scholarly journals Single cell analysis reveals human cytomegalovirus drives latently infected cells towards an anergic-like monocyte state

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Miri Shnayder ◽  
Aharon Nachshon ◽  
Batsheva Rozman ◽  
Biana Bernshtein ◽  
Michael Lavi ◽  
...  
Keyword(s):  
Immune Response ◽  
Single Cell ◽  
Human Cytomegalovirus ◽  
Single Cell Analysis ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Viral Transcript ◽  
Hematopoietic Stem ◽  
Latently Infected ◽  
Stem And Progenitor Cells

Human cytomegalovirus (HCMV) causes a lifelong infection through establishment of latency. Although reactivation from latency can cause life-threatening disease, our molecular understanding of HCMV latency is incomplete. Here we use single cell RNA-seq analysis to characterize latency in monocytes and hematopoietic stem and progenitor cells (HSPCs). In monocytes, we identify host cell surface markers that enable enrichment of latent cells harboring higher viral transcript levels, which can reactivate more efficiently, and are characterized by reduced intrinsic immune response that is important for viral gene expression. Significantly, in latent HSPCs, viral transcripts could be detected only in monocyte progenitors and were also associated with reduced immune-response. Overall, our work indicates that regardless of the developmental stage in which HCMV infects, HCMV drives hematopoietic cells towards a weaker immune-responsive monocyte state and that this anergic-like state is crucial for the virus ability to express its transcripts and to eventually reactivate.

scholarly journals Single-cell RNA-seq reveals a distinct transcriptome signature of aneuploid hematopoietic cells

Blood ◽  
2017 ◽  
Vol 130 (25) ◽  
pp. 2762-2773 ◽  
Author(s):  
Xin Zhao ◽  
Shouguo Gao ◽  
Zhijie Wu ◽  
Sachiko Kajigaya ◽  
Xingmin Feng ◽  
...  
Keyword(s):  
Immune Response ◽  
Single Cell ◽  
Progenitor Cells ◽  
Rna Seq ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Key Points ◽  
Stem And Progenitor Cells ◽  
Diploid Cells ◽  
Transcriptome Signature

Key Points We distinguished aneuploid cells from diploid cells within the hematopoietic stem and progenitor cells using scRNA-seq. Monosomy 7 cells showed downregulated pathways involved in immune response and maintenance of DNA stability.

Single Cell Proteomics Reveals Novel Cytokine-Producing Function of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 26-26
Author(s):  
Jimmy L. Zhao ◽  
Chao Ma ◽  
Ryan O'Connell ◽  
Dinesh S. Rao ◽  
James Heath ◽  
...  
Keyword(s):  
Stem Cells ◽  
Immune Response ◽  
Single Cell ◽  
Progenitor Cells ◽  
Cytokine Production ◽  
Conflicts Of Interest ◽  
Severe Neutropenia ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 26 During infection, hematopoietic stem and progenitor cells (HSPCs) are called upon to proliferate and differentiate to produce more innate and adaptive immune cells to combat infection. Traditionally, HSPCs are thought to respond to depletion of downstream hematopoietic cells during infection. More recent evidence suggests that HSPCs may respond directly to infection and pro-inflammatory cytokines. However, little is known about the direct immune response of HSPCs and the molecular signaling regulating this response upon sensing an infection. In this study, we have combined transgenic and genetic knockout mouse models with a novel single cell barcode proteomics microchip technology to tackle these questions. We show that although long-term hematopoietic stem cells (HSCs) (defined by Lineage-cKit+Sca1+CD150+CD48-) do not secrete cytokines upon toll-like receptor (TLR) stimulation, short-term HSCs and multipotent progenitor cells (MPPs) (defined by Lineage-cKit+Sca1+, referred to as LKS thereafter) can produce copious amounts of cytokines upon direct TLR-4 and TLR-2 stimulation, indicating that LKS cells can directly participate in an immune response by producing a myriad of cytokines, upon a bacterial infection. Within the population of LKS cells we detect multiple functional subsets of cells, specialized in producing myeloid-like, lymphoid-like or both types of cytokines. Moreover, we show that the cytokine production by LKS cells is regulated by the NF-κB activity, as p50-deficient LKS cells show reduced cytokine production while microRNA-146a (miR-146a)-deficient LKS cells show significantly increased cytokine production. As long-term HSCs differentiate, they start to gain effector immune function much earlier than we had originally anticipated. In light of this finding, we should start to view the stepwise differentiation scheme of HSCs, and perhaps all other stem cells, as a strategy to sequentially gain functional capacity, instead of simply losing stemness and self-renewal ability. The remarkable ability of LKS cells to produce copious amounts of cytokines in response to bacteria may provide some protective immunity during severe neutropenia and lymphopenia or in the early stage of HSC transplantation. This study further extends the functions of NF-κB to include the regulation of primitive hematopoietic stem and progenitor cells and provides direct evidence of the bacteria-responding ability of HSPCs through the TLR/NF-κB axis. The single cell barcode proteomics technology can be widely applied to study proteomics of other rare cells or heterogeneous cell population at a single cell level. Disclosures: No relevant conflicts of interest to declare.

scholarly journals HSV-1 single cell analysis reveals anti-viral and developmental programs activation in distinct sub-populations

2019 ◽  
Author(s):  
Nir Drayman ◽  
Parthiv Patel ◽  
Luke Vistain ◽  
Savaş Tay
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Viral Gene Expression ◽  
Population Level ◽  
Cellular Reprogramming ◽  
Viral Gene ◽  
Beta Catenin ◽  
Infected Cells ◽  
Hsv 1

ABSTRACTViral infection is usually studied at the population level by averaging over millions of cells. However, infection at the single-cell level is highly heterogeneous. Here, we combine live-cell imaging and single-cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of primary human cells. We find extreme variability in the level of viral gene expression among individually infected cells and show that they cluster into transcriptionally distinct sub-populations. We find that anti-viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo cellular reprogramming to an embryonic-like transcriptional state. This reprogramming involves the recruitment of beta-catenin to the host nucleus and viral replication compartments and is required for late viral gene expression and progeny production. These findings uncover the transcriptional differences in cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-1.

scholarly journals Functional single-cell genomics of human cytomegalovirus infection

2019 ◽  
Author(s):  
Marco Y. Hein ◽  
Jonathan S. Weissman
Keyword(s):  
High Resolution ◽  
Single Cell ◽  
Human Cytomegalovirus ◽  
Membrane Trafficking ◽  
Fundamental Problem ◽  
Viral Gene Expression ◽  
Host Factors ◽  
Complex Life Cycle ◽  
Viral Gene ◽  
A Genome

Understanding how host factors and hundreds of viral genes or-chestrate the complex life cycle of herpesviruses represents a fundamental problem in virology. Here, we use CRISPR/Cas9-based screening to scan at high-resolution for functional elements in the genome of human cytomegalovirus (HCMV), and to generate a genome-wide mapping of host dependency and restriction factors. Our data reveal an architecture of functional modules in the HCMV genome, and host factor pathways involved in virus adhesion and entry, membrane trafficking, and innate immune response. Single-cell analysis shows that the large majority of cells follow a stereotypical trajectory in viral gene expression space. Perturbation of host factors does not alter this trajectory, but can accelerate or stall progression. Conversely, perturbation of viral factors creates discrete alternate ‘dead-end’ trajectories. Our results reveal a fundamental dichotomy between the roles of host and viral factors in orchestrating viral replication and more generally provide a road map for high-resolution dissection of host-pathogen interactions.

scholarly journals Profiling the Blood Compartment of Hematopoietic Stem Cell Transplant Patients During Human Cytomegalovirus Reactivation

2021 ◽  
Vol 10 ◽  
Author(s):  
Biana Bernshtein ◽  
Aharon Nachshon ◽  
Miri Shnayder ◽  
Lauren Stern ◽  
Selmir Avdic ◽  
...  
Keyword(s):  
Immune Response ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Human Cytomegalovirus ◽  
Digital Pcr ◽  
Cell Transplant ◽  
Viral Transcript ◽  
Peripheral Blood Mononuclear ◽  
Hematopoietic Stem ◽  
Blood Compartment

Human cytomegalovirus (HCMV) is a widespread pathogen establishing a latent infection in its host. HCMV reactivation is a major health burden in immunocompromised individuals, and is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Here we determined HCMV genomic levels using droplet digital PCR in different peripheral blood mononuclear cell (PBMC) populations in HCMV reactivating HSCT patients. This high sensitivity approach revealed that all PBMC populations harbored extremely low levels of viral DNA at the peak of HCMV DNAemia. Transcriptomic analysis of PBMCs from high-DNAemia samples revealed elevated expression of genes typical of HCMV specific T cells, while regulatory T cell enhancers as well as additional genes related to immune response were downregulated. Viral transcript levels in these samples were extremely low, but remarkably, the detected transcripts were mainly immediate early viral genes. Overall, our data indicate that HCMV DNAemia is associated with distinct signatures of immune response in the blood compartment, however it is not necessarily accompanied by substantial infection of PBMCs and the residual infected PBMCs are not productively infected.

scholarly journals A Novel Viral Transcript with Homology to Human Interleukin-10 Is Expressed during Latent Human Cytomegalovirus Infection

2004 ◽  
Vol 78 (3) ◽  
pp. 1440-1447 ◽  
Author(s):  
Christina Jenkins ◽  
Allison Abendroth ◽  
Barry Slobedman
Keyword(s):  
Human Cytomegalovirus ◽  
Mononuclear Cells ◽  
Latent Infection ◽  
Interleukin 10 ◽  
Productive Infection ◽  
Viral Gene ◽  
Immune Recognition ◽  
Viral Transcript ◽  
Latently Infected ◽  
Latent Phase

ABSTRACT Human cytomegalovirus (CMV) establishes latent infections in hematopoietic cells such as granulocyte-macrophage progenitors (GM-Ps). During latency the virus is sequestered in a nonreplicating state, although limited transcriptional activity has been previously reported. In this study we sought to further examine viral gene expression during the latent phase of infection. Using an experimental model of latency, primary human GM-Ps were latently infected with CMV strain Toledo and extracted RNA subjected to reverse transcription-PCR by using CMV gene-specific primers. Using this approach, we detected transcription from the UL111.5A region of the viral genome. This transcription was also detected in GM-Ps latently infected with AD169 and Towne strains, indicating that expression was CMV strain independent. Significantly, we detected UL111.5A-region transcripts in mononuclear cells from healthy bone marrow and mobilized peripheral blood allograft donors, demonstrating expression during natural latent infection. Mapping experiments with RNA extracted from latently infected GM-Ps revealed the expression of a novel UL111.5A region transcript with a splicing pattern that differed from that reported during productive infection of permissive cells. This UL111.5A region transcript expressed during latent infection is predicted to encode a 139-amino-acid protein with homology to the potent immunosuppressor interleukin-10 (IL-10) and to the viral IL-10 homolog that is expressed during productive CMV infection. Expression of a latency-associated cmvIL-10 may confer upon the virus an ability to avoid immune recognition and clearance during the latent phase of infection.

scholarly journals Single-cell analysis at the protein level delineates intracellular signaling dynamic during hematopoiesis

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jinheng Wang ◽  
Chenggong Tu ◽  
Hui Zhang ◽  
Yongliang Huo ◽  
Eline Menu ◽  
...  
Keyword(s):  
Single Cell ◽  
Protein Level ◽  
Intracellular Signaling ◽  
Single Cell Analysis ◽  
Cell Analysis ◽  
Proteomics Data ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Fate Decision ◽  
Substantial Heterogeneity

Abstract Background Hematopoietic stem and progenitor cell (HSPC) subsets in mice have previously been studied using cell surface markers, and more recently single-cell technologies. The recent revolution of single-cell analysis is substantially transforming our understanding of hematopoiesis, confirming the substantial heterogeneity of cells composing the hematopoietic system. While dynamic molecular changes at the DNA/RNA level underlying hematopoiesis have been extensively explored, a broad understanding of single-cell heterogeneity in hematopoietic signaling programs and landscapes, studied at protein level and reflecting post-transcriptional processing, is still lacking. Here, we accurately quantified the intracellular levels of 9 phosphorylated and 2 functional proteins at the single-cell level to systemically capture the activation dynamics of 8 signaling pathways, including EGFR, Jak/Stat, NF-κB, MAPK/ERK1/2, MAPK/p38, PI3K/Akt, Wnt, and mTOR pathways, during mouse hematopoiesis using mass cytometry. Results With fine-grained analyses of 3.2 million of single hematopoietic stem and progenitor cells (HSPCs), and lineage cells in conjunction with multiparameter cellular phenotyping, we mapped trajectories of signaling programs during HSC differentiation and identified specific signaling biosignatures of cycling HSPC and multiple differentiation routes from stem cells to progenitor and lineage cells. We also investigated the recovery pattern of hematopoietic cell populations, as well as signaling regulation in these populations, during hematopoietic reconstruction. Overall, we found substantial heterogeneity of pathway activation within HSPC subsets, characterized by diverse patterns of signaling. Conclusions These comprehensive single-cell data provide a powerful insight into the intracellular signaling-regulated hematopoiesis and lay a solid foundation to dissect the nature of HSC fate decision. Future integration of transcriptomics and proteomics data, as well as functional validation, will be required to verify the heterogeneity in HSPC subsets during HSC differentiation and to identify robust markers to phenotype those HSPC subsets.

scholarly journals Single-cell analysis reveals divergent responses of human dendritic cells to the MVA vaccine

2021 ◽  
Vol 14 (697) ◽  
pp. eabd9720
Author(s):  
Marius Döring ◽  
Kevin De Azevedo ◽  
Guillermo Blanco-Rodriguez ◽  
Francesca Nadalin ◽  
Takeshi Satoh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Innate Immunity ◽  
Dendritic Cells ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Costimulatory Molecule ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Infected Cells ◽  
Innate Immunity Pathway

Modified vaccinia Ankara (MVA) is a live, attenuated human smallpox vaccine and a vector for the development of new vaccines against infectious diseases and cancer. Efficient activation of the immune system by MVA partially relies on its encounter with dendritic cells (DCs). MVA infection of DCs leads to multiple outcomes, including cytokine production, activation of costimulatory molecules for T cell stimulation, and cell death. Here, we examined how these diverse responses are orchestrated in human DCs. Single-cell analyses revealed that the response to MVA infection in DCs was limited to early viral gene expression. In response to the early events in the viral cycle, we found that DCs grouped into three distinct clusters. A cluster of infected cells sensed the MVA genome by the intracellular innate immunity pathway mediated by cGAS, STING, TBK1, and IRF3 and subsequently produced inflammatory cytokines. In response to these cytokines, a cluster of noninfected bystander cells increased costimulatory molecule expression. A separate cluster of infected cells underwent caspase-dependent apoptosis. Induction of apoptosis persisted after inhibition of innate immunity pathway mediators independently of previously described IRF-dependent or replication-dependent pathways and was a response to early MVA gene expression. Together, our study identified multiple mechanisms that underlie the interactions of MVA with human DCs.

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