PMC

(a) Ly6C and MHCII expression by colonic F4/80⁺CD11b⁺ cells from adult CX3CR1^+/gfp mice. (b) CX3CR1 and CCR2 expression by Ly6C^hiMHCII⁻, Ly6C^hiMHCII⁺ and Ly6C⁻ subsets from CX3CR1^+/gfp and CCR2^+/rfp mice respectively. (c) Absolute numbers of colonic Ly6C^hiMHCII⁻, Ly6C⁺MHCII⁺ and Ly6C⁻ subsets from newborn (day 0-1), day 7, day 14, day 21 and 7-8 week old CX3CR1^+/gfp mice. (d) MHCII expression by F4/80⁺CD11b⁺Ly6C⁻ colonic LP cells from newborn (day 0-1), day 7, day 14, day 21 and 7-8 week old CX3CR1^+/gfp mice. (e) Numbers of Ly6C^hiMHCII⁻, Ly6C⁺MHCII⁺ and Ly6C⁻ subsets in colon of newborn (day 0-1), day 7, day 14, day 21, 7 week old and in 9-12 month old adult Ccr2^−/− mice, shown as ratios to the equivalent populations in age-matched Ccr2^+/+ mice. (f) Representative F4/80 and CD11b expression by live CD45⁺SiglecF⁻Ly6G⁻CD11c^lo colonic LP cells and Ly6C and MHCII expression by total F4/80⁺CD11b⁺ cells from 7 week old Ccr2^+/+ or Ccr2^−/− mice. (g) Mean proportions and absolute numbers of Ly6C^hiMHCII⁻, Ly6C⁺MHCII⁺ and Ly6C⁻ subsets in colon of 7 week old Ccr2^+/+ or Ccr2^−/− mice. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test). (h) Representative F4/80 and CD11b expression by live CD45⁺SiglecF⁻Ly6G⁻CD11c^lo liver cells from 8 week old Ccr2^+/+ or Ccr2^−/− mice. Bar charts show the absolute numbers of F4/80^hiCD11b^lo and F4/80^loCD11b⁺ cells from livers of 8wk old Ccr2^+/+ or Ccr2^−/− mice. **P<0.01 (Student’s t-test). Data are representative of 3 independent experiments (a, f, g, h and Cx3cr1^+/gfp mice in b), or from one experiment (Ccr2^+/rfp mice in b), or are pooled from two independent time course experiments (c, d, e). Aged mice were examined in one experiment. Results in c, e, g, h are means ± 1 SD of 6-8 (c, e) or 3-4 mice (g, h) per group.

(a) Representative chimerism of Ly6C^hi blood monocytes and Ly6C^hiMHCII⁻, Ly6C⁺MHCII⁺ and Ly6C⁻ subsets of colonic F4/80⁺ CD11b⁺ cells from the CD45.1⁺ partner of wild-type:wild-type parabionts 10 weeks after joining. Scatter plot shows the proportions of non-host derived cells amongst B and T lymphocytes and Ly6C^hi monocytes in blood, and colonic Ly6C^hiMHCII⁻, Ly6C⁺MHCII⁺ and Ly6C⁻ subsets from individual CD45.1⁺ (open circles) and CD45.2⁺ (filled circles) parabionts. (b) Representative dot plots of the chimerism of Ly6C^hi blood monocytes and of the colonic Ly6C^hiMHCII⁻, Ly6C⁺MHCII⁺ and Ly6C⁻ subsets from wild-type (CD45.2⁺):Ccr2^−/− (CD45.1⁺) mixed BM chimeric mice. Bar chart shows the mean proportions of cells derived from Ccr2^−/− CD45.1⁺ BM amongst CD3⁺ T cells, CD19⁺ B cells, Ly6C^hi monocytes and Ly6G⁺ neutrophils in blood, and amongst the colonic Ly6C^hiMHCII⁻, Ly6C⁺MHCII⁺ and Ly6C⁻ subsets, together with F4/80⁺ CD11b⁺ MHCII⁻ SSC^hi eosinophils in the colon of WT (CD45.1⁺):Ccr2^−/− (CD45.2⁺) mixed BM chimeric mice. ***P<0.0001 (Student’s t-test). (c) Representative non-host chimerism amongst Ly6C⁻ colonic mφ and F4/80^hi CD11b^lo liver Kupffer cells from the WT partner (left plots) and the Ccr2^−/− partner (right plots) of WT-Ccr2^−/− parabiotic mice 10 weeks after establishment of parabiosis. Bar chart shows the mean proportion of non-host chimerism amongst Ly6C⁻ colonic mφ and F4/80^hi CD11b^lo liver Kupffer cells in the WT and Ccr2^−/− partner of WT-Ccr2^−/− parabiotic mice. ***P<0.0001 (Student’s t-test). Data are from one experiment (a), or from one of two independent experiments (b), or are pooled from three experiments (c). Results are the means ± 1 SD of 3 (b) or 4-6 mice per group (c).

(a) Expression of Ki67 by F4/80⁺ CD11b⁺ Ly6C⁻ colonic mφ from early neonate (day 3), day 14, day 21, day 35 and 9-12 week old adult CX3CR1^+/gfp mice. (b, c) Proportions of Ki67⁺ F4/80⁺ CD11b⁺ Ly6C⁻ mφ (b) and Ki67⁺ CD11c^hi F4/80⁻ DC (c) in colon of mice of different ages. The results are of 4-7 individual mice pooled from 2 independent experiments. Each dot represents an individual mouse. ***P<0.0001 (One way ANOVA followed by Bonferroni’s multiple comparison test). (d) Proportion of BrdU⁺ cells amongst CD64⁺ Ly6C^hiMHCII⁻, Ly6C⁺MHCII⁺, Ly6C⁻ cells and CD64⁻ CD11b⁺ DC from the colons of resting mice, mice administered 2% DSS for 3 days (colitis) and mice given DSS for 3 days followed by 3 days of normal water (recovery). Mice were administered BrdU 3 hours prior to sacrifice and the dotted line represents the limit of detection as shown by BrdU staining in a non-DNase treated control sample. Data are pooled from two independent experiments with 4-7 seven mice at each time point (a, b, c), or are from one of two independent experiments with 4 mice per group (d). Each circle represents an individual mouse.

(a) Non-host chimerism of CD64⁺/F4/80⁺ Ly6C⁻ colonic mφ in Ccr2^−/− partners of WT-Ccr2^−/− parabiotic mice given broad spectrum antibiotics for the first 4 weeks after surgery (Abx. 4wks), compared with the group of mice shown in Fig. 5c that received antibiotics for the entire 8 weeks of the study (continuous Abx). **P<0.01 (Student’s t-test). (b) Absolute numbers of the Ly6C^hiMHCII⁻, Ly6C⁺MHCII⁺ and Ly6C⁻ subsets of F4/80⁺ CD11b⁺ cells per colon of adult conventionally housed (CNV) and germ free (GF) mice. *P<0.05, **P<0.005 (Mann Whitney). (c) Representative dot plots of Ly6C and MHCII expression by F4/80⁺ CD11b⁺ colonic LP cells from 3 week old Rag1^−/− CNV and GF mice. Scatter plots show the absolute numbers of the Ly6C^hiMHCII⁻, Ly6C⁺MHCII⁺ and Ly6C⁻ subsets of F4/80⁺ CD11b⁺ cells per colon of individual 3 week old CNV and GF mice. *P<0.05, ***P<0.0001 (Mann Whitney). (d) Representative expression of MHCII by F4/80⁺ CD11b⁺ Ly6C⁻ colonic LP cells from 3 week old mice and mean proportion of F4/80⁺ CD11b⁺ Ly6C⁻ MHCII⁺ cells amongst total F4/80⁺ CD11b⁺ colonic LP cells. *P<0.05 (Student’s t-test). Data are means + 1 SD of results pooled from two independent experiments using 3 (a) or 10 mice per group (b), or are from one of two independent experiments with 6 mice per group (c, d).

(a) YFP expression by total live CD45⁺ colonic leukocytes (left panel) from 9 day old progeny of Csf1r-mer-iCre-mer Rosa26-LSL-YFP mice injected with tamoxifen at E8.5 to pulse label CSF1R⁺ YS macrophages (left panel); expression of F4/80 and CD11b by total YFP⁺ cells (right panel). (b) Representative YFP expression by F4/80⁺ CD11b⁺ colonic LP cells from 9 day or 12 week old progeny of Csf1r-mer-iCre-mer Rosa26-LSL-YFP mice treated as in a. Bar chart shows the mean frequency of YFP⁺ cells amongst F4/80^hi CD11b^lo liver Kupffer cells, total F4/80⁺ CD11b⁺ colonic LP cells and amongst Ly6C^hi blood monocytes from 9 day or 12 week old progeny of Csf1r-mer-iCre-mer Rosa26-LSL-YFP mice treated as above. **P<0.01, ***P<0.0001 (Student’s t-test). (c) Absolute number of eYFP-expressing F4/80⁺ CD11b⁺ colonic LP cells from 9 day or 12 week old adult progeny of Csf1r-mer-iCre-mer Rosa26-LSL-YFP mice. (d) Representative eYFP expression (upper panels) and mean eYFP expression (lower panel) by total CD45⁺ blood leukocytes, Ly6C^hi blood monocytes, F4/80^hi CD11b^lo liver KC and colonic F4/80⁺ CD11b⁺ cells from 8 week old progeny of Flt3-Cre Rosa26-LSL-YFP mice. **P<0.005 (Student’s t-test). (e) Proportions of CD45.1⁺CD45.2⁺ residual host cells amongst Ly6C^hi blood monocytes, total F4/80⁺ CD11b⁺ colonic LP cells, F4/80^hi CD11b^lo liver Kupffer cells and F4/80^hi CD11b^lo splenic macrophages of WT:Ccr2^−/− mixed BM chimeric mice 8 weeks after reconstitution. a, b, c: Data are means + 1 SD and are pooled from two independent experiments with 3 or 4 mice per group (12 week old), or from one experiment with 3 mice (9 day old). d, e: Data are means + 1 SD and are from one of two independent experiments with 3 mice.

(a) F4/80 and CD11b expression by live CD45⁺SiglecF⁻Ly6G⁻CD11c^lo colonic LP cells from neonatal (day 2), day 14, day 21, day 35 and 9 week old CX3CR1^+/gfp mice. (b) Absolute numbers of total F4/80⁺ CD11b⁺ cells per colon in day 2, day 14, day 21, day 35 and 9 week old CX3CR1^+/gfp mice. (c) CD64 and CX3CR1-GFP expression by F4/80⁺ CD11b⁺ colonic LP cells from newborn (day 2; black line) and 8 week old (green line) CX3CR1^+/gfp mice. Shaded histogram represents isotype control. (d) Phagocytic activity of F4/80⁺ CD11b⁺ colonic LP cells from newborn (day 2) or adult CX3CR1^+/gfp mice measured by the uptake of pHrodo Escherichia coli bioparticles for 30-60 mins at 37°C or 4°C as a control (shaded histograms). (e) Morphological appearance of F4/80⁺ CD11b⁺ colonic mφ from newborn and adult mice. Scale bar 25μm. (f) qRT-PCR of mRNA for IL10 and TNFα by F4/80^hi LP mφ from newborn (day 1) or adult mice, and BM-derived mφ (BMM). Results are mean expression relative to cyclophilin A (CPA) + 1SD obtained using the 2^−dΔC(t), with BMM set to 1. **P<0.01 (Student’s t-test). (g, h) Representative plots and mean frequencies of F4/80^hi CD11b^lo (red) and F4/80^lo CD11b⁺ (blue) subsets of total colonic LP mφ from newborn (day 2), day 14, day 21, day 35 and 9 week old CX3CR1^+/gfp mice (g) or from 8 week old wild-type liver (h). Data are from one of 3 (a, b, g, h), or 2 independent experiments with two mice (c), of two independent experiments with 3 mice per group (d), or are representative images from two independent experiments (e). Results in b, g, h are means ± 1 SD of 6 (neonate timepoint) or 4 (day 14, 21 and 35, and 9 week old timepoints) mice per group. Data from E19.5 embryos are from two experiments each using cells pooled from 8 mice. For qRT-PCR analysis (f), data represent 3 (neonate) or 9 (adult) biological replicates using RNA pooled from 1-2 adults or 5 neonates.