• Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry.

      Winkels, Holger; Ehinger, Erik; Vassallo, Melanie; Buscher, Konrad; Dinh, Huy Q; Kobiyama, Kouji; Hamers, Anouk A J; Cochain, Clément; Vafadarnejad, Ehsan; Saliba, Antoine-Emmanuel; et al. (Amercan Heart Association, 2018-03-15)
      Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed Apoe-/- and Ldlr-/- mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe-/- and Ldlr-/- mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients.
    • Einzelzell-RNA-Sequenzierung beleuchtet den Infektionsprozess

      Saliba, Antoine-Emmanuel; Westermann, Alexander J.; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Schneider-Straße 2, 97080 Würzburg. Germany. (2017-10-11)
    • Eleven grand challenges in single-cell data science.

      Lähnemann, David; Köster, Johannes; Szczurek, Ewa; McCarthy, Davis J; Hicks, Stephanie C; Robinson, Mark D; Vallejos, Catalina A; Campbell, Kieran R; Beerenwinkel, Niko; Mahfouz, Ahmed; et al. (BMC, 2020-02-07)
      The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands-or even millions-of cells analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.
    • Genome organization and DNA accessibility control antigenic variation in trypanosomes.

      Müller, Laura S M; Cosentino, Raúl O; Förstner, Konrad U; Guizetti, Julien; Wedel, Carolin; Kaplan, Noam; Janzen, Christian J; Arampatzi, Panagiota; Vogel, Jörg; Steinbiss, Sascha; et al. (2018-01-01)
      Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.
    • A genome-wide transcriptomic analysis of embryos fathered by obese males in a murine model of diet-induced obesity.

      Bernhardt, Laura; Dittrich, Marcus; El-Merahbi, Rabih; Saliba, Antoine-Emmanuel; Müller, Tobias; Sumara, Grzegorz; Vogel, Jörg; Nichols-Burns, Stefanie; Mitchell, Megan; Haaf, Thomas; et al. (Nature Pulishing Group, 2021-01-21)
      Paternal obesity is known to have a negative impact on the male's reproductive health as well as the health of his offspring. Although epigenetic mechanisms have been implicated in the non-genetic transmission of acquired traits, the effect of paternal obesity on gene expression in the preimplantation embryo has not been fully studied. To this end, we investigated whether paternal obesity is associated with gene expression changes in eight-cell stage embryos fathered by males on a high-fat diet. We used single embryo RNA-seq to compare the gene expression profile of embryos generated by males on a high fat (HFD) versus control (CD) diet. This analysis revealed significant upregulation of the Samd4b and Gata6 gene in embryos in response to a paternal HFD. Furthermore, we could show a significant increase in expression of both Gata6 and Samd4b during differentiation of stromal vascular cells into mature adipocytes. These findings suggest that paternal obesity may induce changes in the male germ cells which are associated with the gene expression changes in the resulting preimplantation embryos.
    • Initial HCV infection of adult hepatocytes triggers a temporally structured transcriptional program containing diverse pro- and anti-viral elements.

      Tegtmeyer, Birthe; Vieyres, Gabrielle; Todt, Daniel; Lauber, Chris; Ginkel, Corinne; Engelmann, Michael; Herrmann, Maike; Pfaller, Christian K; Vondran, Florian W R; Broering, Ruth; et al. (ASM, 2021-03-03)
      Transcriptional profiling provides global snapshots of virus-mediated cellular reprogramming, which can simultaneously encompass pro- and antiviral components. To determine early transcriptional signatures associated with HCV infection of authentic target cells, we performed ex vivo infections of adult primary human hepatocytes (PHHs) from seven donors. Longitudinal sampling identified minimal gene dysregulation at six hours post infection (hpi). In contrast, at 72 hpi, massive increases in the breadth and magnitude of HCV-induced gene dysregulation were apparent, affecting gene classes associated with diverse biological processes. Comparison with HCV-induced transcriptional dysregulation in Huh-7.5 cells identified limited overlap between the two systems. Of note, in PHHs, HCV infection initiated broad upregulation of canonical interferon (IFN)-mediated defense programs, limiting viral RNA replication and abrogating virion release. We further find that constitutive expression of IRF1 in PHHs maintains a steady-state antiviral program in the absence of infection, which can additionally reduce HCV RNA translation and replication. We also detected infection-induced downregulation of ∼90 genes encoding components of the EIF2 translation initiation complex and ribosomal subunits in PHHs, consistent with a signature of translational shutoff. As HCV polyprotein translation occurs independently of the EIF2 complex, this process is likely pro-viral: only translation initiation of host transcripts is arrested. The combination of antiviral intrinsic and inducible immunity, balanced against pro-viral programs, including translational arrest, maintains HCV replication at a low-level in PHHs. This may ultimately keep HCV under the radar of extra-hepatocyte immune surveillance while initial infection is established, promoting tolerance, preventing clearance and facilitating progression to chronicity.IMPORTANCEAcute HCV infections are often asymptomatic and therefore frequently undiagnosed. We endeavored to recreate this understudied phase of HCV infection using explanted PHHs and monitored host responses to initial infection. We detected temporally distinct virus-induced perturbations in the transcriptional landscape, which were initially narrow but massively amplified in breadth and magnitude over time. At 72 hpi, we detected dysregulation of diverse gene programs, concurrently promoting both virus clearance and virus persistence. On the one hand, baseline expression of IRF1 combined with infection-induced upregulation of IFN-mediated effector genes suppresses virus propagation. On the other, we detect transcriptional signatures of host translational inhibition, which likely reduces processing of IFN-regulated gene transcripts and facilitates virus survival. Together, our data provide important insights into constitutive and virus-induced transcriptional programs in PHHs, and identifies simultaneous antagonistic dysregulation of pro-and anti-viral programs which may facilitate host tolerance and promote viral persistence.
    • Letter by Cochain et al Regarding Article, "Transcriptome Analysis Reveals Nonfoamy Rather Than Foamy Plaque Macrophages Are Proinflammatory in Atherosclerotic Murine Models".

      Cochain, Clément; Saliba, Antoine-Emmanuel; Zernecke, Alma; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
    • Longitudinal Multi-omics Analyses Identify Responses of Megakaryocytes, Erythroid Cells, and Plasmablasts as Hallmarks of Severe COVID-19.

      Bernardes, Joana P; Mishra, Neha; Tran, Florian; Bahmer, Thomas; Best, Lena; Blase, Johanna I; Bordoni, Dora; Franzenburg, Jeanette; Geisen, Ulf; Josephs-Spaulding, Jonathan; et al. (Elsevier (Cell Press), 2020-11-26)
      Temporal resolution of cellular features associated with a severe COVID-19 disease trajectory is needed for understanding skewed immune responses and defining predictors of outcome. Here, we performed a longitudinal multi-omics study using a two-center cohort of 14 patients. We analyzed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. Validation was performed in two independent cohorts of COVID-19 patients. Severe COVID-19 was characterized by an increase of proliferating, metabolically hyperactive plasmablasts. Coinciding with critical illness, we also identified an expansion of interferon-activated circulating megakaryocytes and increased erythropoiesis with features of hypoxic signaling. Megakaryocyte- and erythroid-cell-derived co-expression modules were predictive of fatal disease outcome. The study demonstrates broad cellular effects of SARS-CoV-2 infection beyond adaptive immune cells and provides an entry point toward developing biomarkers and targeted treatments of patients with COVID-19.
    • Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia.

      Chumduri, Cindrilla; Gurumurthy, Rajendra Kumar; Berger, Hilmar; Dietrich, Oliver; Kumar, Naveen; Koster, Stefanie; Brinkmann, Volker; Hoffmann, Kirstin; Drabkina, Marina; Arampatzi, Panagiota; et al. (Nature research, 2021-01-18)
      The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.
    • Salmonella persisters undermine host immune defenses during antibiotic treatment.

      Stapels, Daphne A C; Hill, Peter W S; Westermann, Alexander J; Fisher, Robert A; Thurston, Teresa L; Saliba, Antoine-Emmanuel; Blommestein, Isabelle; Vogel, Jörg; Helaine, Sophie; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (American Association for the Advancement of Science, 2018-12-07)
      Many bacterial infections are hard to treat and tend to relapse, possibly due to the presence of antibiotic-tolerant persisters. In vitro, persister cells appear to be dormant. After uptake of Salmonella species by macrophages, nongrowing persisters also occur, but their physiological state is poorly understood. In this work, we show that Salmonella persisters arising during macrophage infection maintain a metabolically active state. Persisters reprogram macrophages by means of effectors secreted by the Salmonella pathogenicity island 2 type 3 secretion system. These effectors dampened proinflammatory innate immune responses and induced anti-inflammatory macrophage polarization. Such reprogramming allowed nongrowing Salmonella cells to survive for extended periods in their host. Persisters undermining host immune defenses might confer an advantage to the pathogen during relapse once antibiotic pressure is relieved.
    • scSLAM-seq reveals core features of transcription dynamics in single cells.

      Erhard, Florian; Baptista, Marisa A P; Krammer, Tobias; Hennig, Thomas; Lange, Marius; Arampatzi, Panagiota; Jürges, Christopher S; Theis, Fabian J; Saliba, Antoine-Emmanuel; Dölken, Lars; et al. (Springer-Nature, 2019-01-01)
      Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease1. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further key limitation of scRNA-seq analysis is that the RNA profile of each individual cell can be analysed only once. Here we introduce single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing (scSLAM-seq), which integrates metabolic RNA labelling2, biochemical nucleoside conversion3 and scRNA-seq to record transcriptional activity directly by differentiating between new and old RNA for thousands of genes per single cell. We use scSLAM-seq to study the onset of infection with lytic cytomegalovirus in single mouse fibroblasts. The cell-cycle state and dose of infection deduced from old RNA enable dose-response analysis based on new RNA. scSLAM-seq thereby both visualizes and explains differences in transcriptional activity at the single-cell level. Furthermore, it depicts 'on-off' switches and transcriptional burst kinetics in host gene expression with extensive gene-specific differences that correlate with promoter-intrinsic features (TBP-TATA-box interactions and DNA methylation). Thus, gene-specific, and not cell-specific, features explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations.
    • Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

      Schulte-Schrepping, Jonas; Reusch, Nico; Paclik, Daniela; Baßler, Kevin; Schlickeiser, Stephan; Zhang, Bowen; Krämer, Benjamin; Krammer, Tobias; Brumhard, Sophia; Bonaguro, Lorenzo; et al. (Elsevier /Cell Press), 2020-08-05)
      Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
    • Single-cell RNA-sequencing reports growth-condition-specific global transcriptomes of individual bacteria.

      Imdahl, Fabian; Vafadarnejad, Ehsan; Homberger, Christina; Saliba, Antoine-Emmanuel; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Nature research, 2020-08-17)
      Bacteria respond to changes in their environment with specific transcriptional programmes, but even within genetically identical populations these programmes are not homogenously expressed1. Such transcriptional heterogeneity between individual bacteria allows genetically clonal communities to develop a complex array of phenotypes1, examples of which include persisters that resist antibiotic treatment and metabolically specialized cells that emerge under nutrient-limiting conditions2. Fluorescent reporter constructs have played a pivotal role in deciphering heterogeneous gene expression within bacterial populations3 but have been limited to recording the activity of single genes in a few genetically tractable model species, whereas the vast majority of bacteria remain difficult to engineer and/or even to cultivate. Single-cell transcriptomics is revolutionizing the analysis of phenotypic cell-to-cell variation in eukaryotes, but technical hurdles have prevented its robust application to prokaryotes. Here, using an improved poly(A)-independent single-cell RNA-sequencing protocol, we report the faithful capture of growth-dependent gene expression patterns in individual Salmonella and Pseudomonas bacteria across all RNA classes and genomic regions. These transcriptomes provide important reference points for single-cell RNA-sequencing of other bacterial species, mixed microbial communities and host-pathogen interactions.
    • Time-Resolved scRNA-Seq Tracks the Adaptation of a Sensitive MCL Cell Line to Ibrutinib Treatment.

      Fuhr, Viktoria; Vafadarnejad, Ehsan; Dietrich, Oliver; Arampatzi, Panagiota; Riedel, Angela; Saliba, Antoine-Emmanuel; Rosenwald, Andreas; Rauert-Wunderlich, Hilka; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (MDPI, 2021-02-25)
      Since the approval of ibrutinib for relapsed/refractory mantle cell lymphoma (MCL), the treatment of this rare mature B-cell neoplasm has taken a great leap forward. Despite promising efficacy of the Bruton tyrosine kinase inhibitor, resistance arises inevitably and the underlying mechanisms remain to be elucidated. Here, we aimed to decipher the response of a sensitive MCL cell line treated with ibrutinib using time-resolved single-cell RNA sequencing. The analysis uncovered five subpopulations and their individual responses to the treatment. The effects on the B cell receptor pathway, cell cycle, surface antigen expression, and metabolism were revealed by the computational analysis and were validated by molecular biological methods. The observed upregulation of B cell receptor signaling, crosstalk with the microenvironment, upregulation of CD52, and metabolic reprogramming towards dependence on oxidative phosphorylation favor resistance to ibrutinib treatment. Targeting these cellular responses provide new therapy options in MCL.