Assuntos
Neoplasias Colorretais/sangue , Proteína-Lisina 6-Oxidase/sangue , Adulto , Idoso , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Inquéritos e QuestionáriosRESUMO
A fire in the operating theatres could be disastrous. Colin Weston describes an exercise in a simulated fire at the John Radcliffe Hospital, Oxford, which produced some important lessons.
Assuntos
Planejamento em Desastres , Incêndios , Salas Cirúrgicas , Inglaterra , SegurançaRESUMO
Transhydrogenase undergoes conformational changes to couple the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The protein comprises three components: dI, which binds NAD(H); dIII, which binds NADP(H); and dII, which spans the membrane. Experiments using isothermal titration calorimetry, analytical ultracentrifugation, and small angle x-ray scattering show that, as in the crystalline state, a mixture of recombinant dI and dIII from Rhodospirillum rubrum transhydrogenase readily forms a dI(2)dIII(1) heterotrimer in solution, but we could find no evidence for the formation of a dI(2)dIII(2) tetramer using these techniques. The asymmetry of the complex suggests that there is an alternation of conformations at the nucleotide-binding sites during proton translocation by the complete enzyme. The characteristics of nucleotide interaction with the isolated dI and dIII components and with the dI(2)dIII(1) heterotrimer were investigated. (a) The rate of release of NADP(+) from dIII was decreased 5-fold when the component was incorporated into the heterotrimer. (b) The binding affinity of one of the two nucleotide-binding sites for NADH on the dI dimer was decreased about 17-fold in the dI(2)dIII(1) complex; the other binding site was unaffected. These observations lend strong support to the alternating-site mechanism.