scholarly journals DDX58(RIG-I)-related disease is associated with tissue-specific interferon pathway activation

2021 ◽  
pp. jmedgenet-2020-107447
Author(s):  
Lev Prasov ◽  
Brenda L Bohnsack ◽  
Antonette S El Husny ◽  
Lam C Tsoi ◽  
Bin Guan ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Skin Rash ◽  
Open Angle Glaucoma ◽  
Response To Therapy ◽  
Functional Characterisation ◽  
Single Cell Rna Sequencing ◽  
Rna Ligands

BackgroundSingleton-Merten syndrome (SGMRT) is a rare immunogenetic disorder that variably features juvenile open-angle glaucoma (JOAG), psoriasiform skin rash, aortic calcifications and skeletal and dental dysplasia. Few families have been described and the genotypic and phenotypic spectrum is poorly defined, with variants in DDX58 (DExD/H-box helicase 58) being one of two identified causes, classified as SGMRT2.MethodsFamilies underwent deep systemic phenotyping and exome sequencing. Functional characterisation with in vitro luciferase assays and in vivo interferon signature using bulk and single cell RNA sequencing was performed.ResultsWe have identified a novel DDX58 variant c.1529A>T p.(Glu510Val) that segregates with disease in two families with SGMRT2. Patients in these families have widely variable phenotypic features and different ethnic background, with some being severely affected by systemic features and others solely with glaucoma. JOAG was present in all individuals affected with the syndrome. Furthermore, detailed evaluation of skin rash in one patient revealed sparse inflammatory infiltrates in a unique distribution. Functional analysis showed that the DDX58 variant is a dominant gain-of-function activator of interferon pathways in the absence of exogenous RNA ligands. Single cell RNA sequencing of patient lesional skin revealed a cellular activation of interferon-stimulated gene expression in keratinocytes and fibroblasts but not in neighbouring healthy skin.ConclusionsThese results expand the genotypic spectrum of DDX58-associated disease, provide the first detailed description of ocular and dermatological phenotypes, expand our understanding of the molecular pathogenesis of this condition and provide a platform for testing response to therapy.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation

2019 ◽  
Vol 31 (1) ◽  
pp. 118-138 ◽  
Author(s):  
Sébastien J. Dumas ◽  
Elda Meta ◽  
Mila Borri ◽  
Jermaine Goveia ◽  
Katerina Rohlenova ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Oxidative Phosphorylation ◽  
Single Cell ◽  
Rna Sequencing ◽  
Water Deprivation ◽  
Metabolic Adaptation ◽  
Single Cell Rna Sequencing

BackgroundRenal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown.MethodsWe inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo.ResultsWe identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and—surprisingly—oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration.ConclusionsThis study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.

scholarly journals The novel BET inhibitor UM-002 reduces glioblastoma cell proliferation and invasion

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna M. Jermakowicz ◽  
Matthew J. Rybin ◽  
Robert K. Suter ◽  
Jann N. Sarkaria ◽  
Zane Zeier ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Adult Brain ◽  
Bet Inhibitor ◽  
Bet Inhibitors ◽  
Single Cell Rna Sequencing

AbstractBromodomain and extraterminal domain (BET) proteins have emerged as therapeutic targets in multiple cancers, including the most common primary adult brain tumor glioblastoma (GBM). Although several BET inhibitors have entered clinical trials, few are brain penetrant. We have generated UM-002, a novel brain penetrant BET inhibitor that reduces GBM cell proliferation in vitro and in a human cerebral brain organoid model. Since UM-002 is more potent than other BET inhibitors, it could potentially be developed for GBM treatment. Furthermore, UM-002 treatment reduces the expression of cell-cycle related genes in vivo and reduces the expression of invasion related genes within the non-proliferative cells present in tumors as measured by single cell RNA-sequencing. These studies suggest that BET inhibition alters the transcriptional landscape of GBM tumors, which has implications for designing combination therapies. Importantly, they also provide an integrated dataset that combines in vitro and ex vivo studies with in vivo single-cell RNA-sequencing to characterize a novel BET inhibitor in GBM.

scholarly journals TMIC-59. INVESTIGATING PHYSIOLOGY OF THERAPY RESISTANT, TUMOR-INITIATING GBM CELLS IN HYPOXIC NICHES USING SPATIALLY-SENSITIVE HYPOXIA REPORTER SYSTEM AND SINGLE-CELL RNA SEQUENCING

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi260-vi261
Author(s):  
Anirudh Sattiraju ◽  
Valerie Marallano ◽  
Roland Friedel ◽  
Hongyan Zou
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Reporter System ◽  
T Cell Infiltration ◽  
Single Cell Rna Sequencing ◽  
Stem Cell Lines

Abstract Glioblastoma (GBM) is the most common and lethal brain cancer that invariably recurs after therapy due to presence of resistant GBM cells within hypoxic and peri-necrotic regions. Eradicating such GBM cells, which constitute a major source of tumor recurrence, is important to curb disease relapse. An endogenously expressed, spatially sensitive hypoxia reporter would therefore be a valuable tool to evaluate hypoxic zones in GBM in detail, and to measure the efficacy of hypoxia-activated drugs. For this purpose, we engineered a lentiviral vector that carries a hypoxia reporter, consisting of HIF response elements (HRE) that drive expression of UnaG fluorescent protein, which fluoresces independent of oxidative maturation. We validated the sensitivity of our reporter in vitro using U87MG, GBM2, and patient-derived GBM stem cell lines, and we performed intracranial transplantations of GBM cells in SCID mice to identify cells undergoing hypoxic stress in in vivo microenvironment. In addition, GL261 murine GBM cells with hypoxia reporter were intracranially implanted in C57BL/6 mice as syngeneic model for studies on immune responses. Brains from our transplant studies were dissociated and single-cell RNA sequencing (Drop-Seq) was performed to investigate heterogeneity in response to hypoxia within GBM cells and the cellular composition of microenvironment. We will also apply a hypoxia-activated prodrug, Evofosfamide (Evo), in our ongoing studies that can potentially eradicate hypoxic tumor cells and increase T cell infiltration and reverse immune suppression. As hypoxic niches are thought to confer resistance to radiation therapy (XRT), combining XRT with Evo could thus improve therapy efficacy. Our hypoxia gene reporter, combined with single-cell transcriptomics, could therefore serve as an effective tool to enable fundamental investigation of GBM microenvironment and could be used to evaluate therapies targeting tumor microenvironment to enhance GBM patient survival.

scholarly journals Single Cell RNA Sequencing Identifies Subsets of Hepatic Stellate Cells and Myofibroblasts in Liver Fibrosis

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 503 ◽  
Author(s):  
Oliver Krenkel ◽  
Jana Hundertmark ◽  
Thomas Ritz ◽  
Ralf Weiskirchen ◽  
Frank Tacke
Keyword(s):  
Liver Fibrosis ◽  
Single Cell ◽  
Rna Sequencing ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Marker Genes ◽  
Gene And Protein Expression ◽  
Single Cell Rna Sequencing

Activation of hepatic stellate cells (HSCs) and their trans-differentiation towards collagen-secreting myofibroblasts (MFB) promote liver fibrosis progression. During chronic liver disease, resting HSCs become activated by inflammatory and injury signals. However, HSCs/MFB not only produce collagen, but also secrete cytokines, participate in metabolism, and have biomechanical properties. We herein aimed to characterize the heterogeneity of these liver mesenchymal cells by single cell RNA sequencing. In vivo resting HSCs or activated MFB were isolated from C57BL6/J mice challenged by carbon tetrachloride (CCl4) intraperitoneally for 3 weeks to induce liver fibrosis and compared to in vitro cultivated MFB. While resting HSCs formed a homogenous population characterized by high platelet derived growth factor receptor β (PDGFRβ) expression, in vivo and in vitro activated MFB split into heterogeneous populations, characterized by α-smooth muscle actin (α-SMA), collagens, or immunological markers. S100 calcium binding protein A6 (S100A6) was a universal marker of activated MFB on both the gene and protein expression level. Compared to the heterogeneity of in vivo MFB, MFB in vitro sequentially and only transiently expressed marker genes, such as chemokines, during culture activation. Taken together, our data demonstrate the heterogeneity of HSCs and MFB, indicating the existence of functionally relevant subsets in hepatic fibrosis.

Myeloid cells in liver and bone marrow acquire a functionally distinct inflammatory phenotype during obesity-related steatohepatitis

Gut ◽  
2019 ◽  
Vol 69 (3) ◽  
pp. 551-563 ◽  
Author(s):  
Oliver Krenkel ◽  
Jana Hundertmark ◽  
Ali T Abdallah ◽  
Marlene Kohlhepp ◽  
Tobias Puengel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fatty Liver ◽  
Single Cell ◽  
Rna Sequencing ◽  
Myeloid Cells ◽  
Alcoholic Fatty Liver ◽  
Single Cell Rna Sequencing ◽  
Inflammatory Phenotype

ObjectiveBone marrow-derived myeloid cells accumulate in the liver as monocytes and macrophages during the progression of obesity-related non-alcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH). Myeloid cells comprise heterogeneous subsets, and dietary overnutrition may affect macrophages in the liver and bone marrow. We therefore aimed at characterising in depth the functional adaptations of myeloid cells in fatty liver.DesignWe employed single-cell RNA sequencing to comprehensively assess the heterogeneity of myeloid cells in the liver and bone marrow during NAFLD, by analysing C57BL/6 mice fed with a high-fat, high-sugar, high-cholesterol ‘Western diet’ for 16 weeks. We also characterised NAFLD-driven functional adaptations of macrophages in vitro and their functional relevance during steatohepatitis in vivo.ResultsSingle-cell RNA sequencing identified distinct myeloid cell clusters in the liver and bone marrow. In both compartments, monocyte-derived populations were largely expanded in NASH-affected mice. Importantly, the liver myeloid compartment adapted a unique inflammatory phenotype during NAFLD progression, exemplarily characterised by downregulated inflammatory calprotectin (S100A8/A9) in macrophage and dendritic cell subsets. This distinctive gene signature was also found in their bone marrow precursors. The NASH myeloid phenotype was principally recapitulated by in vitro exposure of bone marrow-derived macrophages with fatty acids, depended on toll-like receptor 4 signalling and defined a characteristic response pattern to lipopolysaccharide stimulation. This imprinted and stable NASH myeloid immune phenotype functionally determined inflammatory responses following acute liver injury (acetaminophen poisoning) in vivo.ConclusionLiver myeloid leucocytes and their bone marrow precursors adapt a common and functionally relevant inflammatory signature during NAFLD progression.

scholarly journals Single-cell RNA sequencing reveals that targeting HSP90 suppresses PDAC progression by restraining mitochondrial bioenergetics

Oncogenesis ◽  
2021 ◽  
Vol 10 (3) ◽  
Author(s):  
Li-Peng Hu ◽  
Kai-Xia Zhou ◽  
Yan-Miao Huo ◽  
De-Jun Liu ◽  
Qing Li ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Treatment Strategies ◽  
Clinical Analysis ◽  
Ductal Adenocarcinoma ◽  
Mechanistic Study ◽  
Hsp90 Inhibition ◽  
Single Cell Rna Sequencing

AbstractPancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, which lacks effective treatment strategies. There is an urgent need for the development of new strategies for PDAC therapy. The genetic and phenotypic heterogeneity of PDAC cancer cell populations poses further challenges in the clinical management of PDAC. In this study, we performed single-cell RNA sequencing to characterize PDAC tumors from KPC mice. Functional studies and clinical analysis showed that PDAC cluster 2 cells with highly Hsp90 expression is much more aggressive than the other clusters. Genetic and pharmacologic inhibition of Hsp90 impaired tumor cell growth both in vitro and in vivo. Further mechanistic study revealed that HSP90 inhibition disrupted the interaction between HSP90 and OPA1, leading to a reduction in mitochondrial cristae amount and mitochondrial energy production. Collectively, our study reveals that HSP90 might be a potential therapeutic target for PDAC.

EPCO-10. INTRATUMORAL HETEROGENEITY OF ENVIRONMENT-INDUCED EXPRESSION PROGRAMS IN GLIOMA PATIENTS AND DERIVED MODEL SYSTEMS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi3-vi3
Author(s):  
Nicholas Bayley ◽  
Christopher Tse ◽  
Henan Zhu ◽  
Lisa Ta ◽  
Lynn Baufeld ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Metabolic Flux ◽  
Model Systems ◽  
Molecular Heterogeneity ◽  
Cellular Interactions ◽  
Tumor Subtypes ◽  
Single Cell Rna Sequencing

Abstract Bulk tumor and single-cell RNA sequencing have revealed the remarkable molecular heterogeneity and plasticity of gliomas. This has led to the creation of tumor subtypes and cellular states describing inter- and intra-tumoral heterogeneity, respectively. While there has been great interest in creating and revising new classifications, the biological reasons for this degree of heterogeneity and the selective pressures driving it remain to be fully described. The brain tumor microenvironment (TME) is a complex, regionally heterogeneous ecosystem of communicating normal and malignant cell types and scavenge-able nutrients and metabolites. We hypothesized that distinct cellular interactions and metabolic flux in the TME may drive the inter- and intra-tumoral heterogeneity of gliomas. To identify tumorigenic programs impacted by environmental context we performed bulk RNA sequencing of over 35 triplets of patient glioma samples and their matched derivative models established in direct orthotopic mouse xenografts (DPDOX) and conventional gliomasphere cultures (GS). This analysis revealed environment-specific programs including in vivo immune and neuroglial signaling, in vitro lipid metabolism, and cell migration altered in model systems. These environmental programs are enriched in specific tumor subtypes and neuroglial signaling programs lost in vitro are dynamically upregulated upon re-transplantation in vivo. To further investigate associations between tumor cellular state and environment-driven programs we performed single-cell RNA sequencing of 3 patient and model system triplets. By annotating with brain cell atlases from previous single-cell characterizations, 6 major clusters and over 20 sub-clusters of tumor cell states and hybrid states were identified. Overlaying results from bulk sequencing revealed cell state-specific expression of environment-induced programs. Further, these cellular state “niches” diverge in model environments suggesting that environmental factors modulate not only the composition of cellular states but also their biological roles within gliomas.

scholarly journals Functionally heterogeneous human satellite cells identified by single cell RNA sequencing

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Emilie Barruet ◽  
Steven M Garcia ◽  
Katharine Striedinger ◽  
Jake Wu ◽  
Solomon Lee ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Satellite Cell ◽  
Satellite Cells ◽  
Cell Pool ◽  
Single Cell Rna Sequencing ◽  
Cell Subpopulations ◽  
Functional Relevance

Although heterogeneity is recognized within the murine satellite cell pool, a comprehensive understanding of distinct subpopulations and their functional relevance in human satellite cells is lacking. We used a combination of single cell RNA sequencing and flow cytometry to identify, distinguish, and physically separate novel subpopulations of human PAX7+ satellite cells (Hu-MuSCs) from normal muscles. We found that, although relatively homogeneous compared to activated satellite cells and committed progenitors, the Hu-MuSC pool contains clusters of transcriptionally distinct cells with consistency across human individuals. New surface marker combinations were enriched in transcriptional subclusters, including a subpopulation of Hu-MuSCs marked by CXCR4/CD29/CD56/CAV1 (CAV1+). In vitro, CAV1+ Hu-MuSCs are morphologically distinct, and characterized by resistance to activation compared to CAV1- Hu-MuSCs. In vivo, CAV1+ Hu-MuSCs demonstrated increased engraftment after transplantation. Our findings provide a comprehensive transcriptional view of normal Hu-MuSCs and describe new heterogeneity, enabling separation of functionally distinct human satellite cell subpopulations.

scholarly journals Single-cell RNA sequencing reveals in vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2020 ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Phenotypic Profile ◽  
Single Cell Rna Sequencing

ABSTRACTThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we used single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induced transcriptional shifts by antigenic stimulation in vitro and took advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allowed identification of SARS-CoV-2-reactive TCRs and revealed phenotypic effects introduced by antigen-specific stimulation. We characterized transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and showed correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

Sign in / Sign up

Export Citation Format

Share Document