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| Status |
Public on Dec 01, 2020 |
| Title |
Characterization of the immunologic impact of sarcoidosis in peripheral blood mononuclear cells via single-cell RNA-seq |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Sarcoidosis is a systemic inflammatory disease characterized by infiltration of immune cells into granulomas. Previous gene expression studies using heterogeneous cell mixtures lack insight into cell-type-specific immune dysregulation. We performed the first single-cell RNA-sequencing study of sarcoidosis in peripheral immune cells in 48 patients and controls. Following unbiased clustering, differentially expressed genes were identified for 18 cell types and bioinformatically assessed for function and pathway enrichment. Our results reveal persistent activation of circulating classical monocytes with subsequent upregulation of trafficking molecules. Specifically, classical monocytes upregulated distinct markers of activation including adhesion molecules, pattern recognition receptors, and chemokine receptors, as well as enrichment of immunoregulatory pathways HMGB1, mTOR, and ephrin receptor signaling. Predictive modeling implicated TGFβ and mTOR signaling as drivers of persistent monocyte activation. Additionally, sarcoidosis T cell subsets displayed patterns of dysregulation. CD4 naïve T cells were enriched for markers of apoptosis and Th17/Treg differentiation, while effector T cells showed enrichment of anergy-related pathways. Differentially expressed genes in regulatory T cells suggested dysfunctional p53, cell death, and TNFR2 signaling. Using more sensitive technology and more precise units of measure, we identify cell-type specific, novel inflammatory and regulatory pathways. Based on our findings, we suggest a novel model involving four convergent arms of dysregulation: persistent hyperactivation of innate and adaptive immunity via classical monocytes and CD4 naïve T cells, regulatory T cell dysfunction, and effector T cell anergy. We further our understanding of the immunopathology of sarcoidosis and point to novel therapeutic targets.
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| Overall design |
We performed single-cell RNA-sequencing study of sarcoidosis in peripheral immune cells in 35 patients and 13 controls. Cell capture was performed using the 10x Genomics Chromium system followed by sequencing on an Illumina HiSeq 3000, each according to the respective manufacturer’s protocol. Briefly, peripheral blood mononuclear cells (PBMCs) and neutrophils were collected using LSM (Gibco) and Polymorphprep™ (Alere Technologies, Oslo, Norway). Fresh PBMCs (target 4,000/patient) and neutrophils (target 1,000/patient) were combined and loaded at a concentration of 1000 cells/µL into a 10x Genomics Single Cell A v2 chip into the Chromium controller. Following single cell emulsion generation, uniquely identifiable first strand template cDNA was generated by emulsion PCR and after emulsion was broken, the second cDNA strand was generated and Illumina compatible adapters were ligated. Following qPCR quantification, final libraries were loaded onto single respective lanes of a HiSeq 3000 using read lengths of 26 bp for the first read, 98 bp for the second read, and an 8 base index read. Upon completion of sequencing the raw bcl files were processed using the 10x Genomics Cell Ranger (v3.0.2) informatics pipeline.
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| Contributor(s) |
Montgomery CG, Garman L, Pelikan RC, Glenn SB |
| Citation(s) |
33363531 |
| Submission date |
Jun 06, 2019 |
| Last update date |
Jan 04, 2021 |
| Contact name |
Courtney G Montgomery |
| Organization name |
Oklahoma Medical Research Foundation
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| Department |
Genes and Human Disease Research Program
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| Lab |
Montgomery Lab
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| Street address |
825 NE 13th St
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| City |
Oklahoma City |
| State/province |
OK |
| ZIP/Postal code |
73104 |
| Country |
USA |
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| Platforms (1) |
| GPL21290 |
Illumina HiSeq 3000 (Homo sapiens) |
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| Samples (53)
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| Relations |
| BioProject |
PRJNA547634 |
| SRA |
SRP200695 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE132338_SingleCellExperiment.RDS.gz |
1.0 Gb |
(ftp)(http) |
RDS |
| GSE132338_matrix_colData.txt.gz |
1.5 Mb |
(ftp)(http) |
TXT |
| GSE132338_matrix_counts.mtx.gz |
119.1 Mb |
(ftp)(http) |
MTX |
| GSE132338_matrix_rowData.mtx.gz |
71.4 Kb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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