Fig 1.
Leishmania infection is associated with differential regulation of host immune response pathways in vivo.
(A) Multi-Dimensional Scaling (MDS) plot showing the gene expression profile between naive and L. major-infected ears. (B) Volcano plot of all DEGs in naive and infected ears. Red dots represent DEGs with increased RNA abundance using a log2FC >1 and p-value <0.05. Blue represents DEGs with decreased RNA abundance using a log2FC <−1 and p-value <0.05. Only annotated genes are shown in plot. FC, fold-change. (C-D) Bulk RNA-Seq analysis indicates DEGs that are highly correlated with signaling pathways. KEGG enrichment analysis of top 20 upregulated (C), and top 20 downregulated (D) pathways enriched among the DEGs between naive and L. major-infected ears (pathways selected by significant and FC > 1.5, list includes rank, significance and adjusted average). Data are shown from 4 naïve control and 6 L. major infected mice.
Table 1.
Differentially expressed genes (DEGs) enriched for top 10 KEGG pathways following L. major infection.
Fig 2.
Heat map analysis of host transcriptional responses to L. major infection in vivo.
Heat maps of DEGs in infected ears compared to naive controls. The DEGs involved in the host immune response pathways by KEGG enrichment analysis whether upregulated (A, B, and C) or downregulated (D) in the infected ears presented as heat maps. Hierarchical clustering of the expression profile was grouped according to functional categories. Heat maps indicate the FC of gene expression in L. major-infected ears >2-fold (red) or <2-fold (blue). Data are shown from 4 naïve control and 6 L. major infected mice.
Fig 3.
scRNA-Seq reveals cellular heterogeneity including distinct resident and recruited cell types in the skin following L. major infection.
(A) C57BL/6 mice were infected or not with L. major parasites in the ear, and ears were digested to isolate RNA for scRNA-Seq analysis. Schematic of cell isolation, cell processing, capture by droplet-based device. (B) Uniform Manifold Approximation and Projection (UMAP) plot revealed cellular heterogeneity with 35 distinct clusters of cells identified and color-coded (both naive and infected groups combined). Seurat’s FindClusters function was used to identity each cell cluster and cell type designation is located to the right.
Fig 4.
Cell type identification and cluster-specific gene expression.
(A) Relative expression of 35 different cell type cluster-specific genes shown as dot plots with two genes per cluster. Dot size indicates the percentage of cells expressing each gene, the dot color indicates the expression level, and ordering is performed from low to high expressing cells. (B) Feature plots of expression distribution for selected cluster-specific genes used to define the cell types. Expression levels for each marker is color-coded and overlaid onto UMAP plot. Cells with the highest expression level are colored black.
Fig 5.
Presence of L. major transcripts in multiple cell types.
(A) Differential expression of L. major parasite transcripts in 35 different cell types. Number of cells associated with parasite transcripts is shown in violin plots. (B) Table summarizes the percentages of individual cells associated with L. major transcripts.
Fig 6.
scRNA-Seq analysis reveals an enhanced immune cell recruitment to the inflamed tissue during L. major infection.
(A) scRNA-Seq UMAP plots of naive and infected ears showing resident and recruited cell populations/clusters. (B, D, F) Representative flow cytometry plots showing the percentage of CD64+ macrophages (B), Ly6C+ inflammatory monocytes (D), and Ly6G+ neutrophils (F) of naive and infected ears at 4 week p.i. Cells were gated on total, live, singlets, CD45+ CD11b+ cells. (C, E, G) Cell numbers of CD64+ macrophages (C), inflammatory monocytes (E), neutrophils (G) from naive and infected ears. (H-J) ECs were gated on total, live, singlets, CD31+ cells. Dermal BECs and LECs were separated by podoplanin expression during FACS analysis. (H) Representative flow cytometry dot plots showing the percentages of BECs and LECs from naive and infected ears 4 week p.i. Corresponding cell numbers of BECs (I) and LECs (J) from naive and infected ears. Data are representative of at least two independent experiments involving 10–17 mice. Data are presented as mean ±SEM. **p < 0.005, ***p < 0.0005, ****p < 0.0001, unpaired t-test.
Fig 7.
Differentially expressed genes in selected immune cell types during L. major infection.
Volcano plot showing the DEGs in macrophages (A), resident macrophages (B), inflammatory monocyte (C), neutrophils (D), BECs (E), and LECs (F). Colored dots indicate genes at least 2 (natural log ~0.693) fold increased (red) or decreased (blue) in infected cells relative to naive cells with an adjusted p-value < 0.05.
Table 2.
List of top 10 DEGs enriched in macrophages (A) and resident macrophages (B) between infected vs naïve controls.
Table 3.
List of top 10 DEGs enriched in inflammatory monocytes (A) and neutrophils (B) clusters between infected vs naïve controls.
Table 4.
List of top 10 DEGs enriched in BEC (A) and LEC (B) clusters following L. major infection.
Table 5.
List of top 5 upstream regulators identified in macrophages by IPA analysis for L. major infected ears.
Table 6.
List of top 5 upstream regulators identified in BECs by IPA analysis for L. major infected ears.
Table 7.
List of top 5 upstream regulators identified in LECs by IPA analysis for L. major infected ears.
Fig 8.
Signaling pathways and molecular networks within individual cell types predicted during L. major infection by Ingenuity Pathway Analysis.
(A-B) Top 5 differentially regulated canonical pathways and their individual heat maps in macrophages following L. major infection. (C-D) Top 5 differentially regulated canonical pathways and their individual heat maps in BECs. (E-F) Top 5 differentially regulated canonical pathways and their individual heat maps in LECs. The color intensity represents the degree of expression. A red-green color scale was used to reflect the standardized gene expression with red representing high expression and green representing low expression. Cut-off values are adjusted p-value < 0.05.
Fig 9.
Ribosomal protein subunit large (Rpl) and small (Rps) transcripts are expressed at decreased levels at the site of L. major infection.
C57BL/6 mice were infected with L. major parasites in the ear dermis, and ears were analyzed by quantitative real-time PCR at 4 wk p.i. for the expression of Rpl4 (A), Rpl12 (B), Rps18 (C), and Rps19 (D). Relative mRNA expression was normalized to the housekeeping gene RPS11 is presented as the mean ± SEM with 20–25 total mice. Data are representative of at least 4 independent experiments. * p<0.05, ** p<0.005, *** p<0.0005 t-test.