Figure 3.
Long-lived macrophages are present in the LP and S/M. (A) Left: Representative flow cytometry plots showing the 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 small intestine of Cx3cr1creER:R26-yfp mice, 5 days after tamoxifen treatment. Right: 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 small intestine of Cx3cr1creER:R26-yfp mice, 5 days after tamoxifen treatment. (B) Left: Representative flow cytometry plots showing the 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 small intestine of Cx3cr1creER:R26-yfp mice, 32 wk after tamoxifen treatment. Right: 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 small intestine of Cx3cr1creER:R26-yfp mice, 32 wk after tamoxifen treatment. (C) Left: Representative flow cytometry plots showing expression of CD4, Tim-4, CD163, or CD63 in YFP+ and YFP− macrophages of the small intestine from Cx3cr1creER:R26-yfp, 32 wk after tamoxifen treatment. Right: Frequency of YFP+ and YFP– macrophages expressing CD4, Tim-4, CD163, or CD63 in the small intestine of Cx3cr1creER:R26-yfp mice, 32 wk after tamoxifen treatment. (D) Frequency of total YFP+ macrophages expressing CD163, CD4, and Tim-4 in the small intestine of Cx3cr1creER:R26-yfp mice, 32 wk after tamoxifen treatment. (E) Left: Representative immunofluorescence of small intestine section from Cx3cr1creER:R26-yfp, 32 wk after tamoxifen treatment. DAPI (blue), IBA1 (red), and YFP (green). Scale bar = 100 μm. (A–C) Numbers in flow cytometry plots denote the percentages of cells within the gate. (A–D) Data (n = 6–8 per group) are pooled from two to three independent experiments. 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 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 and vertical bar graphs showing Y F P positive cells within C D 4 and T i m-4 subsets of C D 163 positive and negative macrophages at early time point. Panel B: Flow cytometry plots and vertical bar graphs showing persistence of Y F P labeled macrophage subsets across C D 4 and T i m-4 populations at later time point. Panel C: Flow cytometry plots and vertical bar graphs showing expression of C D 4, T i m-4, C D 163, and C D 63 in Y F P positive and negative macrophages. Panel D: Vertical bar graph showing frequency of Y F P positive macrophages expressing combinations of C D 4, T i m-4, and C D 163. Panel E: Immunofluorescence images showing localization of macrophages with DAPI, I B A 1, and Y F P staining in intestinal tissue.

Long-lived macrophages are present in the LP and S/M. (A) Left: Representative flow cytometry plots showing the 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 small intestine of Cx3cr1creER:R26-yfp mice, 5 days after tamoxifen treatment. Right: 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 small intestine of Cx3cr1creER:R26-yfp mice, 5 days after tamoxifen treatment. (B) Left: Representative flow cytometry plots showing the 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 small intestine of Cx3cr1creER:R26-yfp mice, 32 wk after tamoxifen treatment. Right: 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 small intestine of Cx3cr1creER:R26-yfp mice, 32 wk after tamoxifen treatment. (C) Left: Representative flow cytometry plots showing expression of CD4, Tim-4, CD163, or CD63 in YFP+ and YFP− macrophages of the small intestine from Cx3cr1creER:R26-yfp, 32 wk after tamoxifen treatment. Right: Frequency of YFP+ and YFP– macrophages expressing CD4, Tim-4, CD163, or CD63 in the small intestine of Cx3cr1creER:R26-yfp mice, 32 wk after tamoxifen treatment. (D) Frequency of total YFP+ macrophages expressing CD163, CD4, and Tim-4 in the small intestine of Cx3cr1creER:R26-yfp mice, 32 wk after tamoxifen treatment. (E) Left: Representative immunofluorescence of small intestine section from Cx3cr1creER:R26-yfp, 32 wk after tamoxifen treatment. DAPI (blue), IBA1 (red), and YFP (green). Scale bar = 100 μm. (A–C) Numbers in flow cytometry plots denote the percentages of cells within the gate. (A–D) Data (n = 6–8 per group) are pooled from two to three independent experiments. 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 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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