Publications by authors named "Ehsan Vafadarnejad"

9 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

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

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

The rad2 gene of haloarchaeum Halobacterium salinarum is functional in the repair of ultraviolet light induced DNA photoproducts.

Microbiol Res 2015 Apr 7;173:44-9. Epub 2015 Feb 7.

Department of Agricultural Biotechnology, College of Science, University of Payam-e-Noor, Tehran, Iran.

There are a lot of bacterial and eukaryotic DNA repair gene homologs among sequenced archaeal genomes but there is little information about DNA repair mechanisms and the interaction of involved repair proteins. In order to study DNA repair mechanisms in the third domain of life, we studied these processes in the model archaeon, Halobacterium salinarum. H. salinarum has homologs of eukaryotic nucleotide excision repair genes such as rad2 gene. A functional analysis of rad2 was performed by knocking down of this gene. We introduced an antisense RNA expression vector into the cells and the sensitivity of transformants against ultraviolet light exposure was measured to determine whether rad2 gene performs any role in the repair of the DNA lesions induced by UV light or not. Our data suggests that rad2 is functional in this pathway and knocked down strains were unable to completely repair the UV induced DNA damages. In this study, for the first time antisense RNA is used for functional analysis of a gene in H. salinarum and it is shown that antisense RNA could be used as a reliable genetic tool for understanding of the archaeal genetics.
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http://dx.doi.org/10.1016/j.micres.2015.01.012DOI Listing
April 2015