RESUMO
The role and molecular mechanisms of intermittent fasting (IF) in non-alcoholic steatohepatitis (NASH) and its transition to hepatocellular carcinoma (HCC) are unknown. Here, we identified that an IF 5:2 regimen prevents NASH development as well as ameliorates established NASH and fibrosis without affecting total calorie intake. Furthermore, the IF 5:2 regimen blunted NASH-HCC transition when applied therapeutically. The timing, length, and number of fasting cycles as well as the type of NASH diet were critical parameters determining the benefits of fasting. Combined proteome, transcriptome, and metabolome analyses identified that peroxisome-proliferator-activated receptor alpha (PPARα) and glucocorticoid-signaling-induced PCK1 act co-operatively as hepatic executors of the fasting response. In line with this, PPARα targets and PCK1 were reduced in human NASH. Notably, only fasting initiated during the active phase of mice robustly induced glucocorticoid signaling and free-fatty-acid-induced PPARα signaling. However, hepatocyte-specific glucocorticoid receptor deletion only partially abrogated the hepatic fasting response. In contrast, the combined knockdown of Ppara and Pck1 in vivo abolished the beneficial outcomes of fasting against inflammation and fibrosis. Moreover, overexpression of Pck1 alone or together with Ppara in vivo lowered hepatic triglycerides and steatosis. Our data support the notion that the IF 5:2 regimen is a promising intervention against NASH and subsequent liver cancer.
Assuntos
Carcinoma Hepatocelular , Jejum , Neoplasias Hepáticas , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica , PPAR alfa , Fosfoenolpiruvato Carboxiquinase (GTP) , PPAR alfa/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Humanos , Camundongos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Masculino , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Transdução de Sinais , Jejum IntermitenteRESUMO
Low-grade chronic inflammation is a hallmark of ageing, associated with impaired tissue function and disease development. However, how cell-intrinsic and -extrinsic factors collectively establish this phenotype, termed inflammaging, remains poorly understood. We addressed this question in the mouse intestinal epithelium, using mouse organoid cultures to dissect stem cell-intrinsic and -extrinsic sources of inflammaging. At the single-cell level, we found that inflammaging is established differently along the crypt-villus axis, with aged intestinal stem cells (ISCs) strongly upregulating major histocompatibility complex class II (MHC-II) genes. Importantly, the inflammaging phenotype was stably propagated by aged ISCs in organoid cultures and associated with increased chromatin accessibility at inflammation-associated loci in vivo and ex vivo, indicating cell-intrinsic inflammatory memory. Mechanistically, we show that the expression of inflammatory genes is dependent on STAT1 signaling. Together, our data identify that intestinal inflammaging in mice is promoted by a cell-intrinsic mechanism, stably propagated by ISCs, and associated with a disbalance in immune homeostasis.
Assuntos
Mucosa Intestinal , Intestinos , Camundongos , Animais , Células-Tronco , Fenótipo , InflamaçãoRESUMO
BACKGROUND & AIMS: As pancreatic ductal adenocarcinoma (PDAC) continues to be recalcitrant to therapeutic interventions, including poor response to immunotherapy, albeit effective in other solid malignancies, a more nuanced understanding of the immune microenvironment in PDAC is urgently needed. We aimed to unveil a detailed view of the immune micromilieu in PDAC using a spatially resolved multimodal single-cell approach. METHODS: We applied single-cell RNA sequencing, spatial transcriptomics, multiplex immunohistochemistry, and mass cytometry to profile the immune compartment in treatment-naïve PDAC tumors and matched adjacent normal pancreatic tissue, as well as in the systemic circulation. We determined prognostic associations of immune signatures and performed a meta-analysis of the immune microenvironment in PDAC and lung adenocarcinoma on single-cell level. RESULTS: We provided a spatially resolved fine map of the immune landscape in PDAC. We substantiated the exhausted phenotype of CD8 T cells and immunosuppressive features of myeloid cells, and highlighted immune subsets with potentially underappreciated roles in PDAC that diverged from immune populations within adjacent normal areas, particularly CD4 T cell subsets and natural killer T cells that are terminally exhausted and acquire a regulatory phenotype. Differential analysis of immune phenotypes in PDAC and lung adenocarcinoma revealed the presence of extraordinarily immunosuppressive subtypes in PDAC, along with a distinctive immune checkpoint composition. CONCLUSIONS: Our study sheds light on the multilayered immune dysfunction in PDAC and presents a holistic view of the immune landscape in PDAC and lung adenocarcinoma, providing a comprehensive resource for functional studies and the exploration of therapeutically actionable targets in PDAC.
Assuntos
Adenocarcinoma de Pulmão , Carcinoma Ductal Pancreático , Doenças do Sistema Imunitário , Neoplasias Pancreáticas , Humanos , Multiômica , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/tratamento farmacológico , Análise de Célula Única , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
Chronic liver disease and hepatocellular carcinoma (HCC) are life-threatening diseases with limited treatment options. The lack of clinically relevant/tractable experimental models hampers therapeutic discovery. Here, we develop a simple and robust human liver cell-based system modeling a clinical prognostic liver signature (PLS) predicting long-term liver disease progression toward HCC. Using the PLS as a readout, followed by validation in nonalcoholic steatohepatitis/fibrosis/HCC animal models and patient-derived liver spheroids, we identify nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of advanced liver disease and HCC chemoprevention. Moreover, perturbation studies combined with single cell RNA-Seq analyses of patient liver tissues uncover hepatocytes and HRH2+, CLEC5Ahigh, MARCOlow liver macrophages as potential nizatidine targets. The PLS model combined with single cell RNA-Seq of patient tissues enables discovery of urgently needed targets and therapeutics for treatment of advanced liver disease and cancer prevention.
Assuntos
Descoberta de Drogas , Fígado/patologia , Modelos Biológicos , Animais , Carcinogênese/patologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Quimioprevenção , Estudos de Coortes , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Hepacivirus/fisiologia , Hepatite C/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Vigilância Imunológica/efeitos dos fármacos , Inflamação/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Knockout , Nizatidina/farmacologia , Prognóstico , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Lymphotoxin ß-receptor (LTßR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures1,2, which are associated with severe chronic inflammatory diseases that span several organ systems3-6. How LTßR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTßR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTßR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTßR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTßR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTßR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFß signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/ß-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTßR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures1 and inhibition of apoptosis with tissue-regenerative strategies.
Assuntos
Pulmão/efeitos dos fármacos , Pulmão/fisiologia , Receptor beta de Linfotoxina/antagonistas & inibidores , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/agonistas , Imunidade Adaptativa , Envelhecimento/metabolismo , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Apoptose/efeitos dos fármacos , Enfisema/metabolismo , Feminino , Humanos , Imunidade Inata , Pulmão/metabolismo , Receptor beta de Linfotoxina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumaça/efeitos adversos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND & AIMS: Cholangiocyte proliferation and ductular reaction contribute to the onset and progression of liver diseases. Little is known about the role of the transcription factor nuclear factor-κB (NF-κB) in this process. We investigated the activities of the RELB proto-oncogene NF-κB subunit in human cholangiocytes and in mouse models of liver disease characterized by a ductular reaction. METHODS: We obtained liver tissue samples from patients with primary sclerosing cholangitis, primary biliary cholangitis, hepatitis B or C virus infection, autoimmune hepatitis, alcoholic liver disease, or without these diseases (controls) from a tissue bank in Germany. Tissues were analyzed by immunohistochemistry for levels of RELB and lymphotoxin ß (LTB). We studied mice with liver parenchymal cell (LPC)-specific disruption of the cylindromatosis (CYLD) lysine 63 deubiquitinase gene (Cyld), with or without disruption of Relb (CyldΔLPC mice and Cyld/RelbΔLPC mice) and compared them with C57BL/6 mice (controls). Mice were fed 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or standard chow diets to induce biliary injury or were given injections of CCl4 to induce non-cholestatic liver fibrosis. Liver tissues were analyzed by histology, immunohistochemistry, immunoblots, in situ hybridization, and quantitative real-time polymerase chain reaction. Cholangiocytes were isolated from normal human liver, incubated with LTB receptor agonist, and transfected with small interfering RNAs to knock down RELB. RESULTS: In liver tissues from patients with primary sclerosing cholangitis, primary biliary cholangitis, chronic infection with hepatitis B or C virus, autoimmune hepatitis, or alcoholic liver disease, we detected increased nuclear translocation of RELB and increased levels of LTB in cholangiocytes that formed reactive bile ducts compared with control liver tissues. Human cholangiocytes, but not those with RELB knockdown, proliferated with exposure to LTB. The phenotype of CyldΔLPC mice, which included ductular reaction, oval cell activation, and biliary fibrosis, was completely lost from Cyld/RelbΔLPC mice. Compared with livers from control mice, livers from CyldΔLPC mice (but not Cyld/RelbΔLPC mice) had increased levels of mRNAs encoding cytokines (LTB; CD40; and tumor necrosis factor superfamily [TNFSF] members TNFSF11 [RANKL], TNFSF13B [BAFF], and TNFSF14 [LIGHT]) produced by reactive cholangiocytes. However, these strains of mice developed similar levels of liver fibrosis in response to CCl4 exposure. CyldΔLPC mice and Cyld/RelbΔLPC mice had improved liver function on the DDC diet compared with control mice fed the DDC diet. CONCLUSION: Reactive bile ducts in patients with chronic liver diseases have increased levels of LTB and nuclear translocation of RELB. RELB is required for the ductular reaction and development of biliary fibrosis in CyldΔLPC mice. Deletion of RELB and CYLD from LPCs protects mice from DDC-induced cholestatic liver fibrosis.