Molecular Signatures of Dendritic Cell Activation upon TNF Stimulation: A Multi-Omics Study in Rheumatoid Arthritis

Abstract

Dendritic cells (DCs) play a central role in the immunopathogenesis of rheumatoid arthritis (RA), yet their regulation by tumor necrosis factor alpha (TNF) and associated receptors remains poorly characterized. We applied a single-cell multi-omics approach (CITE-seq) to profile peripheral blood mononuclear cells (PBMCs) from RA patients and healthy donors, before and after in vitro TNF stimulation. Using integrated analysis of surface protein expression and transcriptomic data, we focused on phenotypic and transcriptional changes in dendritic cell populations. DCs from RA patients exhibited elevated surface expression of CD14 and CD16, indicative of an inflammatory phenotype, and showed marked responsiveness to TNF. Upon stimulation, RA-derived DCs upregulated genes involved in antigen presentation (CD83, LAMP3), lymph node migration (CCR7, ADAM19), and inflammation (TRAF1, IL24) whereas such activation was absent in healthy controls. Our data reveal a TNF-responsive, pro-inflammatory transcriptional program in dendritic cells from RA patients and underscore the relevance of the TNF receptor profile in shaping DC function. These findings provide new insights into the immunobiology of RA and identify dendritic cells as potential targets for personalized immunomodulatory therapy.

Keywords: CITE-seq; TNF-alpha; TNFR1; TNFR2; dendritic cells; immune regulation; inflammation; multi-omics; rheumatoid arthritis; single-cell.

Figure 3

Differential gene expression analysis. (A) Volcano plot showing differential gene expression between TNF-stimulated and unstimulated dendritic cells. Green dots represent genes with |log₂ fold change| < 0.58; blue dots correspond to genes with p-value > 0.05; red dots indicate significantly up- or downregulated genes with |log₂ fold change| > 0.58 and adjusted p-value < 0.05; grey dots represent non-significant changes. (B) Heatmap displaying the expression levels of differentially expressed genes across experimental groups. Red indicates higher expression; blue indicates lower expression. (C) Violin plots showing the expression dynamics of selected genes in dendritic cells from RA patients (Rheu), before and after TNF stimulation. Norm—healthy donors; Rheu—rheumatoid arthritis patients; TNF—tumor necrosis factor alpha.

Figure 1

Identification of the dendritic cell cluster and analysis of TNF receptor expression. (A) UMAP projection of all cells, highlighting the dendritic cell cluster in red. (B) UMAP projection showing the distribution of dendritic cells across experimental groups. (C) Violin plots showing surface protein expression levels of TNFR1p and TNFR2p (top panel) and transcript levels of TNFRSF1A and TNFRSF1B (bottom panel) in dendritic cells from each group. (D) Violin plot of TNFR2/TNFR1 surface protein expression ratio in dendritic cells from RA patients (Rheu) and healthy donors (Norm), before and after TNF stimulation (TNF).

Figure 2

Analysis of relative TNFR1p and TNFR2p expression. Violin plots showing the TNFR2/TNFR1 surface expression ratio across immune cell types in the healthy donor group (A) and in the rheumatoid arthritis patient group (B).