Mapping the heterogeneity of pDCs and Monocytes of systemic sclerosis at single cell level [Bulk RNA-seq]

Systemic sclerosis (SSc) is an uncommon disease characterized by elevated autoantibody production, vasculopathy and fibrosis of the skin and internal organs. pDCs are the core cell type to produce type I IFN and contributes to the ISG signature and SSc progression. Using single-cell RNA sequencing, we profiled 32529 pDCs enriched and T cell depleted peripheral blood mononuclear cells (PBMCs) from 4 patients with SSc and 4 matched controls. Increased CD16+ monocytes and decreased cDCs are the major changes in the cell composition between SSc and heathy controls. Increased expression of interferon-stimulated genes (ISGs) and apoptotic genes distinguished cells from patients with SSc from healthy control cells. The high ISG expression signature (ISGhi) derived from a small number of transcriptionally defined subpopulations within major cell types, including monocytes, natural killer cells, conventional and plasmacytoid dendritic cells, B cells. Profiling of 10976 pDCs revealed a newly identified PTGDS+ population and a clear increased ISG hi clusters in SSc pDCs. Profiling of 13317 Monocytes revealed increased CD16+ Monocytes and a clear increased ISG hi clusters in SSc monocytes. We then compared the TLRs-IFN response of the pDCs from SSc and Healthy control. RNASeq revealed SSc pDCs enables an unexpected TLR8-IFN signaling to increase the IFN signature. This study lays the groundwork for resolving the origin of the SSc transcriptional signatures and the disease heterogeneity towards precision medicine applications. Overall design: 40 ml blood were collected from 4 SSc patients and pDCs were purified from PBMCs. 1-3 x 104 purified pDCs were cultured in 96 U-bottom well with tissue culture medium or TLR8L (200 µg/ml) for 6h. Total RNA was isolated using RNeasy Plus Mini kit. NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (NEB E6420S) were used to prepare Illumina-compatible sequencing libraries. Quality of all RNA were evaluated with BioAnalyser 2100 (Agilent). Pair-end reads were obtained on an Illumina HiSeq 4000 in the Weill Cornell Epigenomics Core Facility at the depth of 16-27 million fragments per sample. Sequencing quality was measured with fastp 19. Reads were then mapped reads in genes counted against the human genome (hg38) with STAR aligner and Gencode v21 20, 21. Differential gene (DEGs) expression analysis was performed in R 22 using the edgeR package 23. Genes with low expression levels (< 3 cpm) were filtered from all downstream analyses. The Benjamini–Hochberg false discovery rate procedure was used to calculate the FDR.