Date: Sunday, October 21, 2018
Session Type: ACR Concurrent Abstract Session
Session Time: 4:30PM-6:00PM
Background/Purpose: Towards understanding the molecular mechanisms that link maternal anti-Ro antibodies to the development of conduction system disease in a second trimester fetus, single cell (scRNA-seq) and bulk RNA-seq were applied to a fetal heart dying with complete congenital heart block (CHB) and a gestational age-matched healthy heart from an elective termination.
Methods: The CHB heart was obtained from a 20-week fetus identified to have complete block at 19 weeks. The mother (35 y/o Asian with SS on no hydroxychloroquine) declined dexamethasone or IVIG and elected to terminate, thus no exposure to maternal medications confounded interpretation of findings. Both hearts were obtained under identical conditions. Freshly collected single-cell suspensions were generated using a Langendorff preparation with cannulation and perfusion of the aorta with collagenase and trypsin enzymes. Two approaches were taken to mine the transcriptome in the resulting cell suspensions: agnostic evaluation applying 10X Genomics platform-based scRNA-seq and low input RNA-seq of flow sorted cells upon leukocytes (DAPI negative, CD45+) and fibroblasts (DAPI negative, CD45-, podoplanin-positive).
Results: For scRNA-seq, we obtained 2,693 and 5,408 high-quality scRNA-seq profiles from the control and CHB hearts, respectively. We applied a graph-based clustering method and identified 13 and 14 major clusters of cells from the control and CHB hearts, respectively, as visualized by t-distributed stochastic neighbor embedding (t-SNE). Differential gene expression analysis guided by established lineage markers revealed four cardiomyocyte clusters (CM1-CM4), three fibroblast clusters (FB1-FB3), endothelial cells (EC), erythroblasts (EB), macrophages (MAC), dendritic cells (DC), T cells (TC) and B cells (BC). Ranked by abundance, the control heart exhibited CM>FB>EC>MAC>DC>EB, BC, TC; the CHB heart exhibited CM>FB>EC, MAC>TC, BC, EB. The CHB heart also contained natural killer cells (NK) and mast cells (MC, lowest abundance). Given the high abundance of MACs among the immune cells (control:108;CHB:606) and the consistent identification of MACs on histologic analysis of CHB hearts, differential expression analysis demonstrated overexpression of interferon-induced genes (4-fold or greater, i.e. log2(CHB-control)>2) in CHB MACs. In CHB, most cell types expressed high levels of ISG1, IFITM1 and IFITM3, whereas in the control only IFITM3 showed widespread expression. For SIGLEC1, expression was restricted to MACs and was expressed by 18% of CHB MACs and only 6% of control MACs. While the transcriptome using low input RNA-seq of anti-CD45 flow-sorted CHB leukocytes did not allow granular analysis of leukocyte subpopulations, expression of SIGLEC1 and interferon-related genes were increased in CHB versus control. Applying 10X Genomics, proliferating fibroblasts expressed MKI67 and TOP2A in CHB but not control fibroblasts.
Conclusion: This unprecedented opportunity to obtain CHB tissue absent any exposure to maternal medications support scRNA-seq’s utility to survey landscape and heterogeneity not possible with low input RNA-seq of flow-sorted cells. IFN- and SIGLEC1-positive macrophages may contribute to fibrosis.
To cite this abstract in AMA style:Suryawanshi H, Buyon JP, Chang M, Tuschl T, Clancy RM. 10X Genomics-Based Single-Cell RNA-Seq and Low Input RNA-Seq Identify a Transcriptional Landscape Supporting Interferon in the Pathogenesis of Autoimmune-Associated Congenital Heart Block [abstract]. Arthritis Rheumatol. 2018; 70 (suppl 10). https://acrabstracts.org/abstract/10x-genomics-based-single-cell-rna-seq-and-low-input-rna-seq-identify-a-transcriptional-landscape-supporting-interferon-in-the-pathogenesis-of-autoimmune-associated-congenital-heart-block/. Accessed January 27, 2021.
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