Single-cell multi-omics reveals the TNF-α activation threshold for Classical Monocytes by studying healthy donors and rheumatoid arthritis patients

Abstract

Introduction: Tumor Necrosis Factor Alpha is a known pro-inflammatory cytokine that plays a key role in the pathogenesis of rheumatoid arthritis. Anti-cytokine therapies targeting Tumor Necrosis Factor Alpha have greatly succeeded in treating rheumatoid arthritis in many patients. Despite these developments, many of the mechanisms of Tumor Necrosis Factor Alpha action have yet to be uncovered.

Methods: In this study, we incubated PBMCs from healthy donors and rheumatoid arthritis patients with Tumor Necrosis Factor Alpha and then performed their single-cell multi-omics analysis via BD Rhapsody.

Results: We have observed that Classical Monocytes have responded to the Tumor Necrosis Factor Alpha stimulation the most and that there was an activational threshold for such response that was dependent on the TNFR2 protein expression level.

Discussion: The profiling of TNFR2 protein expression level on immune cell populations can be a good predictive factor for the assessment of their activation by Tumor Necrosis Factor Alpha.

Keywords: CITE-seq; ScRNA-seq; TNF-α; TNFR1; TNFR2; response to TNF-α; rheumatoid arthritis.

Figure 1

Overview of the experiment. This figure was created via BioRender.

Figure 2

Integrated surface protein and whole transcriptome analysis of the normal (n = 3) and rheumatoid arthritis (n = 3) single peripheral blood mononuclear cells. (A) UMAP plot of the clusters; (B) Stacked bar plot of the percentages of cells per cluster per experimental group, clusters are color-labeled following the subFig A; (C) Feature plots of the surface protein marker expression; (D) Violin plots of the surface protein marker expression per cluster, clusters are color-labeled following the subFig A.

Figure 3

Response to TNF by the healthy donors (n = 3) and rheumatoid arthritis (RA) patients (n = 3) Classical Monocytes without the TNF stimulation. (A) AUCell TNF signaling pathway response detection plot, (B) Pearson correlation analysis of the TNF signaling pathway response activity AUCell scores, cell types, and TNFR1 and TNFR2 protein expression, (C) Violin plots of the TNFR1 and TNFR2 protein expression, (D) Dot plot of the TNF response genes, TNF response AUC scores, and TNF receptor protein expression, mean marker expression values were Z-score transformed, the blue color represents the lowest marker expression whereas the red color represents the maximum marker expression, dot size represents the percentage of Classical Monocytes positive for the marker, samples are split into biogroups by vertical dotted lines, (E) Pearson correlation analysis of the mean TNF signaling pathway response activity AUCell scores and normalized mean TNFR1 and TNFR2 protein expression (PEX) pre- and post TNF incubation for each sample, (F) linear regression of the normalized mean TNFR2 protein expression pre TNF incubation and the mean TNF signaling pathway response activity AUCell scores post TNF incubation, dotted lines depict TNF response activation threshold; (G) conversion between the TNFR2 normalized protein expression and the TNFR2 molecule counts, dotted lines depict the TNF response activation threshold, curve depicts the fitted second-degree polynomial.

Figure 4

Differential gene expression analysis of the Classical Monocytes of healthy controls (n = 3) and rheumatoid arthritis (RA) patients (n = 3). (A) Volcano plot of the differentially expressed genes; (B) Gene Ontology Biological Process overrepresentation analysis of the up-regulated genes. Red corresponds to the lowest q-value, blue corresponds to the highest q-value, and the dot size reflects the percentage of genes in the analysis from the full set of genes in the Gene Ontology Biological Process database; (C) Dot plot of the differentially expressed genes, mean marker expression values were Z-score transformed, the blue color represents the lowest marker expression whereas the red color represents the maximum marker expression, dot size represents the percentage of Classical Monocytes positive for the marker.