Article Figures & Data
Figures
- Figure 1. Annotation and cellular composition of major intestinal cells following infection and mock infection.
(A) IFN-α/β/γR KO mice (8–10 wk old) were intraperitoneally infected with 2 × 106 focus-forming units of DENV-3 P12/08 and euthanized at day 4 post-infection (p.i.). The small intestines were collected from infected (T13, T14, T15, and T18) and mock-infected mice (T34, T35, T36, and T37) for analysis. (B) Uniform Manifold Approximation and Projection plot of the four DV3P12/08-infected mice (n = 4) and mock-infected control (n = 4), which are colored by cell type. (C) Uniform Manifold Approximation and Projection plot showing integrated data, with cells colored according to sample origin (red for infected and green for mock-infected-mice). (D) Bar plot showing relative abundance of major cell populations in each sample. EEC, endocrine cell. (E) Top 10 enriched pathways identified by the KEGG pathway enrichment analysis of differentially expressed genes between infected and uninfected samples.
- Figure S1. Heatmap of the top-expressed genes in each cell type.
Expression level was calculated as the mean expression values of each gene within each the corresponding group. EEC, enteroendocrine cell.
- Figure 2. Il17a is expressed in Th17 and γδ T cells.
(A) Violin plot showing IIl17a (left) or Il17f (right) expression levels of individual mouse infected with DV3P12/08 (T13, T14, T15, and T18) or mock-infected (T34, T35, T36, T37). (B) Uniform Manifold Approximation and Projection plot showing cell distribution of IFN-α/β/γR KO mice infected with DV3P12/08 (n = 4), colored Il17a (left) or Il17f (right) expression levels; cells with no expression are noncolored (gray), and those with measurable expression levels are colored on a scale from white (low) to red (high). (C) Dot plot showing Il17a and Il17f expression across intestinal cell types (n = 4). (D) Sub-clusters of intestinal T cells, including innate lymphoid cells (ILCs) from IFN-α/β/γR KO mice infected with DV3P12/08 (n = 4) (left) or mock-infected (n = 4) (right). (E) Relative abundance of T cell and ILC populations in each sample. (F) Uniform Manifold Approximation and Projection plot showing Il-17a (left) and Il17f (right) expression in T cells (n = 8). (G) Dot plot showing Il17a and Il17f expression in T cell and ILCs sub-clusters (n = 4). (H) Dot plot representing the expression of cytokines and their receptor genes, with dot color intensity indicating scaled mean expression. Scaling was relative to the expression of each gene for all cells in each annotation selection, that is, cells associated with each column label in the dot plot (n = 4).
- Figure S2. Annotation of major intestinal cells following infection.
Uniform Manifold Approximation and Projection plot of single-cell transcriptomes of the intestinal cells from of IFN-α/β/γR KO mice infected with DV3P12/08 (n = 4), which are colored by cell type.
- Figure S3. Annotation of T-cell subtypes.
Dot plot showing the expression levels of gene markers used to annotate T-cell subtypes from the four DV3P12/08-infected mice (n = 4) and mock-infected control (n = 4).
- Figure 3. CCR6-CCL20 axis.
(A) Violin plot showing Ccr6 expression scores for individual mouse infected with DV3P12/08 (T13, T14, T15, and T18) or mock-infected (T34, T35, T36, T37). (B) Uniform Manifold Approximation and Projection (UMAP) plot showing cell distribution of IFN-α/β/γR KO mice infected with DV3P12/08 (n = 4), colored Ccr6 expression level; cells with no expression are noncolored (gray), and those with measurable expression levels are colored on a scale from white (low) to red (high). (C) Dot plot showing the scaled expression of Ccr6 gene across all cell clusters, colored by the average expression of Ccr6 (n = 4). (D) UMAP plot of Ccr6 gene expression in T cell and ILC sub-clusters (n = 4). (E) Dot plot showing Ccr6 expression levels in T cell and ILCs sub-clusters of infected mice (n = 4). (F) Violin plot showing Ccl20 expression levels for individual mouse infected with DV3P12/08 (T13, T14, T15, and T18) or mock-infected (T34, T35, T36, T37). (G) Dot plot showing Ccl20 expression in cells derived from DV3P12/08-infected mice (n = 4). (H) UMAP plot representing Ccl20 expression in small intestine cells of infected mice (n = 4).
- Figure 4. Il6 and receptor expression in IL-6-producing cells.
(A) Violin plot showing IIl6 expression scores for individual mice infected with DV3P12/08 (T13, T14, T15, and T18) or mock-infected (T34, T35, T36, T37). (B) Uniform Manifold Approximation and Projection plot showing integrated single-cell intestinal cell profiles from the four DV3P12/08-infected mice. Il6 gene expression was color-coded from low to high expression (grey to red, respectively) (right) (n = 4). (C) Dot plot showing the scaled expression of Il6 gene across all cell types, colored by the average expression of Il6 (n = 4). (D) Dot plot showing the scaled expression of Tnfrsf1a, Tnfrsf1b, Il17ra, Il1r1, Il1r2, and Il6ra across cell types, with color intensity indicating expression levels (n = 4).
- Figure 5. Il1β expression in macrophages and neutrophils.
(A) Violin plot showing Il1β expression scores for individual mouse infected with DV3P12/08 (T13, T14, T15, and T18) or mock-infected (T34, T35, T36, T37). (B) Uniform Manifold Approximation and Projection (UMAP) plot showing integrated single-cell intestinal cell profiles from the four DV3P12/08-infected mice. Il1β gene expression was color-coded from low to high expression (grey to red, respectively) (right) (n = 4). (C) Dot plot showing the scaled expression of Il1β in all cell types, colored by the average expression of Il1β (n = 4). (D) Sub-clusters of the intestinal macrophages (Mφ) from IFN-α/β/γR KO mice infected with DV3P12/08 (n = 4) and mock-infected (n = 4). UMAP plot showing integrated data and colored according to sample origin (red for infected and green for mock-infected). (E) Relative abundance of M1-like- and M2-like macrophages, monocytes, and DCs in each sample from the individual mouse infected with DV3P12/08 (T13, T14, T15, and T18) or mock-infected (T34, T35, T36, T37). (F) Dot plot showing the expression levels of gene markers used to annotate M1-like- and M2-like macrophages, monocytes, and DCs (n = 4). (G) Dot plot showing Il1β gene expression in M1-like- and M2-like macrophages, monocytes, and DCs (n = 4). (H) UMAP plot of antiviral gene expression in M1-like- and M2-like macrophages, monocytes, and DCs (n = 4). (I) Dot plot showing cytokine receptor gene expressions in M1-like- and M2-like macrophages, monocytes, and DCs (n = 4). Dot color represents scaled mean expression.
- Figure S4. Inflammatory score of cell groups.
Violin plot showing the inflammatory response pathway (GO:0006954) score across different cell populations of IFN-α/β/γR KO mice infected with DV3P12/08 (n = 4). The pathway score was calculated based on the average gene expression associated with that pathway. EECs, enteroendocrine cell; LE cells, lymphatic endothelial cells; SM cells, smooth muscle cells.
- Figure S5. Antiviral gene expression in macrophages.
Dot plot showing the mean expression of genes involved in the antiviral pathway in each group of macrophages from IFN-α/β/γR KO mice infected with DV3P12/08 (n = 4).
- Figure 6. Il1β expression in neutrophil sub-clusters.
(A) Sub-clusters of intestinal neutrophils from IFN-α/β/γR KO mice infected with DV3P12/08 (n = 4) (left) and trajectory analysis of neutrophils (right). (B) Dot plot showing representative gene expression in neutrophil sub-clusters (n = 4). (C) Dot plot showing Il1β gene expression in neutrophil sub-clusters (n = 4). (D) Dot plot showing cytokine receptor gene expression in neutrophil sub-clusters (n = 4). Dot color represents scaled mean expression.
- Figure S6. Matrix plot and developmental trajectory of neutrophil clusters of IFN-α/β/γR KO mice infected with DV3P12/08.
(A) Matrix plot showing the expression of genes used to define developmental stages of each neutrophil clusters according to Xie et al (2020). The x-axis represents marker genes, and the y-axis shows the neutrophil clusters. Each cell is color-coded according to the mean log-normalized expression level of the gene in the corresponding cluster. (B) Force-directed graph layouts of neutrophil clusters colored as per Leiden clustering. Pseudotime progression was calculated with immature neutrophils as root cells. Marker genes associated with immature neutrophils (Chil3, Camp, Ltf, and Mmp9) and mature neutrophils (Csf3r and Il1β) overlaid to show their distribution along the developmental trajectory.
- Figure S7. Nlrp3 expression in macrophages and neutrophils from of IFN-α/β/γR KO mice infected with DV3P12/08.
(A) Uniform Manifold Approximation and Projection plot of single-cell transcriptomes of the intestinal cells (n = 4). Plot is color-coded according to the expression levels of Nlrp3 expression (red). (B) Dot plot showing the Nlrp3 expression of across identified intestinal cell types (n = 4).
- Figure 7. Il23a expression in M1-like macrophages.
(A) Dot plot showing scaled expression of Il23a gene, colored by the average Il23a expression in all cell types (n = 4). (B) Dot plot depicting Il23a gene expression in M1-like- and M2-like macrophages, monocytes, and DCs (n = 4). Dot color represents scaled mean expression.
- Figure 8. Matrix metalloproteinase (Mmp)-8 expression in neutrophils.
(A) Dot plot showing Mmp expression in cells derived from the small intestines of mice infected with DV3P12/08 (n = 4). Dot color represents scaled mean expression. Scaling was relative to each gene’s expression for all cells in each annotation selection, that is, cells associated with each column label in the dot plot. (B) Uniform Manifold Approximation and Projection plot showing sub-clusters (profile) of neutrophil populations from the small intestines of DV3P12/08-infected mice (n = 4). (C) Dot plot depicting cytokine receptor and Mmp8 gene expression (n = 4). (D) Dot color represents scaled mean expression. Scaling was relative to the expression of each gene for all cells in each annotation selection, that is, cells associated with each column label in the dot plot (n = 4).
- Figure S8. Antiviral gene expression in neutrophils from of IFN-α/β/γR KO mice infected with DV3P12/08.
(A) Uniform Manifold Approximation and Projection showing the antiviral pathway score levels in neutrophils. The scores were calculated based on the average gene expression of genes contained in the associated with antiviral pathway. (B) Dot plot showing the mean expression of genes involved in the antiviral pathway-related genes within each group of neutrophils.
- Figure S9. Cytokine and chemokine receptor and ligand expression in intestinal cells from of IFN-α/β/γR KO mice infected with DV3P12/08.
(A, B, C, D) Dot plots showing the expression profiles of Il17ra and Il17rc (A); Csf1r, Csf2ra, Csf2rb, and Csf3r (B); Csf1, Csf2, and Csf3 (C); and Cxcrs (D) in all cell types.
- Figure 9. Schematic depiction of the pathogenic mechanism induced by DENV infection.
(1) Activation of M1-like macrophages during DENV infection leads to the production of TNF-α and IL-23. (2) TNF-α stimulates M2-like macrophages and dendritic cells (DCs), resulting in IL-1β production. (3) IL-1β and IL-23 induce IL-17A/F secretion by ILC3s, γδ T cells, and Th17 cells. (4) IL-17A/F, in synergy with other cytokines, such as TNF-α and IL-1β, activate endothelial cells, epithelial cells, fibroblasts, and pericytes, and induce IL-6 production. (5) IL-17A/F promotes the recruitment and activation of neutrophils. (6) IL-17A/F stimulates M2-like macrophages. (7) IL-17A/F functions in B and plasma cells. (8) IL-6 enhances neutrophil activation, whereas neutrophils are further stimulated by CXCL2, G-CSF, and IL-1β produced by neutrophils themselves.
Tables
Name Il6-expressing cells Tnfrsf1a Tnfrsf1b Il1r1 Il1r2 Il17ra Il6ra Endothelial cells 257 107 74 131 23 31 14 % 41.6 28.8 51.0 8.9 12.1 5.4 Fibroblast 495 156 75 286 40 74 32 % 31.5 15.2 57.8 8.1 14.9 6.5 Pericytes 102 39 11 31 9 8 5 % 38.2 10.8 30.4 8.8 7.8 4.9 Macrophages 707 264 184 48 224 177 69 % 37.3 26.0 6.8 31.7 25.1 9.8
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