Single cell transcriptome sequencing indicates the cellular heterogeneity of small intestine tissue in celiac disease

Abstract

Celiac disease (CeD) is an autoimmune small intestinal disease caused by gluten protein ingestion by genetically susceptible individuals. Genome-wide association studies and transcriptomic data have limited capacity to capture intercellular genetic variations. We aimed to construct a single cell transcriptome spectrum, analyze the immune microenvironment and cellular heterogeneity, discover disease-related specific genes and markers, and explore the pathogenesis of CeD. This study performed single cell RNA sequencing (scRNA-seq) on three small intestine biopsies from patients with CeD and three matched healthy Chinese controls. Immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR) were used to validate potential diagnostic biomarkers of disease-differential genes. A total of 10 cell subpopulations were annotated, including three types of epithelial and stromal cells and seven types of immune cells. IHC revealed a pronounced overexpression of T cell disease-differential genes, TRAT1, BCL11B, and ETS1 in intraepithelial lymphocytes in the CeD group. Further clinical validation using qPCR confirmed that ETS1 (P = 0.010), TRAT1 (P < 0.001), and BCL11B (P = 0.036) were enriched in the CeD small intestinal tissue. The CD28/CTLA-4 pathway regulates the homeostasis of Treg cells. The IFITs family genes may serve as marker genes for antiviral specific CD4⁺ T cell subsets. CeD-derived subsets of CD8⁺ T cells frequently express genes associated with cytotoxicity, including IFNG, GZMK, GZMH, GZMB, SH2D1A, PRF1, and NKG7, as well as genes related to T cell exhaustion, such as PDCD10, CTLA4, TIGIT, PDCD1, and DUSP4. Inflammation and infection pathways were enriched in different cell populations. A single cell expression profile of CeD small intestinal tissue was successfully constructed using scRNA-seq in this study. New biomarkers for CeD-specific histopathology and potential therapeutic targets were discovered, and the biomarkers observed between inflammation and infection pathways were closely related to the onset of CeD.

Keywords: Celiac disease; Immune microenvironment; Marker; Single cell RNA sequencing.

Fig. 1

Single cell expression profiling and cell typing in the small intestine of CeD and Ctrl samples. (A). Schematic representation of the analytical workflow for scRNA-seq in CeD. (B). UMAP clustering scatter plot delineating cell population distributions, encompassing 21,080 cells from Ctrl (n = 3) and CeD (n = 3) small intestinal tissue specimens. (C). Violin plots depicting the expression profiles of marker genes across identified cell types. (D). Barplot depicting the relative abundances of distinct cell populations within each sample. (E). Heatmap of differentially expressed genes of each cell type showing the top 10 genes.

Fig. 2

Presents the single cell landscape of the T-cell compartment in the small intestine for both CeD and Ctrl samples. (A). UMAP visualization of T cell subsets. (B). UMAP visualization of CD4⁺ T cell and CD8⁺ T cell subsets. (C). UMAP visualization illustrating the distribution of T cell subsets between CeD and Ctrl groups. (D). Marker gene expression is depicted for CD4⁺ T cells, CD8⁺ T cells, and Treg cells, with darker colors indicating higher gene expression levels. E. The bubble plot displays KEGG analysis results for differentially expressed genes related to T cell diseases.

Fig. 3

Expression validation for marker genes. (A) Immunohistochemical staining of T cell disease differential genes in small intestine. Magnification: 40×. (B) The expression level of T cell disease differential genes ETS1, TRAT1, and BCL11B in small intestine between CeD and Ctrl groups.

Fig. 4

The expression profile and cell typing of CD4⁺ T cells in small intestine tissue in CeD and Ctrl groups. (A) UMAP clustering scatter plot of CD4⁺ T cell subsets. (B) UMAP clustering scatter plot of the distribution of CD4⁺ T cell subsets among different groups. (C) Bar chart of CD4⁺ T cell subsets proportion in each sample. (D) Violin diagram of CD4⁺ T cell subsets marker gene. (E) GO analysis of C3_CD4⁺ T cell subsets. (F) GO analysis of C4_CD4⁺ T cell subsets.

Fig. 5

The expression profile and cell typing of CD8⁺ T cells in small intestine tissue in CeD and Ctrl groups. (A) UMAP clustering scatter plot of CD8⁺ T cell subsets. (B) UMAP clustering scatter plot of the distribution of CD8⁺ T cell subsets among different groups. (C) Bar chart of CD8⁺ T cell subsets proportion in each sample. (D) Violin diagram of CD8⁺ T cell subsets marker gene. (E) KEGG enrichment analysis of marker genes for the C4_CD8⁺ T cell subsets. (F) KEGG enrichment analysis of marker genes for the C5_CD8⁺ T cell subsets.

Fig. 6

Bubble chart of KEGG analysis for disease differential genes across various cell types. (A) KEGG Analysis Bubble Chart of DEGs in NK Cells. (B) KEGG Analysis Bubble Chart of DEGs in MC Cells. (C) KEGG Analysis Bubble Chart of DEGs in ILC Cells. (D) KEGG Analysis Bubble Chart of DEGs in PC Cells. (E.) KEGG Analysis Bubble Chart of DEGs in B Cells. (F) KEGG Analysis Bubble Chart of DEGs in myeloid cells.

Fig. 7

Heat map of GWAS-related CeD non-HLA susceptibility genes and differentially expressed genes among different cell groups.