Integrative analysis of single-cell RNA-seq and gut microbiome metabarcoding data elucidates macrophage dysfunction in mice with DSS-induced ulcerative colitis

Abstract

Ulcerative colitis (UC) is a significant inflammatory bowel disease caused by an abnormal immune response to gut microbes. However, there are still gaps in our understanding of how immune and metabolic changes specifically contribute to this disease. Our research aims to address this gap by examining mouse colons after inducing ulcerative colitis-like symptoms. Employing single-cell RNA-seq and 16 s rRNA amplicon sequencing to analyze distinct cell clusters and microbiomes in the mouse colon at different time points after induction with dextran sodium sulfate. We observe a significant reduction in epithelial populations during acute colitis, indicating tissue damage, with a partial recovery observed in chronic inflammation. Analyses of cell-cell interactions demonstrate shifts in networking patterns among different cell types during disease progression. Notably, macrophage phenotypes exhibit diversity, with a pronounced polarization towards the pro-inflammatory M1 phenotype in chronic conditions, suggesting the role of macrophage heterogeneity in disease severity. Increased expression of Nampt and NOX2 complex subunits in chronic UC macrophages contributes to the inflammatory processes. The chronic UC microbiome exhibits reduced taxonomic diversity compared to healthy conditions and acute UC. The study also highlights the role of T cell differentiation in the context of dysbiosis and its implications in colitis progression, emphasizing the need for targeted interventions to modulate the inflammatory response and immune balance in colitis.

Fig. 1. Single-cell RNA-seq profiling of epithelial cell in DSS-induced mouse colitis.

a Mice were treated with 3% DSS for 6 days and evaluated at day 11 for acute colitis. The relative body weight of mice treated with 3% DSS and DW. Colitis scores were determined based on clinical parameters such as weight loss, stool consistency, and bleeding. b Histological appearance of the colon at day 11 in mice with 3% DSS compared with DW. c Scheme of the chronic colitis (CC) model induced by 3% DSS. Relative body weight changes during the experiment were measured. Colitis scores were determined based on clinical parameters such as weight loss, stool consistency, and bleeding. d Histological appearance of the colon at the endpoint of the experiment in mice with 3% DSS compared with DW. e UMAP plots displaying cell populations from all participant mice colored by major cell type (upper plot) and by condition (bottom panel; HC indicates healthy conditions, AC acute colitis, and CC chronic colitis. f Bar plots showing proportion of major cell type in each condition. g Dot plot depicting the expression profiles of cell barrier-related genes in epithelial cell population. h Western blotting results showing the protein level expression of intestinal barrier status markers. DW distilled water, DSS dextran sodium sulfate. The uncropped/unedited blot images were added in the Supplementary Information (Supplementary Fig. S5).

Fig. 2. Inferred intercellular interactions in DSS-induced UC.

a Circle plots showing the number of interactions between different cell types. Linewidth corresponds to the number of interactions while line color is the source cell type. b–d Ligand-receptor pairs detected with CellPhoneDB are displayed in a bubble plot for HC (B), AC (C) and CC (D). In b–d, the illustrations on the left were created with BioRender.com.

Fig. 3. Analysis of myeloid cell subsets in healthy and DSS-induced UC.

a UMAP plot of scRNA-seq data for myeloid cell clusters in HC (n = 3), AC (n = 3), and CC (n = 4). b Bar plot showing cell type enrichment analysis. c Dot blot showing expression of DEGs for M1 macrophages in HC, AC, and CC. d Violin plots of Il1b expression in heterogenous polarized macrophages in AC, CC, and HC. e Western blotting result showing the IL-1β protein level expression in macrophages and colon tissue lysates. f Functional annotation enrichment analyses of upregulated genes in AC and CC. The uncropped/unedited blot images were added in the Supplementary Information (Supplementary Fig. S5).

Fig. 4. Enhancement of Nampt and NOX2-complex association led to IL-1β levels in both AC and CC macrophages.

a Schematic diagram of eNAMPT and NOX2 complex interaction. The illustrations were created with BioRender.com. b Box plot showing the expression profile of genes encoding Nampt and NOX2 subunits in HC, AC, and CC macrophages. c, d Western blotting results indicating the upregulation of Nampt, Cybb, Cyba, Ncf1, Ncf2, and Ncf4 in colon macrophages and colon lysates. e Box plots depicting the expression profiles of NLRP3 inflammasome priming and activation genes in macrophages, as determined by scRNA-seq in AC, CC, and HC. The uncropped/unedited blot images were added in the Supplementary Information (Supplementary Fig. S5).

Fig. 5. Analysis of commensal microbiota from the progression of DSS-induced colitis.

a Multi-dimensional Scaling (MDS) plots showing clustering of microbiome profiles based on 16s rRNA sequencing. b Box plot showing the diversity index. c, d Cladogram showing different abundant taxa between samples from different groups (c). The upper panel of d illustrates AC versus HC, while the bottom panel of d shows CC versus HC. The node size represents the difference in relative abundance. p, phylum; c class, o order, f family, g genus. e Bar plot showing the relative abundance and distribution of the family in HC, DSS-induced AC (11 days), and DSS-induced CC (33 days). f Bar plot showing the relative abundance of three microbiota families, Lactobacillaceae, Turicibacteraceae, and Bacteroidaceae, in HC, AC, and CC. g Heatmap of top17 KEGG pathways for HC, AC, and CC based on the normalized enrichment score (NES).

Fig. 6. Analysis of T cell subsets in healthy and DSS-induced UC.

a UMAP plot of scRNA-seq data for T cell clusters in HC (n = 3), AC (n = 3), and CC (n = 4). b, cBox plots showing cell-type enrichment analysis in DSS-induced UC mouse and human UC patients in SCP259 datasets, respectively. d, e Dot blot showing gene expression for Treg, Th17, and Tfh in DSS-induced UC mouse and UC patients in SCP259 datasets, respectively.