Supplementary figure 1

1. Supplementary figure 1 a Steady state CD64 MHCII F4/80 CD24 CD11c FMO b Macrophages CD11b+ DCs CD103+ DCs c IL-10 TNFa 80 25 n.s. * 20 60 * * 15 % of subset % of subset 40 uninfected 10 +anti-IL10R Hh 20 5 0 0 MHCII+ Macrophages MHCII+ Macrophages Monocytes Monocytes IL-6 Nos2 40 5 ** 4 ** 30 3 * % of subset 20 relative to Hprt mRNA expression 2 10 n.s. 1 0 0 MHCII+ Macrophages MHCII+ Macrophages Monocytes Monocytes

2. Supplementary figure 2 Total leukocytes Single events a i, ii • MHCII- monocytes • MHCII+ monocytes • CD11b+ DCs • Macrophages • CD103+ CD11b- DCs • CD103+ CD11b+ DCs MHCII FSC-H SSC CD24 Ly6C CD103 CD103 MHCII SSC Ly6C CD45 CD11c CD11b FSC-A Siglec-F F4/80 F4/80 MHCII CD11b CD11b FSC L/D Live CD45+ CD11b+ - eosinophils b i -neutrophils Ly6Clo MHCII+ - CD103 3 1 2 4 Live CD45+ CD11c+ MHCII+ - F4/80 1 5 6 ii 2 3 4 5 6 CX3CR1

3. Supplementary figure 3 a Cell counts (colon) b MHCII+ monocytes CD11b+ DCs CD103+ CD11b- DCs CD103+ CD11b+ DCs Macrophages c Eosinophils Neutrophils CD4+ T cells CD8+ T cells IL-23+ CD11c IL-23+ CD11c IL-23- CD11c IL-23- CD11c e d Neutrophils

4. Supplementary figure 4 a MHCII- monocytes MHCII+ monocytes Neutrophils macrophages CD11b+ DCs x104 cells x104 cells x104 cells x104 cells x104 cells b c d CD103+ CD11b- DCs CD103+ CD11b+ DCs 24 hours Day 4 Uninfected Hh+anti-IL-10R x104 cells

5. Supplementary figure 5 a c Cre-GFP CD11c CD64 F4/80 MHCII+ monocytes macrophages CD103+ CD11b-DCs CD103+ CD11b+ DCs b BMDMs IL-23+ CD11c IL-23- CD11c

6. Supplementary figure 6 a b Steady-state % of CD45+ MHCII+ Macro- Ly6Clo Monocytes phages CX3CR1int Macs/DCs c + Isotype + Isotype Hh+anti-IL-10R d4 Neutrophils + anti-CSF-1R + anti-CSF-1R +anti- CSF-1R +Iso Ly6Clo MHCII+ Macro- CX3CR1int Macs/DCs Monocytes phages

7. Supplementary figure 1. Infection with H. hepaticus and IL-10R treatment promotes the accumulation and pro-inflammatory functions of myeloid cells. CX3CR1GFP/+ mice were infected with H. hepaticus (Hh) combined with anti-IL-10R mAbs and were compared to uninfected controls. Mice were analysed after 2-3 weeks. (a) Absolute numbers of the indicated myeloid subsets in the colonic lamina propria. (b) histograms of the indicated myeloid subsets during steady-state, as defined in Figure 1: MHCII+ monocytes (CD11b+ CX3CR1int Ly6Chi, red subset), macrophages (CD11b+ CX3CR1hi Ly6Clo cells, green subset), CD11b+ DCs (CD11c+ CD103- CD11b+ CX3CR1int cells, grey subset) CD103+ DCs (CD11c+ CD103+ CX3CR1- cells, orange subset). All cells were pre-gated on live CD45+ leukocytes, excluding Ly6G+ neutrophils and Siglec-F+ eosinophils. Fluorescence minus one (FMO) controls for CD11b+ MHCII+ cells are shown. (c) Frequency of intracellular TNF+, IL-6+ andIL-10+ cells among the indicated subsets, as well as mRNA expression of Nos2 assessed by qPCR on FACS-sorted cells. All data are representative of at least two independent experiments. * p<0.05, ** p<0.01, as determined by Mann-Whitney‘s U test.

8. Supplementary figure 2. General gating strategy for the identification of lamina propria myeloid cell subsets in non-CX3CR1GFP/+ mice (a) Representative flow-cytometry gating strategy during mild colitis used throughout the study for the identification of colonic lamina propria myeloid subsets in non-CX3CR1GFP/+ mice. All subsets are gated on single live CD45+ cells. i. For the determination of monocytes, macrophages and CD11b+ DCs, cells were pre-gated on CD11b+. Eosinophils and neutrophils were excluded. Subsets were defined as follow: 1. MHCII- monocytes, 2. MHCII+ monocytes, 3. CD11b+ DCs and 4. Macrophages. ii. For the determination of classical dendritic cells, cells were pre-gated on CD11chi MHCII+ and F4/80- cells. Subsets were defined as: 5. CD103+ CD11b-DCs and 6. CD103+ CD11b+ DCs. (b) Representative histograms showing the corresponding CX3CR1-GFP expression of the subsets defined in a.

9. Supplementary figure 3. Colonic lamina propria cell composition of CD11cIL-23- mice compared to CD11cIL-23+ mice Cellular composition of CD11cIL23- mice compared to CD11cIL23+ littermates at steady state (uninfected, a-c) or during colitis (d-e). (a) Total number of colonic lamina propria cells per mouse. (b) Frequencies of the indicated cells among CD45+ colonic leukocytes. (c) Frequencies of IFN-+, IFN-+ IL-17A+ and IL-17A+ cells among CD4+ T cells upon restimulation with PMA and ionomycin. (d-e) Mice were infected with H. hepaticus (Hh) combined with anti-IL-10R treatment and analysed after 3 weeks. Frequencies and absolute numbers of indicated myeloid subsets among colonic leukocytes are shown. Cellular subsets were defined as shown in Supplementary figure 2a. Each data point represents individual mice and medians are shown. *p<0.05, **p<0.01, as determined by Mann-Whitney‘s U test.

10. Supplementary figure 4. MHCII+ monocytes express Il23a during early colitis CD11cIL-23+ mice were infected with Hh combined with anti-IL-10R mAb treatment and analysed 4 days after infection alongside uninfected controls. (a-b) Absolute numbers of the indicated myeloid subsets in the colonic lamina propria, as defined in Supplementary Figure 2. (c-d) qPCR analysis of Il23a mRNA expression in FACS-sorted lamina propria cells of CX3CR1GFP/+ reporter mice 24 hours (c) and 4 days (d) after induction of colitis. Subsets were defined as described in Figure 1d. Histograms represent the means with SEM of a pooled group of 10-15 mice, sorted in two biological replicates. *p<0.05, **p<0.01, ***p<0.001 as determined by Mann-Whitney‘s U test (a-b) or ANOVA with Bonferroni’s post-test (c-d).

11. Supplementary figure 5. Blockade of IL-10R-signalling increases H. hepaticus-induced expression of Il23a in MHCII+ monocytes/macrophages (a-b) Bone marrow-derived macrophages (BMDM) of individual CD11cIL-23+ and CD11cIL-23- mice were stimulated overnight with live Hh bacteria and/or anti-IL-10R antibody or left unstimulated (ctrl). (a) Representative histograms of surface markers expression relative to fluorescence minus one (FMO) controls. (b) Il23amRNA expression analysed by qPCR of the corresponding BMDM treated as indicated. (c) CD11cIL-23+ mice were infected with Hh in the presence or absence of anti-IL-10R antibody and analysed after four days. Il23a mRNA expression of FACS-sorted myeloid cells was determined by qPCR. Histograms represent the means + SEM of a pooled group of 10-15 mice, sorted in two biological replicates. Statistics comparing Hh only versus Hh+anti-IL-10R groups are shown. Data are representative of two independent experiments. *p<0.05, **p<0.01, ***p<0.001 as determined by ANOVA with Bonferroni’s post-test.

12. Supplementary figure 6. Anti-CSF-1R treatment selectively depletes Ly6Clo CX3CR1+ myeloid cells. Uninfected CX3CR1GFP/+ mice were treated for 4 days with a blocking anti-CSF-1R mAb and compared to isotype control-treated mice. (a-b) Frequencies of the indicated myeloid subsets among CD45+ colonic leukocytes. (c) CX3CR1GFP/+ mice were treated with a blocking anti-CSF-1R mAb or isotype control and analysed 4 days after Hh and anti-IL-10R mAb treatment. Absolute numbers of the indicated subsets in the colonic lamina propria are shown. Data are representative of or pooled from two independent experiments. Data points represent individual mice and medians are shown. ** p<0.01 as determined by Mann-Whitney‘s U test.

13. Supplementary methods: Generation of mouse bone-marrow derived macrophages (BMDM) and H.hepaticus infection assay. Bone-marrow cells were seeded at 500,000 cells in complete RPMI with 15% L929 cell-conditioned medium (LCM) and cultured for 8 days. BMDM cultures were infected with 0.5 OD H. hepaticus in RPMI (MOI = 50) or RPMI only for 24 hours. Anti-IL-10R (1B1.2) mAb was added 2 hours prior to infection at 10 mg/ml. Antibodies, intracellular staining of myeloid cells and qPCR. For intracellular cytokine staining of myeloid cells, colonic leukocytes were pre-incubated for 3.5 hours in complete RPMI medium with 1/1000 Brefeldin A solution (eBioscience). Following surface staining, cells were fixed and permeabilised with Cytofix/Cytoperm solutions Kit (BD Biosciences) according to the manufacturer’s instructions and stained for intracellular IL-10 (JES5-16E3, BD Pharmingen), TNF (MP6-XT22, eBioscience) and IL-6 (MP5-20F3, BD Pharmingen). Fluorescence minus one (FMO) controls were used for gating. TaqMan Gene Expression Assays for mouse Nos2 (00440502_m1; Invitrogen).