Publications by authors named "Antoine-Emmanuel Saliba"

27 Publications

  • Page 1 of 1

Time-Resolved scRNA-Seq Tracks the Adaptation of a Sensitive MCL Cell Line to Ibrutinib Treatment.

Int J Mol Sci 2021 Feb 25;22(5). Epub 2021 Feb 25.

Institute of Pathology, University of Würzburg and Comprehensive Cancer Center (CCC) Mainfranken, 97080 Würzburg, Germany.

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 , and metabolic reprogramming towards dependence on oxidative phosphorylation favor resistance to ibrutinib treatment. Targeting these cellular responses provide new therapy options in MCL.
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http://dx.doi.org/10.3390/ijms22052276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956352PMC
February 2021

Initial HCV infection of adult hepatocytes triggers a temporally structured transcriptional program containing diverse pro- and anti-viral elements.

J Virol 2021 Mar 3. Epub 2021 Mar 3.

Division of Veterinary Medicine, Paul Ehrlich Institute, Langen, Germany

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 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.Acute 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 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.
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http://dx.doi.org/10.1128/JVI.00245-21DOI Listing
March 2021

Homozygous BCMA gene deletion in response to anti-BCMA CAR T cells in a patient with multiple myeloma.

Nat Med 2021 Feb 22. Epub 2021 Feb 22.

Department of Internal Medicine 2, University Hospital of Würzburg, Würzburg, Germany.

B cell maturation antigen (BCMA) is a target for various immunotherapies and a biomarker for tumor load in multiple myeloma (MM). We report a case of irreversible BCMA loss in a patient with MM who was enrolled in the KarMMa trial ( NCT03361748 ) and progressed after anti-BCMA CAR T cell therapy. We identified selection of a clone with homozygous deletion of TNFRSF17 (BCMA) as the underlying mechanism of immune escape. Furthermore, we found heterozygous TNFRSF17 loss or monosomy 16 in 37 out of 168 patients with MM, including 28 out of 33 patients with hyperhaploid MM who had not been previously treated with BCMA-targeting therapies, suggesting that heterozygous TNFRSF17 deletion at baseline could theoretically be a risk factor for BCMA loss after immunotherapy.
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http://dx.doi.org/10.1038/s41591-021-01245-5DOI Listing
February 2021

A genome-wide transcriptomic analysis of embryos fathered by obese males in a murine model of diet-induced obesity.

Sci Rep 2021 Jan 21;11(1):1979. Epub 2021 Jan 21.

Institute of Human Genetics, Julius Maximilians University, 97074, Würzburg, Germany.

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.
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http://dx.doi.org/10.1038/s41598-021-81226-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820458PMC
January 2021

Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia.

Nat Cell Biol 2021 02 18;23(2):184-197. Epub 2021 Jan 18.

Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.

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.
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http://dx.doi.org/10.1038/s41556-020-00619-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878191PMC
February 2021

Advances and challenges in single-cell RNA-seq of microbial communities.

Curr Opin Microbiol 2020 10 4;57:102-110. Epub 2020 Nov 4.

Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Center for Infection Research (HZI), Würzburg, Germany. Electronic address:

Microbes have developed complex strategies to respond to their environment and escape the immune system by individualizing their behavior. While single-cell RNA sequencing has become instrumental for studying mammalian cells, its use with fungi, protozoa and bacteria is still in its infancy. In this review, we highlight the major progress towards mapping the molecular states of microbes at the single-cell level using genome-wide transcriptomics and describe how these technologies can be extended to probe thousands of species at high throughput. We envision that mammalian and microbial single-cell profiling could soon be integrated for the study of microbial communities in health and disease.
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http://dx.doi.org/10.1016/j.mib.2020.10.001DOI Listing
October 2020

Dynamics of Cardiac Neutrophil Diversity in Murine Myocardial Infarction.

Circ Res 2020 Oct 19;127(9):e232-e249. Epub 2020 Aug 19.

Comprehensive Heart Failure Center Wuerzburg (G.R., L.K., A.-P.A.-L., V.A.N., D.J.J.S., C.C.), University Hospital Wuerzburg, Germany.

Rationale: After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage.

Objective: To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction.

Methods And Results: We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G (lymphocyte antigen 6G) neutrophils could be delineated into 6 distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils (, , ), and putative activity of transcriptional regulators involved in hypoxic response () and emergency granulopoiesis (). At 3 and 5 days, 2 major subsets of (enriched for eg, and ) and () neutrophils were found. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis in blood and heart revealed that while circulating neutrophils undergo a process of aging characterized by loss of surface CD62L and upregulation of , heart infiltrating neutrophils acquired a unique SiglecF signature. SiglecF neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecF signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecF neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecF neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation.

Conclusions: Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecF signature.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.317200DOI Listing
October 2020

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

Cell 2020 09 5;182(6):1419-1440.e23. Epub 2020 Aug 5.

Department of Infectious Diseases and Respiratory Medicine, Charité, Universitätsmedizin Berlin, Berlin, Germany; German Center for Lung Research (DZL).

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-DRCD11c 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-DR 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.
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http://dx.doi.org/10.1016/j.cell.2020.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405822PMC
September 2020

Single-cell RNA-sequencing reports growth-condition-specific global transcriptomes of individual bacteria.

Nat Microbiol 2020 10 17;5(10):1202-1206. Epub 2020 Aug 17.

Helmholtz Institute for RNA-based Infection Research, Helmholtz-Center for Infection Research, Würzburg, Germany.

Bacteria respond to changes in their environment with specific transcriptional programmes, but even within genetically identical populations these programmes are not homogenously expressed. Such transcriptional heterogeneity between individual bacteria allows genetically clonal communities to develop a complex array of phenotypes, examples of which include persisters that resist antibiotic treatment and metabolically specialized cells that emerge under nutrient-limiting conditions. Fluorescent reporter constructs have played a pivotal role in deciphering heterogeneous gene expression within bacterial populations 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.
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http://dx.doi.org/10.1038/s41564-020-0774-1DOI Listing
October 2020

Eleven grand challenges in single-cell data science.

Genome Biol 2020 02 7;21(1):31. Epub 2020 Feb 7.

Life Sciences and Health, Centrum Wiskunde & Informatica, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1186/s13059-020-1926-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007675PMC
February 2020

Tracheal brush cells release acetylcholine in response to bitter tastants for paracrine and autocrine signaling.

FASEB J 2020 01 22;34(1):316-332. Epub 2019 Nov 22.

Institute of Anatomy and Cell Biology, Saarland University, Homburg, Germany.

For protection from inhaled pathogens many strategies have evolved in the airways such as mucociliary clearance and cough. We have previously shown that protective respiratory reflexes to locally released bacterial bitter "taste" substances are most probably initiated by tracheal brush cells (BC). Our single-cell RNA-seq analysis of murine BC revealed high expression levels of cholinergic and bitter taste signaling transcripts (Tas2r108, Gnat3, Trpm5). We directly demonstrate the secretion of acetylcholine (ACh) from BC upon stimulation with the Tas2R agonist denatonium. Inhibition of the taste transduction cascade abolished the increase in [Ca] in BC and subsequent ACh-release. ACh-release is regulated in an autocrine manner. While the muscarinic ACh-receptors M3R and M1R are activating, M2R is inhibitory. Paracrine effects of ACh released in response to denatonium included increased [Ca] in ciliated cells. Stimulation by denatonium or with Pseudomonas quinolone signaling molecules led to an increase in mucociliary clearance in explanted tracheae that was Trpm5- and M3R-mediated. We show that ACh-release from BC via the bitter taste cascade leads to immediate paracrine protective responses that can be boosted in an autocrine manner. This mechanism represents the initial step for the activation of innate immune responses against pathogens in the airways.
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http://dx.doi.org/10.1096/fj.201901314RRDOI Listing
January 2020

scSLAM-seq reveals core features of transcription dynamics in single cells.

Nature 2019 07 10;571(7765):419-423. Epub 2019 Jul 10.

Institute for Virology and Immunobiology, Julius-Maximilians-University Würzburg, Würzburg, Germany.

Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease. 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 labelling, biochemical nucleoside conversion 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.
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http://dx.doi.org/10.1038/s41586-019-1369-yDOI Listing
July 2019

persisters undermine host immune defenses during antibiotic treatment.

Science 2018 12;362(6419):1156-1160

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK.

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 species by macrophages, nongrowing persisters also occur, but their physiological state is poorly understood. In this work, we show that persisters arising during macrophage infection maintain a metabolically active state. Persisters reprogram macrophages by means of effectors secreted by the pathogenicity island 2 type 3 secretion system. These effectors dampened proinflammatory innate immune responses and induced anti-inflammatory macrophage polarization. Such reprogramming allowed nongrowing 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.
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http://dx.doi.org/10.1126/science.aat7148DOI Listing
December 2018

Genome organization and DNA accessibility control antigenic variation in trypanosomes.

Nature 2018 11 17;563(7729):121-125. Epub 2018 Oct 17.

Department of Veterinary Sciences, Experimental Parasitology, Ludwig-Maximilians-Universität München, Munich, Germany.

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 host. 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 accessibility. 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 folding. 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.
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http://dx.doi.org/10.1038/s41586-018-0619-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784898PMC
November 2018

Neonatally imprinted stromal cell subsets induce tolerogenic dendritic cells in mesenteric lymph nodes.

Nat Commun 2018 09 25;9(1):3903. Epub 2018 Sep 25.

Department Experimental Immunology, Helmholtz Centre for Infection Research, 38124, Braunschweig, Germany.

Gut-draining mesenteric lymph nodes (mLNs) are important for inducing peripheral tolerance towards food and commensal antigens by providing an optimal microenvironment for de novo generation of Foxp3 regulatory T cells (Tregs). We previously identified microbiota-imprinted mLN stromal cells as a critical component in tolerance induction. Here we show that this imprinting process already takes place in the neonatal phase, and renders the mLN stromal cell compartment resistant to inflammatory perturbations later in life. LN transplantation and single-cell RNA-seq uncover stably imprinted expression signatures in mLN fibroblastic stromal cells. Subsetting common stromal cells across gut-draining mLNs and skin-draining LNs further refine their location-specific immunomodulatory functions, such as subset-specific expression of Aldh1a2/3. Finally, we demonstrate that mLN stromal cells shape resident dendritic cells to attain high Treg-inducing capacity in a Bmp2-dependent manner. Thus, crosstalk between mLN stromal and resident dendritic cells provides a robust regulatory mechanism for the maintenance of intestinal tolerance.
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http://dx.doi.org/10.1038/s41467-018-06423-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156403PMC
September 2018

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

Circ Res 2018 06 15;122(12):1675-1688. Epub 2018 Mar 15.

Institute of Experimental Biomedicine, University Hospital Würzburg, Germany (C.C., A.Z.)

Rationale: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood.

Objective: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis.

Methods And Results: Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed and 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 and 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.

Conclusions: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.312513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993603PMC
June 2018

Single-Cell RNA-Seq Reveals the Transcriptional Landscape and Heterogeneity of Aortic Macrophages in Murine Atherosclerosis.

Circ Res 2018 06 15;122(12):1661-1674. Epub 2018 Mar 15.

From the Institute of Experimental Biomedicine (C.C., A.Z.)

Rationale: It is assumed that atherosclerotic arteries contain several macrophage subsets endowed with specific functions. The precise identity of these subsets is poorly characterized as they have been defined by the expression of a restricted number of markers.

Objective: We have applied single-cell RNA sequencing as an unbiased profiling strategy to interrogate and classify aortic macrophage heterogeneity at the single-cell level in atherosclerosis.

Method And Results: We performed single-cell RNA sequencing of total aortic CD45 cells extracted from the nondiseased (chow fed) and atherosclerotic (11 weeks of high-fat diet) aorta of low-density lipoprotein receptor-deficient () mice. Unsupervised clustering singled out 13 distinct aortic cell clusters. Among the myeloid cell populations, resident-like macrophages with a gene expression profile similar to aortic resident macrophages were found in healthy and diseased aortas, whereas monocytes, monocyte-derived dendritic cells, and 2 populations of macrophages were almost exclusively detectable in atherosclerotic aortas, comprising inflammatory macrophages showing enrichment in and previously undescribed TREM2 (triggered receptor expressed on myeloid cells 2) macrophages showing enrichment in . Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these 3 macrophage subsets and monocyte-derived dendritic cells and uncovered putative functions of each cell type. Notably, TREM2 macrophages seemed to be endowed with specialized functions in lipid metabolism and catabolism and presented a gene expression signature reminiscent of osteoclasts, suggesting a role in lesion calcification. TREM2 expression was moreover detected in human lesional macrophages. Importantly, these macrophage populations were present also in advanced atherosclerosis and in aortas, indicating relevance of our findings in different stages of atherosclerosis and mouse models.

Conclusions: These data unprecedentedly uncovered the transcriptional landscape and phenotypic heterogeneity of aortic macrophages and monocyte-derived dendritic cells in atherosclerotic and identified previously unrecognized macrophage populations and their gene expression signature, suggesting specialized functions. Our findings will open up novel opportunities to explore distinct myeloid cell populations and their functions in atherosclerosis.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.312509DOI Listing
June 2018

Tolerogenic Transcriptional Signatures of Steady-State and Pathogen-Induced Dendritic Cells.

Front Immunol 2018 28;9:333. Epub 2018 Feb 28.

Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany.

Dendritic cells (DCs) are key directors of tolerogenic and immunogenic immune responses. During the steady state, DCs maintain T cell tolerance to self-antigens by multiple mechanisms including inducing anergy, deletion, and Treg activity. All of these mechanisms help to prevent autoimmune diseases or other hyperreactivities. Different DC subsets contribute to pathogen recognition by expression of different subsets of pattern recognition receptors, including Toll-like receptors or C-type lectins. In addition to the triggering of immune responses in infected hosts, most pathogens have evolved mechanisms for evasion of targeted responses. One such strategy is characterized by adopting the host's T cell tolerance mechanisms. Understanding these tolerogenic mechanisms is of utmost importance for therapeutic approaches to treat immune pathologies, tumors and infections. Transcriptional profiling has developed into a potent tool for DC subset identification. Here, we review and compile pathogen-induced tolerogenic transcriptional signatures from mRNA profiling data of currently available bacterial- or helminth-induced transcriptional signatures. We compare them with signatures of tolerogenic steady-state DC subtypes to identify common and divergent strategies of pathogen induced immune evasion. Candidate molecules are discussed in detail. Our analysis provides further insights into tolerogenic DC signatures and their exploitation by different pathogens.
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http://dx.doi.org/10.3389/fimmu.2018.00333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835767PMC
March 2019

New RNA-seq approaches for the study of bacterial pathogens.

Curr Opin Microbiol 2017 02 17;35:78-87. Epub 2017 Feb 17.

RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Straße 2, D-97080 Würzburg, Germany; Helmholtz Institute for RNA-based Infection Research, D-97080 Würzburg, Germany. Electronic address:

Understanding how bacteria cause disease requires knowledge of which genes are expressed and how they are regulated during infection. While RNA-seq is now a routine method for gene expression analysis in bacterial pathogens, the past years have also witnessed a surge of novel RNA-seq based approaches going beyond standard mRNA profiling. These include variations of the technique to capture post-transcriptional networks controlled by small RNAs and to discover associated RNA-binding proteins in the pathogen itself. Dual RNA-seq analyzing pathogen and host simultaneously has revealed roles of noncoding RNAs during infection and enabled the correlation of bacterial gene activity with specific host responses. Single-cell RNA-seq studies have addressed how heterogeneity among individual host cells may determine infection outcomes.
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http://dx.doi.org/10.1016/j.mib.2017.01.001DOI Listing
February 2017

Single-cell RNA-seq ties macrophage polarization to growth rate of intracellular Salmonella.

Nat Microbiol 2016 Nov 14;2:16206. Epub 2016 Nov 14.

RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Straße 2, D-97080 Würzburg, Germany.

Intracellular bacterial pathogens can exhibit large heterogeneity in growth rate inside host cells, with major consequences for the infection outcome. If and how the host responds to this heterogeneity remains poorly understood. Here, we combined a fluorescent reporter of bacterial cell division with single-cell RNA-sequencing analysis to study the macrophage response to different intracellular states of the model pathogen Salmonella enterica serovar Typhimurium. The transcriptomes of individual infected macrophages revealed a spectrum of functional host response states to growing and non-growing bacteria. Intriguingly, macrophages harbouring non-growing Salmonella display hallmarks of the proinflammatory M1 polarization state and differ little from bystander cells, suggesting that non-growing bacteria evade recognition by intracellular immune receptors. By contrast, macrophages containing growing bacteria have turned into an anti-inflammatory, M2-like state, as if fast-growing intracellular Salmonella overcome host defence by reprogramming macrophage polarization. Additionally, our clustering approach reveals intermediate host functional states between these extremes. Altogether, our data suggest that gene expression variability in infected host cells shapes different cellular environments, some of which may favour a growth arrest of Salmonella facilitating immune evasion and the establishment of a long-term niche, while others allow Salmonella to escape intracellular antimicrobial activity and proliferate.
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http://dx.doi.org/10.1038/nmicrobiol.2016.206DOI Listing
November 2016

A protocol for the systematic and quantitative measurement of protein-lipid interactions using the liposome-microarray-based assay.

Nat Protoc 2016 06 5;11(6):1021-38. Epub 2016 May 5.

European Molecular Biology Laboratory (EMBL) Structural and Computational Biology Unit, Heidelberg, Germany.

Lipids organize the activity of the cell's proteome through a complex network of interactions. The assembly of comprehensive atlases embracing all protein-lipid interactions is an important challenge that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the protein-lipid interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled protein (either purified proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify protein-lipid interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol-including device preparation, assay and data analysis-takes 6-8 d. This protocol paves the way for protein-lipid interaction screens to be performed on the proteome and lipidome scales.
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http://dx.doi.org/10.1038/nprot.2016.059DOI Listing
June 2016

The systematic analysis of protein-lipid interactions comes of age.

Nat Rev Mol Cell Biol 2015 12 28;16(12):753-61. Epub 2015 Oct 28.

European Molecular Biology Laboratory (EMBL), Structural and Computational Biology Unit and Molecular Medicine Partnership Unit, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

Lipids tailor membrane identities and function as molecular hubs in all cellular processes. However, the ways in which lipids modulate protein function and structure are poorly understood and still require systematic investigation. In this Innovation article, we summarize pioneering technologies, including lipid-overlay assays, lipid pull-down assays, affinity-purification lipidomics and the liposome microarray-based assay (LiMA), that will enable protein-lipid interactions to be deciphered on a systems level. We discuss how these technologies can be applied to the charting of system-wide networks and to the development of new pharmaceutical strategies.
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http://dx.doi.org/10.1038/nrm4080DOI Listing
December 2015

Lipid Cooperativity as a General Membrane-Recruitment Principle for PH Domains.

Cell Rep 2015 Sep 20;12(9):1519-30. Epub 2015 Aug 20.

Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany; Molecular Medicine Partnership Unit, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany. Electronic address:

Many cellular processes involve the recruitment of proteins to specific membranes, which are decorated with distinctive lipids that act as docking sites. The phosphoinositides form signaling hubs, and we examine mechanisms underlying recruitment. We applied a physiological, quantitative, liposome microarray-based assay to measure the membrane-binding properties of 91 pleckstrin homology (PH) domains, the most common phosphoinositide-binding target. 10,514 experiments quantified the role of phosphoinositides in membrane recruitment. For most domains examined, the observed binding specificity implied cooperativity with additional signaling lipids. Analyses of PH domains with similar lipid-binding profiles identified a conserved motif, mutations in which-including some found in human cancers-induced discrete changes in binding affinities in vitro and protein mislocalization in vivo. The data set reveals cooperativity as a key mechanism for membrane recruitment and, by enabling the interpretation of disease-associated mutations, suggests avenues for the design of small molecules targeting PH domains.
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http://dx.doi.org/10.1016/j.celrep.2015.07.054DOI Listing
September 2015

Single-cell RNA-seq: advances and future challenges.

Nucleic Acids Res 2014 Aug 22;42(14):8845-60. Epub 2014 Jul 22.

Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Straße 2, D-97080 Würzburg, Germany

Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Single-cell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNA-seq protocols have been introduced and are reviewed here-each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field.
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http://dx.doi.org/10.1093/nar/gku555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132710PMC
August 2014

A quantitative liposome microarray to systematically characterize protein-lipid interactions.

Nat Methods 2014 Jan 24;11(1):47-50. Epub 2013 Nov 24.

Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

Lipids have a role in virtually all biological processes, acting as structural elements, scaffolds and signaling molecules, but they are still largely under-represented in known biological networks. Here we describe a liposome microarray-based assay (LiMA), a method that measures protein recruitment to membranes in a quantitative, automated, multiplexed and high-throughput manner.
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http://dx.doi.org/10.1038/nmeth.2734DOI Listing
January 2014

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.

Proc Natl Acad Sci U S A 2010 Aug 2;107(33):14524-9. Epub 2010 Aug 2.

Institut Curie, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Unité Mixte de Recherche 168, 75005 Paris, France.

We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.
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http://dx.doi.org/10.1073/pnas.1001515107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930475PMC
August 2010