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1.
Cell ; 181(7): 1475-1488.e12, 2020 06 25.
Article in English | MEDLINE | ID: mdl-32479746

ABSTRACT

Viruses are a constant threat to global health as highlighted by the current COVID-19 pandemic. Currently, lack of data underlying how the human host interacts with viruses, including the SARS-CoV-2 virus, limits effective therapeutic intervention. We introduce Viral-Track, a computational method that globally scans unmapped single-cell RNA sequencing (scRNA-seq) data for the presence of viral RNA, enabling transcriptional cell sorting of infected versus bystander cells. We demonstrate the sensitivity and specificity of Viral-Track to systematically detect viruses from multiple models of infection, including hepatitis B virus, in an unsupervised manner. Applying Viral-Track to bronchoalveloar-lavage samples from severe and mild COVID-19 patients reveals a dramatic impact of the virus on the immune system of severe patients compared to mild cases. Viral-Track detects an unexpected co-infection of the human metapneumovirus, present mainly in monocytes perturbed in type-I interferon (IFN)-signaling. Viral-Track provides a robust technology for dissecting the mechanisms of viral-infection and pathology.


Subject(s)
Coronavirus Infections/physiopathology , Host-Pathogen Interactions , Pneumonia, Viral/physiopathology , Software , Animals , Betacoronavirus/isolation & purification , COVID-19 , Coinfection/immunology , Coronavirus Infections/immunology , Coronavirus Infections/pathology , Coronavirus Infections/virology , Humans , Interferons/immunology , Lung/pathology , Pandemics , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , SARS-CoV-2 , Sensitivity and Specificity , Sequence Analysis, RNA , Severity of Illness Index , Single-Cell Analysis
2.
Cell ; 182(4): 872-885.e19, 2020 08 20.
Article in English | MEDLINE | ID: mdl-32783915

ABSTRACT

Cell function and activity are regulated through integration of signaling, epigenetic, transcriptional, and metabolic pathways. Here, we introduce INs-seq, an integrated technology for massively parallel recording of single-cell RNA sequencing (scRNA-seq) and intracellular protein activity. We demonstrate the broad utility of INs-seq for discovering new immune subsets by profiling different intracellular signatures of immune signaling, transcription factor combinations, and metabolic activity. Comprehensive mapping of Arginase 1-expressing cells within tumor models, a metabolic immune signature of suppressive activity, discovers novel Arg1+ Trem2+ regulatory myeloid (Mreg) cells and identifies markers, metabolic activity, and pathways associated with these cells. Genetic ablation of Trem2 in mice inhibits accumulation of intra-tumoral Mreg cells, leading to a marked decrease in dysfunctional CD8+ T cells and reduced tumor growth. This study establishes INs-seq as a broadly applicable technology for elucidating integrated transcriptional and intra-cellular maps and identifies the molecular signature of myeloid suppressive cells in tumors.


Subject(s)
Membrane Glycoproteins/metabolism , Neoplasms/pathology , RNA, Small Cytoplasmic/chemistry , Receptors, Immunologic/metabolism , Animals , Arginase/genetics , Arginase/metabolism , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Female , Gene Expression Regulation , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Neoplasms/immunology , Neoplasms/metabolism , RNA, Small Cytoplasmic/metabolism , Receptors, Immunologic/genetics , Sequence Analysis, RNA , Single-Cell Analysis , Transcription Factors/metabolism , Tumor Microenvironment , Tumor Necrosis Factor-alpha/metabolism , p38 Mitogen-Activated Protein Kinases
3.
Cell ; 175(4): 1031-1044.e18, 2018 11 01.
Article in English | MEDLINE | ID: mdl-30318149

ABSTRACT

Lung development and function arises from the interactions between diverse cell types and lineages. Using single-cell RNA sequencing (RNA-seq), we characterize the cellular composition of the lung during development and identify vast dynamics in cell composition and their molecular characteristics. Analyzing 818 ligand-receptor interaction pairs within and between cell lineages, we identify broadly interacting cells, including AT2, innate lymphocytes (ILCs), and basophils. Using interleukin (IL)-33 receptor knockout mice and in vitro experiments, we show that basophils establish a lung-specific function imprinted by IL-33 and granulocyte-macrophage colony-stimulating factor (GM-CSF), characterized by unique signaling of cytokines and growth factors important for stromal, epithelial, and myeloid cell fates. Antibody-depletion strategies, diphtheria toxin-mediated selective depletion of basophils, and co-culture studies show that lung resident basophils are important regulators of alveolar macrophage development and function. Together, our study demonstrates how whole-tissue signaling interaction map on the single-cell level can broaden our understanding of cellular networks in health and disease.


Subject(s)
Basophils/metabolism , Cell Communication , Genomic Imprinting , Macrophages, Alveolar/metabolism , Transcriptome , Animals , Cell Differentiation , Cell Line, Tumor , Cells, Cultured , Female , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Interleukin-33/metabolism , Macrophages, Alveolar/cytology , Male , Mice , Mice, Inbred C57BL , Signal Transduction , Single-Cell Analysis
4.
Proc Natl Acad Sci U S A ; 113(16): E2231-40, 2016 Apr 19.
Article in English | MEDLINE | ID: mdl-27044092

ABSTRACT

Microfluidic organ-on-a-chip technology aims to replace animal toxicity testing, but thus far has demonstrated few advantages over traditional methods. Mitochondrial dysfunction plays a critical role in the development of chemical and pharmaceutical toxicity, as well as pluripotency and disease processes. However, current methods to evaluate mitochondrial activity still rely on end-point assays, resulting in limited kinetic and prognostic information. Here, we present a liver-on-chip device capable of maintaining human tissue for over a month in vitro under physiological conditions. Mitochondrial respiration was monitored in real time using two-frequency phase modulation of tissue-embedded phosphorescent microprobes. A computer-controlled microfluidic switchboard allowed contiguous electrochemical measurements of glucose and lactate, providing real-time analysis of minute shifts from oxidative phosphorylation to anaerobic glycolysis, an early indication of mitochondrial stress. We quantify the dynamics of cellular adaptation to mitochondrial damage and the resulting redistribution of ATP production during rotenone-induced mitochondrial dysfunction and troglitazone (Rezulin)-induced mitochondrial stress. We show troglitazone shifts metabolic fluxes at concentrations previously regarded as safe, suggesting a mechanism for its observed idiosyncratic effect. Our microfluidic platform reveals the dynamics and strategies of cellular adaptation to mitochondrial damage, a unique advantage of organ-on-chip technology.


Subject(s)
Chromans/adverse effects , Lab-On-A-Chip Devices , Liver/metabolism , Mitochondria, Liver/metabolism , Mitochondrial Diseases/metabolism , Oxygen Consumption/drug effects , Thiazolidinediones/adverse effects , Chromans/pharmacology , Hep G2 Cells , Humans , Liver/pathology , Mitochondria, Liver/pathology , Mitochondrial Diseases/chemically induced , Mitochondrial Diseases/pathology , Thiazolidinediones/pharmacology , Troglitazone
5.
J Am Soc Nephrol ; 29(2): 434-448, 2018 02.
Article in English | MEDLINE | ID: mdl-29030466

ABSTRACT

Altered glucose reabsorption via the facilitative glucose transporter 2 (GLUT2) during diabetes may lead to renal proximal tubule cell (RPTC) injury, inflammation, and interstitial fibrosis. These pathologies are also triggered by activating the cannabinoid-1 receptor (CB1R), which contributes to the development of diabetic nephropathy (DN). However, the link between CB1R and GLUT2 remains to be determined. Here, we show that chronic peripheral CB1R blockade or genetically inactivating CB1Rs in the RPTCs ameliorated diabetes-induced renal structural and functional changes, kidney inflammation, and tubulointerstitial fibrosis in mice. Inhibition of CB1R also downregulated GLUT2 expression, affected the dynamic translocation of GLUT2 to the brush border membrane of RPTCs, and reduced glucose reabsorption. Thus, targeting peripheral CB1R or inhibiting GLUT2 dynamics in RPTCs has the potential to treat and ameliorate DN. These findings may support the rationale for the clinical testing of peripherally restricted CB1R antagonists or the development of novel renal-specific GLUT2 inhibitors against DN.


Subject(s)
Diabetic Nephropathies/genetics , Diabetic Nephropathies/metabolism , Glucose Transporter Type 2/genetics , Glucose Transporter Type 2/metabolism , Kidney Tubules, Proximal/pathology , Receptor, Cannabinoid, CB1/metabolism , Albuminuria/urine , Animals , Biological Transport , Blood Glucose/metabolism , Blood Urea Nitrogen , Creatinine/urine , Diabetic Nephropathies/chemically induced , Dogs , Fibrosis , Glucose/metabolism , Glucose Transporter Type 2/antagonists & inhibitors , Insulin/blood , Islets of Langerhans/pathology , Madin Darby Canine Kidney Cells , Male , Mice , Mice, Knockout , Protein Kinase C beta/metabolism , Pyrazoles/pharmacology , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB1/genetics , Streptozocin , Sulfonamides/pharmacology
6.
J Neurosci ; 37(4): 972-985, 2017 01 25.
Article in English | MEDLINE | ID: mdl-28123029

ABSTRACT

The central nervous system (CNS) is endowed with several immune-related mechanisms that contribute to its protection and maintenance in homeostasis and under pathology. Here, we discovered an additional mechanism that controls inflammatory responses within the CNS milieu under injurious conditions, involving CD200 ligand (CD200L) expressed by newly formed endothelial cells. We observed that CD200L is constitutively expressed in the mouse healthy CNS by endothelial cells of the blood-cerebrospinal fluid barrier and of the spinal cord meninges, but not by the endothelium of the blood-spinal cord barrier. Following spinal cord injury (SCI), newly formed endothelial cells, located only at the epicenter of the lesion site, expressed CD200L. Moreover, in the absence of CD200L expression by CNS-resident cells, functional recovery of mice following SCI was impaired. High throughput single-cell flow cytometry image analysis following SCI revealed CD200L-dependent direct interaction between endothelial and local CD200R+ myeloid cells, including activated microglia and infiltrating monocyte-derived macrophages (mo-MΦ). Absence of CD200L signaling, both in vitro and in vivo, resulted in a higher inflammatory response of the encountering macrophages, manifested by elevation in mRNA expression of Tnfα and Il1ß, increased intracellular TNFα immunoreactivity, and reduced expression levels of macrophage factors that are associated with resolution of inflammation, Dectin-1, CD206 (mannose receptor), and IL-4R. Collectively, our results highlight the importance of CD200-mediated immune dialogue between endothelial cells and the local resident microglia and infiltrating mo-MΦ within the lesion area, as a mechanism that contributes to regulation of inflammation following acute CNS injury. SIGNIFICANCE STATEMENT: This manuscript focuses on a novel mechanism of inflammation-regulation following spinal cord injury (SCI), orchestrated by CD200-ligand (CD200L) expressed by newly formed endothelial cells within the lesion site. Our study reveals that, in homeostasis, CD200L is expressed by endothelial cells of the mouse blood-cerebrospinal fluid barrier and of the blood-leptomeningeal barrier, but not by endothelial cells of the blood-spinal cord barrier. Following SCI, newly formed endothelial cells located within the epicenter of the lesion site were found to express CD200L at time points that were shown to be critical for repair. Our results reveal a direct interaction between CD200L+ endothelial cells and CD200R+ microglia and macrophages, resulting in attenuated inflammation, biasing macrophage phenotype toward inflammation-resolving cells, and promotion of functional recovery following SCI.


Subject(s)
Antigens, CD/biosynthesis , Endothelial Cells/metabolism , Macrophages/metabolism , Meninges/metabolism , Microglia/metabolism , Spinal Cord Injuries/metabolism , Animals , Antigens, CD/genetics , Blood-Brain Barrier/cytology , Blood-Brain Barrier/metabolism , Gene Expression , Male , Meninges/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Spinal Cord , Spinal Cord Injuries/pathology
7.
EMBO J ; 33(24): 2906-21, 2014 Dec 17.
Article in English | MEDLINE | ID: mdl-25385836

ABSTRACT

Tissue microenvironment influences the function of resident and infiltrating myeloid-derived cells. In the central nervous system (CNS), resident microglia and freshly recruited infiltrating monocyte-derived macrophages (mo-MΦ) display distinct activities under pathological conditions, yet little is known about the microenvironment-derived molecular mechanism that regulates these differences. Here, we demonstrate that long exposure to transforming growth factor-ß1 (TGFß1) impaired the ability of myeloid cells to acquire a resolving anti-inflammatory phenotype. Using genome-wide expression analysis and chromatin immunoprecipitation followed by next-generation sequencing, we show that the capacity to undergo pro- to anti-inflammatory (M1-to-M2) phenotype switch is controlled by the transcription factor interferon regulatory factor 7 (IRF7) that is down-regulated by the TGFß1 pathway. RNAi-mediated perturbation of Irf7 inhibited the M1-to-M2 switch, while IFNß1 (an IRF7 pathway activator) restored it. In vivo induction of Irf7 expression in microglia, following spinal cord injury, reduced their pro-inflammatory activity. These results highlight the key role of tissue-specific environmental factors in determining the fate of resident myeloid-derived cells under both physiological and pathological conditions.


Subject(s)
Gene Expression Regulation/drug effects , Interferon Regulatory Factor-7/metabolism , Myeloid Cells/drug effects , Myeloid Cells/immunology , Transforming Growth Factor beta1/metabolism , Animals , Chromatin Immunoprecipitation , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Mice, Inbred C57BL
8.
Nat Chem Biol ; 12(12): 1037-1045, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27723751

ABSTRACT

Viruses lack the basic machinery needed to replicate and therefore must hijack the host's metabolism to propagate. Virus-induced metabolic changes have yet to be systematically studied in the context of host transcriptional regulation, and such studies shoul offer insight into host-pathogen metabolic interplay. In this work we identified hepatitis C virus (HCV)-responsive regulators by coupling system-wide metabolic-flux analysis with targeted perturbation of nuclear receptors in primary human hepatocytes. We found HCV-induced upregulation of glycolysis, ketogenesis and drug metabolism, with glycolysis controlled by activation of HNF4α, ketogenesis by PPARα and FXR, and drug metabolism by PXR. Pharmaceutical inhibition of HNF4α reversed HCV-induced glycolysis, blocking viral replication while increasing apoptosis in infected cells showing virus-induced dependence on glycolysis. In contrast, pharmaceutical inhibition of PPARα or FXR reversed HCV-induced ketogenesis but increased viral replication, demonstrating a novel host antiviral response. Our results show that virus-induced changes to a host's metabolism can be detrimental to its life cycle, thus revealing a biologically complex relationship between virus and host.


Subject(s)
Hepacivirus/metabolism , Hepatitis C/metabolism , Hepatitis C/virology , Host-Pathogen Interactions , Receptors, Cytoplasmic and Nuclear/metabolism , Glycolysis , Hepacivirus/drug effects , Hepacivirus/growth & development , Hepatocytes/metabolism , Hepatocytes/virology , Humans
9.
Pharmacology ; 99(3-4): 124-127, 2017.
Article in English | MEDLINE | ID: mdl-27988509

ABSTRACT

BACKGROUND: Rivaroxaban is a member of the novel target-specific oral anticoagulants (TSOACs) family of drugs recently approved for the prevention and treatment of venous thromboembolism events. A major drawback of the drug is its potential for causing severe hemorrhagic events, which may be difficult to treat in an emergency setting due to lack of effective antidote. Here, we describe a case of acute gastrointestinal (GI) hemorrhage leading to complete colon obstruction in a patient treated with rivaroxaban. Summary and Key Messages: The case presented here demonstrates a chain of events originating from an unprovoked intramural bleeding in a patient using rivaroxaban, leading to an organized giant clot formation, and to complete colon obstruction. In the available literature, the specific site of the GI bleeding has not been discussed. A further study is recommended and re-examination of bleeding events and exploration of new cases due to the use of TSOACs can help predict the course and the outcomes of such complications.


Subject(s)
Colon/diagnostic imaging , Factor Xa Inhibitors/adverse effects , Gastrointestinal Hemorrhage/chemically induced , Gastrointestinal Hemorrhage/diagnostic imaging , Intestinal Obstruction/diagnostic imaging , Rivaroxaban/adverse effects , Acute Disease , Aged , Colon/surgery , Diagnosis, Differential , Gastrointestinal Hemorrhage/surgery , Humans , Intestinal Obstruction/surgery , Male
10.
Hepatology ; 62(1): 265-78, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25808545

ABSTRACT

UNLABELLED: The liver is the main organ responsible for the modification, clearance, and transformational toxicity of most xenobiotics owing to its abundance in cytochrome P450 (CYP450) enzymes. However, the scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSCs) represent an excellent source of differentiated hepatocytes; however, current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly, fetal hepatocytes acquire mature CYP450 expression only postpartum, suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K2 and lithocholic acid, a by-product of intestinal flora, activate pregnane X receptor (PXR) and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produce albumin and apolipoprotein B100 at levels equivalent to primary human hepatocytes, while demonstrating an 8-fold induction of CYP450 activity in response to aryl hydrocarbon receptor (AhR) agonist omeprazole and a 10-fold induction in response to PXR agonist rifampicin. Flow cytometry showed that over 83% of cells were albumin and hepatocyte nuclear factor 4 alpha (HNF4α) positive, permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R(2) correlation of 0.94 between TC50 values obtained in stem cell-derived hepatocytes and primary cells, compared to 0.62 for HepG2 cells. Finally, stem cell-derived hepatocytes demonstrate all toxicological endpoints examined, including steatosis, apoptosis, and cholestasis, when exposed to nine known hepatotoxins. CONCLUSION: Our work provides fresh insights into liver development, suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes postpartum. Addition of these cues results in the first functional, inducible, hPSC-derived hepatocyte for predictive toxicology.


Subject(s)
Cell Culture Techniques , Hepatocytes/cytology , Lithocholic Acid/pharmacology , Pluripotent Stem Cells/drug effects , Vitamin K 2/pharmacology , Cell Differentiation , Cells, Cultured , Cytochrome P-450 CYP2C9/metabolism , Cytochrome P-450 CYP3A/metabolism , Embryonic Stem Cells/drug effects , Hepatocytes/drug effects , Hepatocytes/enzymology , Humans , Pregnane X Receptor , Receptors, Steroid/metabolism , Sequence Analysis, RNA , Toxicity Tests, Acute , Vitamin K 2/analogs & derivatives
11.
Arch Toxicol ; 90(5): 1181-91, 2016 May.
Article in English | MEDLINE | ID: mdl-26041127

ABSTRACT

Prediction of drug-induced toxicity is complicated by the failure of animal models to extrapolate human response, especially during assessment of repeated dose toxicity for cosmetic or chronic drug treatments. In this work, we present a 3D microreactor capable of maintaining metabolically active HepG2/C3A spheroids for over 28 days in vitro under stable oxygen gradients mimicking the in vivo microenvironment. Mitochondrial respiration was monitored using two-frequency phase modulation of phosphorescent microprobes embedded in the tissue. Phase modulation is focus independent and unaffected by cell death or migration. This sensitive measurement of oxygen dynamics revealed important information on the drug mechanism of action and transient subthreshold effects. Specifically, exposure to antiarrhythmic agent, amiodarone, showed that both respiration and the time to onset of mitochondrial damage were dose dependent showing a TC50 of 425 µm. Analysis showed significant induction of both phospholipidosis and microvesicular steatosis during long-term exposure. Importantly, exposure to widely used analgesic, acetaminophen, caused an immediate, reversible, dose-dependent loss of oxygen uptake followed by a slow, irreversible, dose-independent death, with a TC50 of 12.3 mM. Transient loss of mitochondrial respiration was also detected below the threshold of acetaminophen toxicity. The phenomenon was repeated in HeLa cells that lack CYP2E1 and 3A4, and was blocked by preincubation with ascorbate and TMPD. These results mark the importance of tracing toxicity effects over time, suggesting a NAPQI-independent targeting of mitochondrial complex III might be responsible for acetaminophen toxicity in extrahepatic tissues.


Subject(s)
Acetaminophen/toxicity , Amiodarone/toxicity , Analgesics, Non-Narcotic/toxicity , Anti-Arrhythmia Agents/toxicity , Bioreactors , Chemical and Drug Induced Liver Injury/etiology , Cytochrome P-450 CYP2E1/metabolism , Hepatocytes/drug effects , Lab-On-A-Chip Devices , Mitochondria, Liver/drug effects , Oxygen Consumption , Acetaminophen/metabolism , Activation, Metabolic , Amiodarone/metabolism , Analgesics, Non-Narcotic/metabolism , Anti-Arrhythmia Agents/metabolism , Biomarkers/metabolism , Cellular Microenvironment , Chemical and Drug Induced Liver Injury/enzymology , Chemical and Drug Induced Liver Injury/pathology , Coculture Techniques , Dose-Response Relationship, Drug , Equipment Design , Hep G2 Cells , Hepatocytes/enzymology , Hepatocytes/pathology , Humans , Mitochondria, Liver/enzymology , Mitochondria, Liver/pathology , Spheroids, Cellular , Time Factors
12.
J Neurosci ; 34(31): 10141-55, 2014 Jul 30.
Article in English | MEDLINE | ID: mdl-25080578

ABSTRACT

Monocyte-derived macrophages (mo-MΦs) and T cells have been shown to contribute to spinal cord repair. Recently, the remote brain choroid plexus epithelium (CP) was identified as a portal for monocyte recruitment, and its activation for leukocyte trafficking was found to be IFN-γ-dependent. Here, we addressed how the need for effector T cells can be reconciled with the role of inflammation-resolving immune cells in the repair process. Using an acute spinal cord injury model, we show that in mice deficient in IFN-γ-producing T cells, the CP was not activated, and recruitment of inflammation-resolving mo-MΦ to the spinal cord parenchyma was limited. We further demonstrate that mo-MΦ locally regulated recruitment of thymic-derived Foxp3(+) regulatory T (Treg) cells to the injured spinal cord parenchyma at the subacute/chronic phase. Importantly, an ablation protocol that resulted in reduced Tregs at this stage interfered with tissue remodeling, in contrast to Treg transient ablation, restricted to the 4 d period before the injury, which favored repair. The enhanced functional recovery observed following such a controlled decrease of Tregs suggests that reduced systemic immunosuppression at the time of the insult can enhance CNS repair. Overall, our data highlight a dynamic immune cell network needed for repair, acting in discrete compartments and stages, and involving effector and regulatory T cells, interconnected by mo-MΦ. Any of these populations may be detrimental to the repair process if their level or activity become dysregulated. Accordingly, therapeutic interventions must be both temporally and spatially controlled.


Subject(s)
Nerve Regeneration/immunology , Recovery of Function/physiology , Spinal Cord Injuries/physiopathology , T-Lymphocytes, Regulatory/physiology , Animals , Antigens, CD/metabolism , CD11c Antigen/genetics , CX3C Chemokine Receptor 1 , Diphtheria Toxin/pharmacology , Disease Models, Animal , Forkhead Transcription Factors/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Macrophages/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myelin-Oligodendrocyte Glycoprotein/immunology , Nerve Regeneration/genetics , Peptide Fragments/immunology , Receptors, Chemokine/genetics , Recovery of Function/genetics , T-Box Domain Proteins/deficiency , T-Box Domain Proteins/genetics , Vaccination
13.
Cancer Cell ; 11(2): 133-46, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17292825

ABSTRACT

Myocardin is known as an important transcriptional regulator in smooth and cardiac muscle development. Here we found that myocardin is frequently repressed during human malignant transformation, contributing to a differentiation defect. We demonstrate that myocardin is a transcriptional target of TGFbeta required for TGFbeta-mediated differentiation of human fibroblasts. Serum deprivation, intact contact inhibition response, and the p16ink4a/Rb pathway contribute to myocardin induction and differentiation. Restoration of myocardin expression in sarcoma cells results in differentiation and inhibition of malignant growth, whereas inactivation of myocardin in normal fibroblasts increases their proliferative potential. Myocardin expression is reduced in multiple types of human tumors. Collectively, our results demonstrate that myocardin is an important suppressive modifier of the malignant transformation process.


Subject(s)
Cell Differentiation , Cell Transformation, Neoplastic , Cyclin-Dependent Kinase Inhibitor p16/antagonists & inhibitors , Fibroblasts/cytology , Nuclear Proteins/antagonists & inhibitors , Trans-Activators/antagonists & inhibitors , Blotting, Western , Cell Adhesion , Cell Proliferation , Cells, Cultured , Colony-Forming Units Assay , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , DNA Methylation , Fibroblasts/metabolism , Fluorescent Antibody Technique , Gene Expression Regulation , Humans , Lung/embryology , Mesoderm/cytology , Mesoderm/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Plasmids , Promoter Regions, Genetic , RNA, Small Interfering/pharmacology , Trans-Activators/genetics , Trans-Activators/metabolism , Transforming Growth Factor beta/pharmacology
14.
Nat Cancer ; 5(5): 742-759, 2024 May.
Article in English | MEDLINE | ID: mdl-38429414

ABSTRACT

Successful immunotherapy relies on triggering complex responses involving T cell dynamics in tumors and the periphery. Characterizing these responses remains challenging using static human single-cell atlases or mouse models. To address this, we developed a framework for in vivo tracking of tumor-specific CD8+ T cells over time and at single-cell resolution. Our tools facilitate the modeling of gene program dynamics in the tumor microenvironment (TME) and the tumor-draining lymph node (tdLN). Using this approach, we characterize two modes of anti-programmed cell death protein 1 (PD-1) activity, decoupling induced differentiation of tumor-specific activated precursor cells from conventional type 1 dendritic cell (cDC1)-dependent proliferation and recruitment to the TME. We demonstrate that combining anti-PD-1 therapy with anti-4-1BB agonist enhances the recruitment and proliferation of activated precursors, resulting in tumor control. These data suggest that effective response to anti-PD-1 therapy is dependent on sufficient influx of activated precursor CD8+ cells to the TME and highlight the importance of understanding system-level dynamics in optimizing immunotherapies.


Subject(s)
CD8-Positive T-Lymphocytes , Immunotherapy , Tumor Microenvironment , Animals , Mice , Immunotherapy/methods , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Tumor Microenvironment/immunology , Humans , Neoplasms/immunology , Neoplasms/therapy , Neoplasms/drug therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Dendritic Cells/immunology , Dendritic Cells/drug effects , Cell Line, Tumor
15.
Nat Biomed Eng ; 7(11): 1493-1513, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37550423

ABSTRACT

The study of cardiac physiology is hindered by physiological differences between humans and small-animal models. Here we report the generation of multi-chambered self-paced vascularized human cardiac organoids formed under anisotropic stress and their applicability to the study of cardiac arrhythmia. Sensors embedded in the cardiac organoids enabled the simultaneous measurement of oxygen uptake, extracellular field potentials and cardiac contraction at resolutions higher than 10 Hz. This microphysiological system revealed 1 Hz cardiac respiratory cycles that are coupled to the electrical rather than the mechanical activity of cardiomyocytes. This electro-mitochondrial coupling was driven by mitochondrial calcium oscillations driving respiration cycles. Pharmaceutical or genetic inhibition of this coupling results in arrhythmogenic behaviour. We show that the chemotherapeutic mitoxantrone induces arrhythmia through disruption of this pathway, a process that can be partially reversed by the co-administration of metformin. Our microphysiological cardiac systems may further facilitate the study of the mitochondrial dynamics of cardiac rhythms and advance our understanding of human cardiac physiology.


Subject(s)
Biochemical Phenomena , Myocytes, Cardiac , Animals , Humans , Myocytes, Cardiac/metabolism , Arrhythmias, Cardiac , Myocardial Contraction/physiology , Organoids
16.
Elife ; 122023 01 27.
Article in English | MEDLINE | ID: mdl-36705566

ABSTRACT

Background: Viral infection is associated with a significant rewire of the host metabolic pathways, presenting attractive metabolic targets for intervention. Methods: We chart the metabolic response of lung epithelial cells to SARS-CoV-2 infection in primary cultures and COVID-19 patient samples and perform in vitro metabolism-focused drug screen on primary lung epithelial cells infected with different strains of the virus. We perform observational analysis of Israeli patients hospitalized due to COVID-19 and comparative epidemiological analysis from cohorts in Italy and the Veteran's Health Administration in the United States. In addition, we perform a prospective non-randomized interventional open-label study in which 15 patients hospitalized with severe COVID-19 were given 145 mg/day of nanocrystallized fenofibrate added to the standard of care. Results: SARS-CoV-2 infection produced transcriptional changes associated with increased glycolysis and lipid accumulation. Metabolism-focused drug screen showed that fenofibrate reversed lipid accumulation and blocked SARS-CoV-2 replication through a PPARα-dependent mechanism in both alpha and delta variants. Analysis of 3233 Israeli patients hospitalized due to COVID-19 supported in vitro findings. Patients taking fibrates showed significantly lower markers of immunoinflammation and faster recovery. Additional corroboration was received by comparative epidemiological analysis from cohorts in Europe and the United States. A subsequent prospective non-randomized interventional open-label study was carried out on 15 patients hospitalized with severe COVID-19. The patients were treated with 145 mg/day of nanocrystallized fenofibrate in addition to standard-of-care. Patients receiving fenofibrate demonstrated a rapid reduction in inflammation and a significantly faster recovery compared to patients admitted during the same period. Conclusions: Taken together, our data suggest that pharmacological modulation of PPARα should be strongly considered as a potential therapeutic approach for SARS-CoV-2 infection and emphasizes the need to complete the study of fenofibrate in large randomized controlled clinical trials. Funding: Funding was provided by European Research Council Consolidator Grants OCLD (project no. 681870) and generous gifts from the Nikoh Foundation and the Sam and Rina Frankel Foundation (YN). The interventional study was supported by Abbott (project FENOC0003). Clinical trial number: NCT04661930.


Subject(s)
COVID-19 , Fenofibrate , Humans , Fenofibrate/therapeutic use , Lipids , PPAR alpha , Prospective Studies , SARS-CoV-2 , Treatment Outcome
17.
J Immunol ; 185(10): 5869-78, 2010 Nov 15.
Article in English | MEDLINE | ID: mdl-20956342

ABSTRACT

Sialylation of tumor cells is involved in various aspects of their malignancy (proliferation, motility, invasion, and metastasis); however, its effect on the process of immunoediting that affects tumor cell immunogenicity has not been studied. We have shown that in mice with impaired immunoediting, such as in IL-1α(-/-) and IFNγ(-/-) mice, 3-methylcholanthrene-induced fibrosarcoma cells are immunogenic and concomitantly bear low levels of surface sialylation, whereas tumor cells derived from wild type mice are nonimmunogenic and bear higher levels of surface sialylation. To study immune mechanisms whose interaction with tumor cells involves surface sialic acid residues, we used highly sialylated 3-methylcholanthrene-induced nonimmunogenic fibrosarcoma cell lines from wild type mice, which were treated with sialidase to mimic immunogenic tumor cell variants. In vivo and in vitro experiments revealed that desialylation of tumor cells reduced their growth and induced cytotoxicity by NK cells. Moreover, sialidase-treated tumor cells better activated NK cells for IFN-γ secretion. The NKG2D-activating receptor on NK cells was shown to be involved in interactions with desialylated ligands on tumor cells, the nature of which is still not known. Thus, the degree of sialylation on tumor cells, which is selected during the process of immunoediting, has possibly evolved as an important mechanism of tumor cells with low intrinsic immunogenicity or select for tumor cells that can evade the immune system or subvert its function. When immunoediting is impaired, such as in IFN-γ(-/-) and IL-1α(-/-) mice, the overt tumor consists of desialylayed tumor cells that interact better with immunosurveillance cells.


Subject(s)
Cytotoxicity, Immunologic/immunology , Fibrosarcoma/immunology , Fibrosarcoma/metabolism , N-Acetylneuraminic Acid/metabolism , Animals , Carcinogens/toxicity , Cell Proliferation , Cell Separation , Female , Fibrosarcoma/chemically induced , Flow Cytometry , Killer Cells, Natural/immunology , Methylcholanthrene/toxicity , Mice , Mice, Inbred BALB C , Mice, Knockout , Microscopy, Confocal , Microscopy, Fluorescence , NK Cell Lectin-Like Receptor Subfamily K/immunology
18.
Lab Chip ; 22(23): 4469-4480, 2022 11 22.
Article in English | MEDLINE | ID: mdl-36281785

ABSTRACT

Aminoglycosides are an important class of antibiotics that play a critical role in the treatment of life-threatening infections, but their use is limited by their toxicity. In fact, gentamicin causes severe nephrotoxicity in 17% of hospitalized patients. The kidney proximal tubule is particularly vulnerable to drug-induced nephrotoxicity due to its role in drug transport. In this work, we developed a perfused vascularized model of human kidney tubuloids integrated with tissue-embedded microsensors that track the metabolic dynamics of aminoglycoside-induced renal toxicity in real time. Our model shows that gentamicin disrupts proximal tubule polarity at concentrations 20-fold below its TC50, leading to a 3.2-fold increase in glucose uptake, and reverse TCA cycle flux culminating in a 40-fold increase in lipid accumulation. Blocking glucose reabsorption using the SGLT2 inhibitor empagliflozin significantly reduced gentamicin toxicity by 10-fold. These results demonstrate the utility of sensor-integrated kidney-on-chip platforms to rapidly identify new metabolic mechanisms that may underly adverse drug reactions. The results should improve our ability to modulate the toxicity of novel aminoglycosides.


Subject(s)
Aminoglycosides , Anti-Bacterial Agents , Humans , Aminoglycosides/toxicity , Aminoglycosides/metabolism , Anti-Bacterial Agents/toxicity , Gentamicins/toxicity , Kidney/metabolism , Kidney Tubules, Proximal/metabolism
19.
Nat Cancer ; 3(3): 303-317, 2022 03.
Article in English | MEDLINE | ID: mdl-35241835

ABSTRACT

Despite their key regulatory role and therapeutic potency, the molecular signatures of interactions between T cells and antigen-presenting myeloid cells within the tumor microenvironment remain poorly characterized. Here, we systematically characterize these interactions using RNA sequencing of physically interacting cells (PIC-seq) and find that CD4+PD-1+CXCL13+ T cells are a major interacting hub with antigen-presenting cells in the tumor microenvironment of human non-small cell lung carcinoma. We define this clonally expanded, tumor-specific and conserved T-cell subset as T-helper tumor (Tht) cells. Reconstitution of Tht cells in vitro and in an ovalbumin-specific αß TCR CD4+ T-cell mouse model, shows that the Tht program is primed in tumor-draining lymph nodes by dendritic cells presenting tumor antigens, and that their function is important for harnessing the antitumor response of anti-PD-1 treatment. Our molecular and functional findings support the modulation of Tht-dendritic cell interaction checkpoints as a major interventional strategy in immunotherapy.


Subject(s)
Lung Neoplasms , Tumor Microenvironment , Animals , Cell Line, Tumor , Dendritic Cells , Immune Checkpoint Inhibitors/pharmacology , Lung Neoplasms/therapy , Mice , T-Lymphocytes, Helper-Inducer
20.
Sci Transl Med ; 13(582)2021 02 24.
Article in English | MEDLINE | ID: mdl-33627489

ABSTRACT

The kidney plays a critical role in fluid homeostasis, glucose control, and drug excretion. Loss of kidney function due to drug-induced nephrotoxicity affects over 20% of the adult population. The kidney proximal tubule is a complex vascularized structure that is particularly vulnerable to drug-induced nephrotoxicity. Here, we introduce a model of vascularized human kidney spheroids with integrated tissue-embedded microsensors for oxygen, glucose, lactate, and glutamine, providing real-time assessment of cellular metabolism. Our model shows that both the immunosuppressive drug cyclosporine and the anticancer drug cisplatin disrupt proximal tubule polarity at subtoxic concentrations, leading to glucose accumulation and lipotoxicity. Impeding glucose reabsorption using glucose transport inhibitors blocked cyclosporine and cisplatin toxicity by 1000- to 3-fold, respectively. Retrospective study of 247 patients who were diagnosed with kidney damage receiving cyclosporine or cisplatin in combination with the sodium-glucose cotransporter-2 (SGLT2) inhibitor empagliflozin showed significant (P < 0.001) improvement of kidney function, as well as reduction in creatinine and uric acid, markers of kidney damage. These results demonstrate the potential of sensor-integrated kidney-on-chip platforms to elucidate mechanisms of action and rapidly reformulate effective therapeutic solutions, increasing drug safety and reducing the cost of clinical and commercial failures.


Subject(s)
Pharmaceutical Preparations , Sodium-Glucose Transporter 2 Inhibitors , Humans , Kidney , Lab-On-A-Chip Devices , Retrospective Studies , Sodium-Glucose Transporter 1
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