Figure S1.
Identification and curation of scRNA-seq clusters from WT and Ccr2−/−mice. (A) Flow cytometry gating strategy for identification of small intestinal macrophages from a WT C57BL/6 mouse. (B) UMAP plots of scRNA-seq data from 5,639 live CD45+Lin−CD11b+MHCII+CD64+ cells from the small intestine of two WT and four Ccr2−/− mice. Left: Colors denote individual cells assigned to the same cluster. Right: Colors denote cells derived from WT (blue) or Ccr2−/− (red) mice. (C) Violin plots showing expression of Adgre1 (F4/80) and Cx3cr1 (CX3CR1) in each cluster. (D) UMAP plots showing expression levels of Mki67, Ifit1, and Spink1. (E) Proportions of each macrophage cluster by mouse genotype. (F) Violin plots showing the similarity score of each cluster against ImmGen datasets for monocytes. (G) Violin plots showing the similarity score of excluded clusters 3, 6, and 10 against each of the other clusters. (H) Representative immunofluorescence images of small intestine section from mice subject to a variety of infectious (H. polygyrus; T. gondii), inflammatory (α-CD3; doxorubicin), or non-homeostatic conditions (Ccr2−/−, germ-free mice, after PLX3397-mediated depletion). DAPI (blue), IBA1 (red), and CD163 (green). Dashed line demarcates the boundary between the S/M, and LP. Scale bar = 100 μm. (I) Quantification of CD163-expressing cells in the LP (upper) and S/M (lower) of the small intestine from GF, Ccr2−/−, and WT mice 7 wk after treatment with PLX3397 diet, 3 day after treatment with α-CD3, 3 day after treatment with doxorubicin, 7 day after infection with H. polygyrus, or 10 days after infection with T. gondii. Data for the SPF group are combined from the uninfected, untreated, and WT controls from all other groups. Data from n = 3–15 per group. Error bars show mean ± SD. Statistical comparisons were performed with a Brown–Forsythe and Welch ANOVA test for parametric data and a Kruskal–Wallis test with Dunn’s multiple comparison test for nonparametric data. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. UMAP, uniform manifold approximation and projection. Refer to the image caption for details. Panel A: Flow cytometry plots showing the gating strategy for isolating intestinal macrophage populations. Panel B: U M A P plots displaying clustering of single-cell transcriptomic data by identity and genotype. Panel C: Violin plots showing expression distribution of A d g r e 1 (F 4 slash 80) and C x 3 c r 1 (C X 3 C R 1) across clusters. Panel D: U M A P feature plots showing expression of M k i 67, I f i t 1, and S p i n k 1 across cell populations. Panel E: Stacked bar charts showing proportions of macrophage clusters across different genotypes. Panel F: Violin plots showing similarity scores of clusters compared to monocyte reference datasets. Panel G: Violin plots showing similarity scores of selected clusters against other cluster identities. Panel H: Immunofluorescence images showing tissue localization of macrophages under different experimental conditions with D A P I, I B A 1, and C D 163 staining. Panel I: Vertical bar graphs quantifying C D 163 positive cells in L P and S slash M regions across different treatment conditions.

Identification and curation of scRNA-seq clusters from WT and Ccr2 −/− mice. (A) Flow cytometry gating strategy for identification of small intestinal macrophages from a WT C57BL/6 mouse. (B) UMAP plots of scRNA-seq data from 5,639 live CD45+Lin−CD11b+MHCII+CD64+ cells from the small intestine of two WT and four Ccr2−/− mice. Left: Colors denote individual cells assigned to the same cluster. Right: Colors denote cells derived from WT (blue) or Ccr2−/− (red) mice. (C) Violin plots showing expression of Adgre1 (F4/80) and Cx3cr1 (CX3CR1) in each cluster. (D) UMAP plots showing expression levels of Mki67, Ifit1, and Spink1. (E) Proportions of each macrophage cluster by mouse genotype. (F) Violin plots showing the similarity score of each cluster against ImmGen datasets for monocytes. (G) Violin plots showing the similarity score of excluded clusters 3, 6, and 10 against each of the other clusters. (H) Representative immunofluorescence images of small intestine section from mice subject to a variety of infectious (H. polygyrus; T. gondii), inflammatory (α-CD3; doxorubicin), or non-homeostatic conditions (Ccr2−/−, germ-free mice, after PLX3397-mediated depletion). DAPI (blue), IBA1 (red), and CD163 (green). Dashed line demarcates the boundary between the S/M, and LP. Scale bar = 100 μm. (I) Quantification of CD163-expressing cells in the LP (upper) and S/M (lower) of the small intestine from GF, Ccr2−/−, and WT mice 7 wk after treatment with PLX3397 diet, 3 day after treatment with α-CD3, 3 day after treatment with doxorubicin, 7 day after infection with H. polygyrus, or 10 days after infection with T. gondii. Data for the SPF group are combined from the uninfected, untreated, and WT controls from all other groups. Data from n = 3–15 per group. Error bars show mean ± SD. Statistical comparisons were performed with a Brown–Forsythe and Welch ANOVA test for parametric data and a Kruskal–Wallis test with Dunn’s multiple comparison test for nonparametric data. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. UMAP, uniform manifold approximation and projection.

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