Single-cell analysis of human PBMCs in healthy and type 2 diabetes populations: dysregulated immune networks in type 2 diabetes unveiled through single-cell profiling

Abstract

Abnormalities in glucose metabolism that precede the onset of type 2 diabetes (T2D) activate immune cells, leading to elevated inflammatory factors and chronic inflammation. However, no single-cell RNA sequencing (scRNA-seq) studies have characterized the properties and networks of individual immune cells in T2D. Here, we analyzed peripheral blood mononuclear cells (PBMCs) from non-diabetes and T2D patients by scRNA-seq. We found that CD14 monocytes in T2D patients were in a pro-inflammatory state and intermediate monocytes expressed more MHC class II genes. In T2D patients, cytotoxic CD4 T cells, effector memory CD8 T cells, and γδ T cells have increased cytotoxicity and clonal expansion. B cells were characterized by increased differentiation into intermediate B cells, plasma cells, and isotype class switching with increased expression of soluble antibody genes. These results suggest that monocytes, T cells, and B cells could interact to induce chronic inflammation in T2D patients with pro-inflammatory characteristics.

Keywords: T cell; cell interaction; monocyte; pro-inflammatory characteristics; single-cell RNA sequencing; type 2 diabetes.

Figure 1

scRNA-seq identified Type 2 diabetes immune cell atlas. (A) Schematic representation of experimental design and techniques used in this study. 34 non-diabetes PBMC samples and 37 type 2 diabetes patients were collected for analysis. (B) UMAP plot of 293,923 PBMC cells shows the major cells types. Each dot represents a single cells, and colors represent different cell types. (C) Dot plot of mean expression of canonical markers across the cell types. (D) The proportion of global cell clusters depending on normal or diabetes using bar plot. (E) The proportion of global cell clusters depending on normal or diabetes using box plot. N.S. not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

T2D monocytes have increased expression of inflammatory molecules and antigen-presenting genes. (A) The UMAP visualization shows the distribution of 56,250 myeloid cells, revealing the presence of six distinct monocyte cell clusters, as well as clusters representing dendritic cells. (B) The expression of myeloid cell marker genes is depicted in the UMAP plot, highlighting their expression patterns across the identified myeloid clusters. (C) The proportion of myeloid clusters depending on normal or diabetes (D) The expression pattern of cytokine gene and MHC class II genes in monocytes. (E) Distributions of CD14 Mono pro-inflammation module score with respect to normal and diabetes. (F) Different patterns heatmap between non-diabetes and T2D patients by pro-inflammatory genes (CXCL8, CCL2, CCL3, CCL5, IL1B, CXCL9, and CXCL10) using pseudobulk differential expression analysis (G) The top 10 enriched biological processes by GO analysis of upregulated DEGs of CD14 Mono of diabetes and normal group. Dot color indicates the statistical significance of enrichment and dot is in proportion of gene ratio of the enriched gene number. (H) Heatmap showing the differential number of interactions between non-diabetes and T2D patient. In the color bar, red (or blue) represents increased (or decreased) signaling in the T2D patient compared to non-diabetes. (I) Scatter plots comparing the outgoing and incoming interaction strength in the 2D space among each cell population. (J) Bubble chart of cell to cell signaling in non-diabetes and T2D patient. N.S. not significant, **p < 0.01, ***p < 0.001.

Figure 3

T2D enhanced cytotoxicity and clonal expansions of cytotoxic T cells. (A) The UMAP visualization shows the distribution of 21,871 T cells, revealing the presence of five distinct T cell clusters, as well as clusters representing NK cells. (B) Dot plot of mean expression of canonical markers across the cell types. (C) The proportion of T cell clusters depending on normal or diabetes. (D) Distributions of CD4_Tcyt, CD8_Tem cytotoxic and gdT module score with respect to normal and diabetes. (E) Distributions of CD8_Tem KLRG1 expression score with respect to normal and diabetes. (F) The UMAP plot specifically focuses on the visualization of marked TCR abundance. The circle size represents the log(Abundance) and color represent normal and diabetes. (G) Distributions of Gini index score of cytotoxic T cells (CD4_Tcyt and CD8_Tem) depending on normal and diabetes. N.S. not significant, *p < 0.05, ***p < 0.001.

Figure 4

Increased Plasma Cell Differentiation and BCR diversity in Diabetes. (A) The UMAP visualization shows the distribution of 21,871 B cells, revealing the presence of five distinct B cell clusters, as well as clusters representing plasma cells and cycling plasma cells. (B) The expression of B cell marker genes is depicted in the UMAP plot, highlighting their expression patterns across the identified B cell clusters. (C) The proportion of B cell clusters depending on normal or diabetes. (D) The UMAP plot specifically focuses on the visualization of marked B cell receptor (BCR) isotypes. The color-coding indicates the presence or absence of matched BCR data, with grey color representing cases where BCR data was not available. (E) The distribution of BCR isotypes within each B cell and plasma cell cluster based on the normal or diabetes. N.S. not significant, *p < 0.05, **p < 0.01, ***p < 0.001.