SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFORMATION doi: /nature11809 Supplementary Figure 1. Antibiotic treatment reduces intestinal bacterial load and allows access of non-pathogenic bacteria to the MLN, inducing intestinal immune responses, without increasing epithelial cell permeability. (a) Intestinal bacterial load from unmanipulated (n=3) or antibiotictreated (n=3) mice was quantified by isolating DNA and performing quantitative PCR for total bacterial 16S rrna genes. Signal was quantified using a standard curve and normalized to mg feces. *P<0.05, one-way ANOVA with Bonferroni correction. Bars show the S.E.M. Data are representative of 2 independent experiments. (b) In vivo cytokine secretion was analyzed by infecting or mockinfecting antibiotic-treated and untreated mice with ΔAroA/ΔinvA Salmonella. 9d later mice were injected with Brefeldin and Salmonella antigens. 6h later cells were isolated from the spleen and MLN and analyzed by flow cytometry. IFNγ vs IL-17 staining is shown for MHC - TCRβ + CD4 + cells. (c) Unmanipulated (-ABX) or antibiotic (+ABX)-treated mice were infected by gavage with non-invasive, nonpathogenic Salmonella (ΔAroA/ΔinvA). Three days later spleens and MLN were analyzed for bacteria. Each point represents a single mouse, data were pooled from two independent experiments. **P<0.001, Mann Whitney test. Bar represents geometric mean. (d) Uninfected or infected unmanipulated (-ABX) (n=5), antibiotic (+ABX)-treated (n=7), or DSS (+DSS)-treated (n=6) mice were fasted overnight before gavaging with FITC-Dextran. Three hours later, the amount of FITC-Dextran in the blood was determined. Results are representative of two independent experiments. *P<0.05, one-way ANOVA with Bonferroni correction. Error bars represent S.E.M. 1

2 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 2. Intestinal colonization by non-invasive Salmonella is insufficient to allow for MLN access and induction of IgA. Animals were treated with ampicillin (Amp), vancomycin (Vanco), metronidazole (Met), or a combination of Amp, Vanco, Met and neomycin (ABX) before they were infected with non-invasive Salmonella. Salmonella titers in the cecum (a) and MLN (b) were determined by plating. ***P<0.0001, Mann-Whitney test, bar represents geometric mean. Each point represents a single animal. (c) Antibiotic (ABX)- or single-antibiotic-treated mice were infected twice with non-pathogenic Salmonella (ΔAroA/ΔinvA). 14 days after the second infection the induction of Salmonella specific IgA antibodies in the feces was analyzed by ELISA. Data show average of 4 mice from each group and are representative of two independent experiments. ***P<0.001, unpaired t test. Error bars represent S.E.M. 2

3 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 3. Commensals limit access to the MLN by commensal bacteria. (a) Untreated (-ABX) or antibiotic-treated (+ABX) B6 mice from Jackson Laboratories were gavaged with E. coli K-12. Two days later MLN were analyzed for presence of bacteria. Each point represents a single mouse. Data were pooled from two independent experiments. **p<0.001, Mann-Whitney test. (b) Untreated or antibiotic-treated Jackson B6 mice were infected twice with E. coli K-12 by gavage and feces were analyzed 14d after second infection for the presence of E. coli K-12-specific IgA by ELISA. Bars are representative of 4 individual mice. Data are representative of 2 independent experiments. ***P<0.001, unpaired t test. 3

4 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 4. Whole bacteria are required to limit MyD88- dependent MLN colonization by non-invasive Salmonella. (a) Nod2 -/-, Nalp3 -/-, Myd88 -/- and wildtype controls were infected with 1X10 8 ΔinvA cfu. 2d later CFU in the MLN were analyzed by plating. ***P>0.0005, Mann-Whitney test. Error bars represent geometric mean. (b) Antibiotic-treated mice were taken off antibiotics for 2 days before gavaging with PBS (PBS) or with cecal contents from unmanipulated mice. At 24h or 2 weeks, the mice were infected with noninvasive Salmonella and bacterial titers in the MLN were determined at 48h. Bars represent 8 individual mice pooled from two independent experiments. (c) Antibiotic-treated mice were left untreated (ABX) or fed heat-killed Salmonella (ABX+HK) 24h before infection with non-invasive Salmonella. 48h later Salmonella in the cecum was quantified by plating. (d) MyD88-deficient mice were gavaged with PBS (NT) or heat-killed Salmonella before infecting with noninvasive Salmonella. After 48h, bacterial titers in the MLN were determined. Bars represent 4 individual mice. (e) Quantitative PCR was performed for RegIIIγ mrna in the terminal ileum of mice left untreated (NT) (n=2), antibiotic-treated (ABX) (n=2), or antibiotic-treated and then gavaged with LPS (n=2) or heat-killed Salmonella (HK ST) (n=2). RegIIIγ expression is expressed as relative to untreated mice. Data are representative of two independent experiments. *P<0.05, unpaired t test. Error bars represent SEM. 4

5 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 5. Depletion of intestinal cell populations in CD11c- DTR mice. Antibiotic-treated wild-type or CD11c-DTR bone marrow chimeras were injected daily for two days with DT. (a) Intestinal lamina propria cells were isolated and analyzed by flow cytometry for MHCII + CD11c + cells to assess depletion. (b) Expression of CD14 and CD103 on gated MHCII + CD11c + cells. Panels show cells from individual mice. 5

6 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 6. Intestinal DC populations in antibiotic treated and untreated mice. (a) Intestinal lamina propria cells from untreated and antibiotictreated Cx3cr1 gfp/+ mice were isolated and analyzed by flow cytometry. Cells were gated on MHC + CD11c + (top row) and analyzed for the expression of CX 3 CR1-GFP, CD14, CD11b, CD103 and F4/80. (b) Lamina propria MHC + CD11c + cells (shown in top left) from Cx3cr1 gfp/+ mice were sorted into the four populations shown based on expression of CX 3 CR1-GFP and CD103 (bottom left). Cells were left untreated (top row) or stimulated for 6 hours with LPS (middle row) at 37 o C and were then stained and analyzed by flow cytometry for expression of CX 3 CR1-GFP, CD103 and CCR7. Comparison of CCR7 expression under different conditions for each population is shown in the bottom row. 6

7 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 7. Diphtheria toxin-mediated depletion of CX 3 CR1 hi myeloid cells in the small intestine. (a) Schematic for generation of CX 3 CR1- STOP-DTR mice. (b) CX 3 CR1-STOP-DTR mice were bred to the CX 3 CR1-GFP mice to track the CX 3 CR1-expressing population. Animals with and without the CD11c-Cre transgene were injected daily for three days with DT. Cells from the small intestinal lamina propria were isolated and analyzed by flow cytometry for the loss of GFP + cell populations. The bottom panels show the expression of CX 3 CR1-GFP versus CD103, CD11b, CD14 and F4/80 among gated MHCII + CD11c + cells shown in the second row. Panels show cells from individual mice. (c) Quantification of depletion of CX 3 CR1 hi cells from 5 mice per group. The data are representative of multiple independent experiments. 7

8 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 8. Increase in inflammatory monocytes after diphtheria toxin-mediated depletion of CD11c + CX 3 CR1 hi cells. CX 3 CR1- STOP-DTR mice with and without CD11c-Cre (n=5 each) were injected daily for three days with DT. Cells were then isolated from the small intestine (SI), spleen, and blood and analyzed by flow cytometry for the presence of monocytes. The number of Ly6c hi (inflammatory) monocytes was compared to that of Ly6c lo (resident) monocytes of the DAPI - Lineage - CD11b + CD11c - Ly6g - population of cells. Absolute cell numbers are shown in the bar graphs on the left and a representative flow plot from each organ is shown on the right. 8

9 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 9. CX 3 CR1 + and not CD103 + cells migrate from the intestine to the MLN in antibiotic-treated mice infected with non-invasive Salmonella. (a) The absolute number of MLN CX 3 CR1 hi, CX 3 CR1 low, CD103 + and CX 3 CR1 - CD103 - (DN) cells in the MHCII + CD11c + population in unmanipulated (-ABX), infected (-ABX/invA), antibiotic treated (+ABX) and antibiotic treated and infected (+ABX/invA) mice. Bars represent the average from 10 mice per condition and were pooled from 3 independent experiments. *P<0.05, **P<0.005, ***P<0.0002, One-way ANOVA with Bonferroni correction. Bars represent S.E.M. (b) Antibiotic-treated Cx3cr1 gfp/+ mice were gavaged with PBS or with heat killed Salmonella. After 24h, mice were gavaged with PBS or non-invasive Salmonella. MLN cells were analyzed by flow cytometry for expression of CX 3 CR1 and CD11b at 48h. Cell plots are gated on MHCII and CD11c. (c) Antibiotic-treated Cx3cr1 gfp/+ mice were gavaged with Salmonella. At 48h, CD19 - TCRβ - MHCII + CD11c + cells expressing CX 3 CR1-GFP, CD103, or neither were sorted from the MLN. Shown is the post sort analysis. Plots are representative of 15 individual mice from 4 independent experiments. (d) Cx3cr1 gfp/+ mice were left untreated (NT) or fed 10µg of R848. Four hours later cells from the afferent intestinal lymph were collected and analyzed by flow cytometry. Top panel shows MHCII and CD11c staining of live cells. Bottom is the expression of CX 3 CR1-GFP versus CD103 among the MHCII + CD11c + cells. 9

10 RESEARCH SUPPLEMENTARY INFORMATION Strain LD50 (oral) Effect of Mutation Attach to Epithelial Cells? Attach to Epithelial Cells? Wildtype 5X10 5 N/A yes yes inva 1X10 7 SPI-1 deficient yes no AroA/invA >10 8 SPI-1 deficient; auxotrophic yes no Supplementary Table 1. Description of Salmonella strains used. Adapted from refs 1,2. References 1 Baumler, A. J., Tsolis, R. M., Valentine, P. J., Ficht, T. A. & Heffron, F. Synergistic effect of mutations in inva and lpfc on the ability of Salmonella typhimurium to cause murine typhoid. Infection and immunity 65, (1997). 2 Penheiter, K. L., Mathur, N., Giles, D., Fahlen, T. & Jones, B. D. Noninvasive Salmonella typhimurium mutants are avirulent because of an inability to enter and destroy M cells of ileal Peyer's patches. Mol Microbiol 24, (1997). 10