Supplementary Figure 1: Transcriptomic Analysis of Colonic and Other Tissue Resident Macrophages

(a) Hierarchical cluster analysis of mature colonic macrophages (CD11b+CD64+Ly6C– MHCII+CX3CR1hi; P4) compared with macrophages from the lung, brain, bone marrow, spleen, peritoneal cavity and dermis based on the 37 genes described as a macrophage ‘core’ signature by Gautier et al. 2012. (b) Genes that are expressed at least twofold higher by mature colonic macrophages compared with any of the tissue resident macrophage populations as in a above. (Student’s t test P≤0.05, fold change ≥ 2.0).

Supplementary Figure 2: Surface protein expression among the P1 to P4 subsets in the colon and small intestine

Flow cytometric analysis of surface protein expression by P1-P4 subsets obtained from the colon and small intestine of unmanipulated Cx3cr1+/GFP reporter mice compared with the appropriate isotype control. The surface proteins are encoded by genes (indicated in brackets) that define the colonic macrophage maturation process (Supplementary Table 1). Data are representative of 3-4 independent experiments.

Supplementary Figure 3: Identification of Dermal and Colonic Macrophage Subsets

(a) Gating strategy to define monocyte-macrophage subsets. Expression of CCR2 and CD64 by CD11b+CD64+EpCAM– cells (left panel) and expression of Ly6C and MHCII by CCR2+(upper right panel) and CCR2–(lower right panel) CD11b+CD64+EpCAM– cells. Dermal monocytes-macrophages were gated according to Tamoutounouret al. 2013. (b) Gating strategy to identify mature colonic CD64+Ly6C– MHCII+macrophages in colon from CD11c-Cre.Tgfbr1fl/fl Rag1–/–mice. Debris, cell aggregates and dead cells were excluded on the basis of FSC and 7-AAD staining. Leukocytes were identified by expression of CD45, before SiglecF+eosinophils and Ly6G+neutrophils were gated out. All other myeloid cells were selected by expression of CD11b and cells of the macrophage lineage identified on the basis of CD64 expression. Mature macrophages were identified in the ‘monocyte waterfall’ as Ly6C– MHCII+cells. Post-sort purity was routinely >98%. Representative purity shown.

Supplementary Figure 4: Analysis of CD11c-Cre.Rosa26-LSL-YFP Mice

(a) Schematic representation of the CD11c-Cre.Rosa26-LSL-YFP strain (b) Representative expression of YFP by Ly6C+MHCII–(P1), Ly6C+MHCII+ (P2), Ly6C–MHCII+ (P3+P4) cells and CD11chiMHCII+F4/80– DC from the colon of unmanipulated CD11c-Cre.Rosa26-LSL-YFP mice (Cre+;black line). Shaded histograms represent YFP fluorescence in Rosa26-LSL-YFP (Cre–) littermate controls. (c) Expression of YFP by Ly6C+MHCII–(P1), Ly6C+MHCII+ (P2), Ly6C–MHCII+ (P3+P4) cells, CD11chiMHCII+F4/80– DC, CD11b+ Ly6C–MHCII–SSChieosinophils, CD3+T cells and CD19+B cells from the colon of unmanipulated CD11c-Cre.Rosa26-LSL-YFP mice. Symbols represent individual animals and the horizontal bar is the mean of 6 mice from one experiment.

Supplementary Figure 5: Dysregulated Monocyte-Macrophage Differentiation in the Absence of TGFR1 Signalling

(a) Frequency of Tgfbr1fl/fl-derived (CD45.2+) cells among Ly6C+MHCII–(P1), Ly6C+MHCII+ (P2), Ly6C–MHCII+ (P3+P4) cells and eosinophils in the colon of WT:Cre–and WT:Cre+mixed BM chimeras. Symbols represent individual animals and horizontal lines represent the mean. Data represent 21 mice per group and are pooled from 5 independent experiments. **P<0.01 as determined by Student’s t-test with Holm-Sidak correction. (b) Frequency of WT-derived (CD45.1+) and Tgfbr1fl/fl-derived (CD45.2+) Ly6C+MHCII–(P1), Ly6C+MHCII+ (P2), Ly6C–MHCII+ (P3+P4) cells amongst CD64+cells in the colon of WT:Cre–and WT:Cre+mixed BM chimeras. Symbols represent individual animals and horizontal lines represent the mean of 21 mice per group pooled from 5 independent experiments. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 as determined by one-way ANOVA followed by Tukey’s multiple comparisons test. (c) Frequency of Tgfbr1fl/fl-derived (CD45.2+) cells among Ly6C+MHCII–(P1), Ly6C+MHCII+ (P2), Ly6C–MHCII+ (P3+P4) cells and eosinophils from the colon of Ccr2–/–:Cre–and Ccr2–/–:Cre+mixed BM chimeras. Data are from 7 (Ccr2–/–:Cre–) or 11 (Ccr2–/–:Cre+) mice per group and are pooled from 3-4 independent experiments.(d) Representative histological appearance of colon from Ccr2–/–:Cre–and Ccr2–/–:Cre+mixed BM chimeras (H&E x 200 magnification). (e)Frequency of CD103+CD11b-, CD103+CD11b+ and CD103-CD11b+ cells among total DCs from the colon of Ccr2–/–:Cre–and Ccr2–/–:Cre+mixed BM chimeras. Data are from 7 (Ccr2–/–:Cre–)or 11 (Ccr2–/–:Cre+)mice per group and are pooled from 3-4 independent experiments.