Figure 7.
Long-lived macrophages are present in the LP and S/M of the colon and are regulated by TGF-β signalling in the upper crypt region. (A) Representative flow cytometry plots showing the frequency of Tim-4−CD4−-, Tim-4−CD4+-, and Tim-4+CD4+-expressing cells within the CD163− and CD163+ subsets of total colonic intestinal macrophages from WT mice. (B) Frequency of Tim-4−CD4−-, Tim-4−CD4+-, and Tim-4+CD4+-expressing cells within the CD163− and CD163+ subsets of colonic macrophages from WT mice. Numbers in flow cytometry plots denote the percentages of cells within the gate. (C) Frequency of YFP-expressing cells within the CD163− and CD163+ subsets of Tim-4−CD4–, Tim-4−CD4+, and Tim-4+CD4+ macrophage of the colon from Cx3cr1creER:R26-yfp mice, 5 days and 32 wk after tamoxifen treatment. (D) Representative immunofluorescence of colon tissue from Cx3cr1creER:R26-yfp mice, 32 wk after tamoxifen treatment. DAPI (blue), IBA1 (red), and YFP (green). (E) Frequency of total YFP+ macrophages expressing CD163, CD4, and Tim-4 in the colon of Cx3cr1creER:R26-yfp mice, 32 wk after tamoxifen treatment. (B, C, and E) Data (n = 5 per group) are pooled from two independent experiments. For clarity, only comparisons to CD163+CD4+Tim4+ populations have been shown in E. (F) Left: Representative immunofluorescence image of colon section from WT mice showing the location of CD163-expressing macrophages. DAPI (blue), IBA1 (red), and CD163 (green). Right: Quantification of CD163+ macrophages in the S/M, and upper (LP-U) and lower (LP-L) layers of the LP. Dashed line demarcates the boundary between the S/M, and LP. Dotted line demarcates the boundary between the upper (LP-U) and lower (LP-L) layers of the LP. (G) Left: Representative immunofluorescence images of the colon showing the location of CD163- and TGF-β1-expressing cells in WT mice. DAPI (blue), CD163 (green), and TGF-β1 (red). Right: Quantification of CD163 and TGF-β1 expression levels in individual cells relative to their displacement from the crypt tip. Data (n = 3 per group) are pooled from two independent experiments. (H) Representative immunofluorescence images of the colon showing the location of CD163-expressing macrophages in Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. DAPI (blue), CD163 (green), and IBA1 (red). Right: Quantification of CD163-expressing cells in the LP and S/M of the colon in Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. (I) Representative immunofluorescence images of the colon showing the location of CD163-expressing macrophages in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. DAPI (blue), CD163 (green), and IBA1 (red). Right: Quantification of CD163-expressing cells in the LP and S/M of the colon in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. (H and I) Data (n = 5–6 per group) are pooled from two independent experiments. (D and F–I) Scale bar = 100 μm. Error bars show mean ± SD. Statistical comparisons between two groups were performed with an unpaired t test with Welch’s correction for parametric data and a Mann–Whitney test for nonparametric unpaired data. Statistical comparisons between more than two groups were performed with a one-way ANOVA, with Tukey’s multiple comparison 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; and ****, P ≤ 0.0001. Refer to the image caption for details. Panel A: Flow cytometry plots showing macrophage subset distribution by L y 6 C (lymphocyte antigen 6 complex), M H C class 2, C D 11 b, C D 4, Tim-4, and C D 163. Panel B: Vertical bar graphs showing frequencies of macrophage subsets across Tim-4 negative C D 4 negative, Tim-4 negative C D 4 positive, and Tim-4 positive C D 4 positive groups in C D 163 positive and C D 163 negative populations. Panel C: Vertical bar graphs showing Y F P within C D 163 positive and C D 163 negative subsets at 5 days and 32 weeks after tamoxifen treatment. Panel D: Immunofluorescence image showing localization of Y F P positive macrophages in colon tissue with I B A 1 and D A P I staining. Panel E: Vertical bar graph showing frequency of Y F P positive macrophages across subsets (C D 4 negative Tim-4 negative, C D 4 positive Tim-4 negative, C D 4 positive Tim-4 positive) in C D 163 positive and C D 163 negative groups. Panel F: Immunofluorescence images and vertical bar graph showing C D 163 positive macrophage distribution across intestinal layers with I B A 1 co-staining and quantification as number of C D 163 positive cells per square millimeter; Panel G: Immunofluorescence images and line graph showing spatial relationship of C D 163 and transforming growth factor beta 1 (T G F-beta 1). Panel H: Immunofluorescence images and vertical bar graphs showing effect of transforming growth factor beta 1 (T g f b 1) gene deletion on C D 163 macrophage distribution. Panel I: Immunofluorescence images and vertical bar graphs showing effect of transforming growth factor beta receptor 2 (T g f b r 2) deletion on macrophage localization.

Long-lived macrophages are present in the LP and S/M of the colon and are regulated by TGF-β signalling in the upper crypt region. (A) Representative flow cytometry plots showing the frequency of Tim-4−CD4−-, Tim-4−CD4+-, and Tim-4+CD4+-expressing cells within the CD163− and CD163+ subsets of total colonic intestinal macrophages from WT mice. (B) Frequency of Tim-4−CD4−-, Tim-4−CD4+-, and Tim-4+CD4+-expressing cells within the CD163− and CD163+ subsets of colonic macrophages from WT mice. Numbers in flow cytometry plots denote the percentages of cells within the gate. (C) Frequency of YFP-expressing cells within the CD163− and CD163+ subsets of Tim-4−CD4–, Tim-4−CD4+, and Tim-4+CD4+ macrophage of the colon from Cx3cr1creER:R26-yfp mice, 5 days and 32 wk after tamoxifen treatment. (D) Representative immunofluorescence of colon tissue from Cx3cr1creER:R26-yfp mice, 32 wk after tamoxifen treatment. DAPI (blue), IBA1 (red), and YFP (green). (E) Frequency of total YFP+ macrophages expressing CD163, CD4, and Tim-4 in the colon of Cx3cr1creER:R26-yfp mice, 32 wk after tamoxifen treatment. (B, C, and E) Data (n = 5 per group) are pooled from two independent experiments. For clarity, only comparisons to CD163+CD4+Tim4+ populations have been shown in E. (F) Left: Representative immunofluorescence image of colon section from WT mice showing the location of CD163-expressing macrophages. DAPI (blue), IBA1 (red), and CD163 (green). Right: Quantification of CD163+ macrophages in the S/M, and upper (LP-U) and lower (LP-L) layers of the LP. Dashed line demarcates the boundary between the S/M, and LP. Dotted line demarcates the boundary between the upper (LP-U) and lower (LP-L) layers of the LP. (G) Left: Representative immunofluorescence images of the colon showing the location of CD163- and TGF-β1-expressing cells in WT mice. DAPI (blue), CD163 (green), and TGF-β1 (red). Right: Quantification of CD163 and TGF-β1 expression levels in individual cells relative to their displacement from the crypt tip. Data (n = 3 per group) are pooled from two independent experiments. (H) Representative immunofluorescence images of the colon showing the location of CD163-expressing macrophages in Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. DAPI (blue), CD163 (green), and IBA1 (red). Right: Quantification of CD163-expressing cells in the LP and S/M of the colon in Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. (I) Representative immunofluorescence images of the colon showing the location of CD163-expressing macrophages in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. DAPI (blue), CD163 (green), and IBA1 (red). Right: Quantification of CD163-expressing cells in the LP and S/M of the colon in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. (H and I) Data (n = 5–6 per group) are pooled from two independent experiments. (D and F–I) Scale bar = 100 μm. Error bars show mean ± SD. Statistical comparisons between two groups were performed with an unpaired t test with Welch’s correction for parametric data and a Mann–Whitney test for nonparametric unpaired data. Statistical comparisons between more than two groups were performed with a one-way ANOVA, with Tukey’s multiple comparison 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; and ****, P ≤ 0.0001.

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