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Objective:  Early treatment-naïve 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 (As 2 O 3 ).    Methods:  We studied the effects of As 2 O 3  on gene expression in early treatment-naïve 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 As 2 O 3  (at 0.1 µM) 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:  As 2 O 3  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. As 2 O 3  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-κB) signaling pathway, tumor necrosis factor α (TNF-α), p53 signaling pathway, and phosphatidylinositol 3′-kinase (PI3K)-Akt signaling pathway were involved in the pathogenesis of early treatment-naïve RA.    Conclusion:  This is the first study investigating the genome-wide effects of As 2 O 3  on the gene expression of treatment-naïve Treg cells. In addition to clear anti-inflammatory and immunoregulation effects, As 2 O 3  affect amino acids and glucose metabolism in Treg cells, an observation that might be particularly important in the metabolic phenotype of treatment-naïve RA.

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