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Tanscriptional profile of primary endothelial cells (HUVEC) in ADA2 depleted setting and effect of Dipyridamole (DPM) treatment

ABSTRACT: Adenosine deaminase 2 deficiency (DADA2) is an inherited disorder which can cause vasculitis of medium-sized blood vessels. The disease is caused by mutations in the CECR1 gene, which encodes a key metabolic enzyme in the purine pathway. It is not clear how DADA2 leads to vasculitis, and there are currently limited treatment options. We, therefore, as a first step aimed to study the altered gene expression profile of primary endothelial cells in ADA2 depleted setting and then assessed the effect of Dipyridamole (DPM), a pharmacological inhibitor of ENT- transporters, on the differentially expressed genes. The whole genome transcriptome analysis revealed a robust induction of an IFNβ-stimulated gene signature in unstimulated siADA2-treated cells. Differentially expressed transcripts in this dataset included genes encoding pro-inflammatory cytokines, chemokines, and innate immune response proteins. Pre-treatment with dipyridamole (DPM), reduced expression of the IFNβ-driven gene signature upon depletion of ADA2.

ORGANISM(S): Homo sapiens

PROVIDER: GSE150540 | GEO | 2020/06/01

REPOSITORIES: GEO

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Project description:Background and methods: Deficiency of adenosine deaminase 2 (DADA2) is the first molecularly described monogenic vasculitis syndrome. DADA2 is caused by autosomal-recessive loss-of-function mutations in the ADA2 gene (previously known as CECR1). Vasculitis, vasculopathy and inflammation are dominant clinical features of this disease; however wide spectrum of clinical manifestations includes immunodeficiency, lymphoproliferation, as well as hematologic manifestations. ADA2 is primarily secreted by stimulated monocytes and macrophages. It has been shown that monocytes differentiation to macrophages, and neutrophils play a pathogenic role in DADA2, but little is known about T lymphocytes in this disease. Results: We performed combined single cell RNA sequencing and TCR sequencing to profile T cell repertoire in ten patients with DADA2. Although there are no significant alterations of T cell subsets, we observed activation of both CD8+ and CD4+ T cells, and inflammatory status in these patients. We did not observe clonal expansion of T cells: majority TCRs are expressed at basal level in patients and healthy donors. TCR usage is individual-specific in patients and not disease-specific, indicating unlikely a common pathogenic background or predisposition to a common pathogen in this cohort of patients. We recognized activation of interferon pathways as signature of T cells, and STAT1 as a hub gene in the gene network of T cell activation and cytotoxicity. Overall, T cells in DADA2 patients have altered cell-cell interactions with monocytes, as compared to that in healthy donors, many of these ligand-receptor interactions likely drive upregulation of STAT1 in both T cells and lymphocytes in patients. Conclusion: In conclusion, our single cell analysis of T cells discloses subsets, clonality and transcriptome of a previously undercharacterized cell type in DADA2. Activation of immune response especially IFN pathways remain the core signature of T cells and monocytes. Modulation of IFN pathway, as well as ligand-receptor pairs between immune cells may contribute a novel and directed therapeutic approach in the treatment of DADA2.

Project description:Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including Enterobactin (Ent). However, Ent is bound by the host protein Lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. In two experiments we treated A549 (lung cancer cell line) cells with Lcn2, Ent, and iron, alone and in combination. In experiment 1, biological duplicates of 4 conditions were used: PBS control, Lcn2, Lcn2+Ent, and Lcn2+Ent+iron. In experiment 2, 4 biological replicates of 4 conditions were used: PBS control, Ent, iron, and Ent+iron. Targets made from the samples were hybridized to Affymetrix Human Gene 1.0 ST arrays to measure transcript abundances. The RMA algorithm was used to estimate transcript levels. Replicate samples were exchangeable, so we fit one-way ANOVA models to log2-transformed data separately to each experiment, and tested for pairwise differences between groups in each experiment, as well as asking if the Ent vs. PBS differences were larger or smaller than the Ent+iron vs. iron differences (Ent by iron interactions). We report results for 29096 probe-sets that were not annotated as positive or negative controls on the array. A supplementary Excel workbook is provided that contains the estimated expression level, some probe-set annotation, and simple statistical analysis for each probe-set. It may be convenient for some users, however obtaining newer probe-set annotation may be advisable. A549 (ATCC CCL-185) cells, a human type II pneumocyte cell line, were cultured in F12K (Invitrogen, Carlsbad, CA) media supplemented with 10% fetal bovine serum (Invitrogen) and 1:100 penicillin streptomycin (Invitrogen). 24-well plates were seeded with A549 cells at a concentration of 35000 cells/well. After two days, cells were weaned from serum and antibiotics overnight. Cells were then stimulated overnight with the indicated combinations of 50 uM Ferric ammonium citrate (FAC) (Sigma, St. Louis, MO), 50 um Ent (Sigma or EMC Microcollections, Tubingen, Germany), and/or 25 uM lipocalin 2 (Lcn2) in F12K media lacking serum or antibiotics. Prior to incubation with cells, siderophore-Lcn2 complexes were prepared by sequential incubation at room temperature of FAC and Ent for 30 minutes followed by addition of Lcn2 and incubation for an additional 30 minutes. In experiment 1, 2 biological replicates of each of these 4 conditions were grown: PBS, 25 uM lipocalin 2 (Lcn2), a combination of 25 uM Lcn2 with 50 uM Enterobactin (Ent), and a combination of Lcn2, Ent and 50 uM Ferric Ammonium Citrate (FAC). In experiment 2, 4 biological replicates of each of these 4 conditions were grown: PBS, 50 uM Ent, 50 uM FAC, and a combination of 50 uM Ent and 50 uM FAC. Cells were harvested and RNA was extracted using an miRNeasy Mini Kit (Qiagen). Biotinylated cDNA targets were prepared according to the Ambion WT expression kit protocol from 150 ng total RNA. There are no batches within each experiment - the samples in each group are exchangeable. Targets were hybridized to Affymetrix Human Gene 1.0 ST arrays. Arrays were scanned and quantified by usual procedures, and transcript abundances for 29096 non-control probe-sets estimated by an RMA algorithm.

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Tanscriptional profile of primary endothelial cells (HUVEC) in ADA2 depleted setting and effect of Dipyridamole (DPM) treatment

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