Single Cell RNA-Seq Analysis Identifies Differentially Expressed Genes of Treg Cell in Early Treatment-Naive Rheumatoid Arthritis By Arsenic Trioxide

Objective: Early treatment-naive rheumatoid arthritis (RA) has defective regulatory T (Treg) cells and increased inflammation response. In this study, we aim to illustrate the regulation of Treg cells in pathogenesis of early rheumatoid arthritis by arsenic trioxide (As2O3). Methods: We studied the effects of As2O3 on gene expression in early treatment-naive RA Treg cells with single cell RNA-seq (scRNA-seq). Treg cells were sorted from peripheral blood mononuclear cells (PBMCs) and purified by fluorescence-activated cell sorting (FACS) and cultured with or without As2O3 (at 0.1 mu M) for 24 h. Total RNA was isolated and sequenced, and functional analysis was performed against the Gene Ontology (GO) database. Results for selected genes were confirmed with RT-qPCR. Results: As2O3 exerts no significant effect on CD4(+) T-cell apoptosis under physical condition, and selectively modulate (CD4+) T cells toward Treg cells not Th17 cells under special polarizing stimulators. As2O3 increased the expression of 200 and reduced that of 272 genes with fold change (FC) 2.0 or greater. Several genes associated with inflammation, Treg-cell activation and differentiation as well as glucose and amino acids metabolism were among the most strongly affected genes. GO function analysis identified top ten ranked significant biological process (BPs), molecular functions (MFs), and cell components (CCs) in treatment and nontreatment Treg cells. In GO analysis, genes involved in the immunoregulation, cell apoptosis and cycle, inflammation, and cellular metabolism were enriched among the significantly affected genes. The KEGG pathway enrichment analysis identified the forkhead box O (FoxO) signal pathway, apoptosis, cytokine-cytokine receptor interaction, cell cycle, nuclear factor-kappa B (NF-kappa B) signaling pathway, tumor necrosis factor alpha (TNF-alpha), p53 signaling pathway, and phosphatidylinositol 3 '-kinase (PI3K)-Akt signaling pathway were involved in the pathogenesis of early treatment-naive RA. Conclusion: This is the first study investigating the genome-wide effects of As2O3 on the gene expression of treatment-naive Treg cells. In addition to clear anti-inflammatory and immunoregulation effects, As2O3 affect amino acids and glucose metabolism in Treg cells, an observation that might be particularly important in the metabolic phenotype of treatment-naive RA.

Automated summary

The study aimed to investigate the regulation of Treg cells in early rheumatoid arthritis pathogenesis through arsenic trioxide (As2O3) and found that As2O3 had significant effects on gene expression in early treatment-naive RA Treg cells. As2O3 modulated CD4(+) T cells towards Treg cells and affected genes associated with inflammation, Treg-cell activation, differentiation, and cellular metabolism. The KEGG pathway analysis identified several pathways involved in the pathogenesis of early treatment-naive RA.