Figure S5.
Characterization of Timd4creTgfbr2fl/fland Cx3cr1creERTgfb1fl/flmice. (A) Frequency of Ly6Chi blood and bone marrow monocytes, Tim-4+ liver Kupffer cells, Tim-4+ large peritoneal macrophages, Tim-4− large peritoneal macrophages, and small peritoneal macrophages expressing YFP. Data (n = 3 per group) are from a single experiment. (B) Expression of Tgfbr2 in six macrophage populations sorted from WT small intestinal tissues by bulk RNA-seq. (C) Representative H&E-stained small intestine from Timd4creTgfbr2fl/fl mice and Tgfbr2fl/fl mice. (D) Representative H&E-stained colon from Timd4creTgfbr2fl/fl mice and Tgfbr2fl/fl mice. (E) Concentration of FITC-dextran in plasma of mice 1 h after oral gavage with FITC-dextran. Data (n = 5 per group) are pooled from two independent experiments. (F) Left: Representative flow cytometry plots showing the frequency of small intestinal macrophages expressing Tim-4 in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. Right: Frequencies of Tim-4+ macrophages in the small intestine of Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. (G) Frequency of Tim-4+CD163− and Tim-4+CD163+ small intestinal macrophages expressing iNOS in response to overnight LPS and IFN-γ stimulation. (H) Left: Representative flow cytometry plots showing the frequency of Tim-4− macrophages expressing CD163. Right: Frequencies of Tim-4− macrophages expressing CD163. (I) Left: Representative flow cytometry plots showing the frequency of Tim-4− macrophages expressing iNOS. Right: Frequencies of Tim-4− macrophages expressing iNOS. (J) Left: Representative flow cytometry plots showing the frequency of Tim-4+ macrophages in the small intestine of Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. Right: Frequencies of Tim-4+ macrophages in the small intestine of Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. (K) Left: Representative immunofluorescence images showing the location of Tim-4– and CD163-expressing cells in the small intestine of Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. DAPI (blue), Tim-4 (green), and CD163 (red). Right: Quantification of Tim-4–expressing cells in the LP of the small intestine in Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. (L) Example of the spatial distribution plots used to measure the distance of individual cells from the serosa in the small intestine. (M) Example of the spatial distribution plots used to measure the distance of individual cells from the serosa in the colon. (N) Quantification of CD163 expression levels in individual cells relative to their displacement from the tip in the colon of adult Cx3cr1creERTgfb1fl/fl mice, 4 wk after tamoxifen dosing, adult Timd4creTgfbr2fl/fl, and respective Cre– littermates. Data (n = 5–6 per group) are pooled from two independent experiments. (C, D, and K) Scale bar = 100 μm. (F–K) Data (n = 4–7 per group) are pooled from two independent experiments. Error bars show mean ± SD. Statistical comparisons between two groups were performed with an unpaired t test with Welch’s correction. *, P ≤ 0.05. Statistical comparisons between more than two groups were performed with a one-way ANOVA, with Tukey’s multiple comparison test. *, P ≤ 0.05; ***, P ≤ 0.001; and ****, P ≤ 0.0001. Refer to the image caption for details. Panel A: Vertical bar graph showing frequency of Y F P positive monocytes and macrophage populations across different tissues. Panel B: Vertical bar graph showing expression levels of T g f b r 2 across multiple macrophage subsets. Panel C: Histological images showing small intestine morphology in different genotypes using staining. Panel D: Histological images showing colon tissue structure across experimental genotypes. Panel E: Vertical bar graph showing F I T C-dextran permeability levels in plasma after oral administration. Panel F: Flow cytometry plots and vertical bar graph showing i N O S expression in macrophage subsets after stimulation. Panel G: Flow cytometry plots and vertical bar graph showing frequency of T i m-4 positive macrophages across genotypes. Panel H: Flow cytometry plots and vertical bar graph showing C D 163 expression in T i m-4 negative macrophages. Panel I: Flow cytometry plots and vertical bar graph showing i N O S expression in T i m-4 negative macrophages. Panel J: Flow cytometry plots and vertical bar graph showing frequency of T i m-4 positive macrophages in different genetic models. Panel K: Immunofluorescence images and vertical bar graph showing localization and quantification of T i m-4 and C D 163 positive cells in intestinal tissue. Panel L: Scatter plot showing spatial distribution of cells relative to intestinal serosal region. Panel M: Scatter plot showing spatial distribution of cells in colon tissue relative to serosa. Panel N: Line graph showing variation of C D 163 expression relative to distance from intestinal tip.

Characterization of Timd4 cre Tgfbr2 fl/fl and Cx3cr1 creER Tgfb1 fl/fl mice. (A) Frequency of Ly6Chi blood and bone marrow monocytes, Tim-4+ liver Kupffer cells, Tim-4+ large peritoneal macrophages, Tim-4− large peritoneal macrophages, and small peritoneal macrophages expressing YFP. Data (n = 3 per group) are from a single experiment. (B) Expression of Tgfbr2 in six macrophage populations sorted from WT small intestinal tissues by bulk RNA-seq. (C) Representative H&E-stained small intestine from Timd4creTgfbr2fl/fl mice and Tgfbr2fl/fl mice. (D) Representative H&E-stained colon from Timd4creTgfbr2fl/fl mice and Tgfbr2fl/fl mice. (E) Concentration of FITC-dextran in plasma of mice 1 h after oral gavage with FITC-dextran. Data (n = 5 per group) are pooled from two independent experiments. (F) Left: Representative flow cytometry plots showing the frequency of small intestinal macrophages expressing Tim-4 in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. Right: Frequencies of Tim-4+ macrophages in the small intestine of Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. (G) Frequency of Tim-4+CD163− and Tim-4+CD163+ small intestinal macrophages expressing iNOS in response to overnight LPS and IFN-γ stimulation. (H) Left: Representative flow cytometry plots showing the frequency of Tim-4− macrophages expressing CD163. Right: Frequencies of Tim-4− macrophages expressing CD163. (I) Left: Representative flow cytometry plots showing the frequency of Tim-4− macrophages expressing iNOS. Right: Frequencies of Tim-4− macrophages expressing iNOS. (J) Left: Representative flow cytometry plots showing the frequency of Tim-4+ macrophages in the small intestine of Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. Right: Frequencies of Tim-4+ macrophages in the small intestine of Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. (K) Left: Representative immunofluorescence images showing the location of Tim-4– and CD163-expressing cells in the small intestine of Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. DAPI (blue), Tim-4 (green), and CD163 (red). Right: Quantification of Tim-4–expressing cells in the LP of the small intestine in Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. (L) Example of the spatial distribution plots used to measure the distance of individual cells from the serosa in the small intestine. (M) Example of the spatial distribution plots used to measure the distance of individual cells from the serosa in the colon. (N) Quantification of CD163 expression levels in individual cells relative to their displacement from the tip in the colon of adult Cx3cr1creERTgfb1fl/fl mice, 4 wk after tamoxifen dosing, adult Timd4creTgfbr2fl/fl, and respective Cre– littermates. Data (n = 5–6 per group) are pooled from two independent experiments. (C, D, and K) Scale bar = 100 μm. (F–K) Data (n = 4–7 per group) are pooled from two independent experiments. Error bars show mean ± SD. Statistical comparisons between two groups were performed with an unpaired t test with Welch’s correction. *, P ≤ 0.05. Statistical comparisons between more than two groups were performed with a one-way ANOVA, with Tukey’s multiple comparison test. *, P ≤ 0.05; ***, P ≤ 0.001; and ****, P ≤ 0.0001.

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