Figure 6.
TGF-β signalling controls the localization, phenotype, and responsiveness of long-lived small intestinal macrophages. (A) Left: Expression of YFP in CD64− and CD64+ cells from total live CD45+ cells in the small intestine. Right: Expression of YFP in Tim-4−CD4−, Tim-4−CD4+, and Tim-4+CD4+ macrophages in the small intestine. (B) Frequency of small intestinal Tim-4−CD4−, Tim-4−CD4+, and Tim-4+CD4+ macrophages expressing YFP. (C) Left: Representative flow cytometry plots showing the frequency of Tim-4+ macrophages expressing CD163 in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. Right: Frequencies of Tim-4+ macrophages expressing CD163 in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. (D) Representative immunofluorescence images of the small intestine showing the location of CD163-expressing macrophages in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. DAPI (blue), IBA1 (red), and CD163 (green). (E) Quantification of CD163-expressing cells in the LP (left) and S/M (right) of the small intestine in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. (F) Left: Representative flow cytometry plots showing the frequency of total macrophages expressing iNOS in response to overnight LPS and IFN-γ stimulation. Right: Frequencies of total macrophages expressing iNOS in response to overnight LPS and IFN-γ stimulation. (G) Left: Representative flow cytometry plots showing the frequency of Tim-4+ macrophages expressing iNOS in response to overnight LPS and IFN-γ stimulation. Right: Frequencies of Tim-4+ macrophages expressing iNOS in response to overnight LPS and IFN-γ stimulation. (H) Left: Representative immunofluorescence images of the small intestine 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 small intestine in Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. (I) Left: Representative immunofluorescence images of the small intestine 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 tip. (D, H, and I) Scale bar = 100 μm. (C, F, and G) Numbers in flow cytometry plots denote the percentages of cells within the gate. Data (n = 6 per group) are pooled from two independent experiments. (B, C, and E) Data (n = 5 per group) are pooled from two independent experiments. (H) Data (n = 5–6 per group) are pooled from two independent experiments. Error bars show mean ± SD. (I) Data (n = 3 per group) are pooled from rom two independent experiments. SM, submucosa; M, muscularis externa; S, serosa. Statistical comparisons were performed with an unpaired t test with Welch’s correction for parametric data and a Mann–Whitney test for nonparametric data. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Refer to the image caption for details. Panel A: Flow cytometry plots showing Y F P expression in C D 64 positive macrophages and across T i m-4 and C D 4 subsets. Panel B: Vertical bar graph showing frequency of Y F P positive macrophage subsets across T i m-4 and C D 4 populations. Panel C: Flow cytometry plots and vertical bar graph showing C D 163 expression in T i m-4 positive macrophages under different genotypes. Panel D: Immunofluorescence images showing localization of C D 163 positive macrophages with DAPI and I B A 1 staining in intestinal tissue. Panel E: Vertical bar graphs quantifying C D 163 positive cells in L P and S / M regions across conditions. Panel F: Flow cytometry plots and vertical bar graph showing i N O S expression in total macrophages after stimulation. Panel G: Flow cytometry plots and vertical bar graph showing i N O S expression in T i m-4 positive macrophages after stimulation. Panel H: Immunofluorescence images and vertical bar graphs showing C D 163 positive macrophage localization across genotypes in intestinal regions. Panel I: Immunofluorescence images and line graph showing spatial relationship between C D 163 and T G F-beta 1 expression across intestinal tissue gradient.

TGF-β signalling controls the localization, phenotype, and responsiveness of long-lived small intestinal macrophages. (A) Left: Expression of YFP in CD64− and CD64+ cells from total live CD45+ cells in the small intestine. Right: Expression of YFP in Tim-4−CD4−, Tim-4−CD4+, and Tim-4+CD4+ macrophages in the small intestine. (B) Frequency of small intestinal Tim-4−CD4−, Tim-4−CD4+, and Tim-4+CD4+ macrophages expressing YFP. (C) Left: Representative flow cytometry plots showing the frequency of Tim-4+ macrophages expressing CD163 in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. Right: Frequencies of Tim-4+ macrophages expressing CD163 in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. (D) Representative immunofluorescence images of the small intestine showing the location of CD163-expressing macrophages in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. DAPI (blue), IBA1 (red), and CD163 (green). (E) Quantification of CD163-expressing cells in the LP (left) and S/M (right) of the small intestine in Timd4creTgfbr2fl/fl and Tgfbr2fl/fl mice. (F) Left: Representative flow cytometry plots showing the frequency of total macrophages expressing iNOS in response to overnight LPS and IFN-γ stimulation. Right: Frequencies of total macrophages expressing iNOS in response to overnight LPS and IFN-γ stimulation. (G) Left: Representative flow cytometry plots showing the frequency of Tim-4+ macrophages expressing iNOS in response to overnight LPS and IFN-γ stimulation. Right: Frequencies of Tim-4+ macrophages expressing iNOS in response to overnight LPS and IFN-γ stimulation. (H) Left: Representative immunofluorescence images of the small intestine 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 small intestine in Cx3cr1creERTgfb1fl/fl and Tgfb1fl/fl mice. (I) Left: Representative immunofluorescence images of the small intestine 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 tip. (D, H, and I) Scale bar = 100 μm. (C, F, and G) Numbers in flow cytometry plots denote the percentages of cells within the gate. Data (n = 6 per group) are pooled from two independent experiments. (B, C, and E) Data (n = 5 per group) are pooled from two independent experiments. (H) Data (n = 5–6 per group) are pooled from two independent experiments. Error bars show mean ± SD. (I) Data (n = 3 per group) are pooled from rom two independent experiments. SM, submucosa; M, muscularis externa; S, serosa. Statistical comparisons were performed with an unpaired t test with Welch’s correction for parametric data and a Mann–Whitney test for nonparametric data. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001.

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