Benakis et al. Supplementary Figure 1

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1 Benakis et al. Supplementary Figure a flora Naive C7BL/ d AC weeks d S rrna sequencing Antibiotic in drinking water Stool pellet collection riginal seeder flora flora Naive C7BL/ d AC weeks d S rrna sequencing Antibiotic in drinking water Stool pellet collection b Color Key low high TU frequency Burkholderiaceae Streptococcaceae Enterobacteriaceae Bifidobacteriaceae unclassified Firmicutes unclassified Bacteria unclassified RF9 S.7 Clostridiaceae unclassifiedrf Anaeroplasmataceae Lactobacillaceae Turicibacteraceae Ruminococcaceae unclassified Clostridiales Lachnospiraceae Erysipelotrichaceae Bacteroidaceae orphyromonadaceae Verrucomicrobiaceae Alcaligenaceae revotellaceae Rikenellaceae unclassified Bacteroidales Helicobacteraceae Desulfovibrionaceae Deferribacteraceae Coprobacillaceae H wild-type wild-type Il -/- wild-type Il -/- Vancomycin wild-type Supplementary Figure In vivo model of intestinal dysbiosis and antibiotic-resistant flora. (a) Upper panel, experimental layout of intestinal dysbiosis. Experimental naive 7-week-old C7BL/ mice are treated for weeks with amoxicillin and clavulanic acid (AC). Mice with a modification of the intestinal microbiota are named AC-sensitive flora mice ( ). Lower panel, colonization of AC resistant flora ( ) from the original seeder mouse to naive 7-week-old C7BL/ mice. Naive mice (black) are co-housed with a seeder mouse (orange) carrying antibiotic resistant microbiota and received antibiotics in the drinking water for weeks. Because of the coprophagic behavior of mice, the resistant flora is successfully transmitted to naive mice. Stool pellets are collected days after co-housing for S ribosomal RNA (rrna) analysis. (b) The heat map depicts family-level operational taxonomic unit (TU) frequencies of bacterial populations observed in non-treated animals (H ),, wild-type and Il -/- K mice, and vancomycin-treated wild-type mice after weeks of treatment. nly TUs with frequency > % were included. Nature Medicine: doi:.8/nm.8

2 Benakis et al. Supplementary Figure a Vanco H C7BL/ 7-week-old Vancomycin or H weeks d H FF antibiotic and Stool collection d MCA b Infarct volume (mm ) 8 ** H Vancomycin H Vanco c Family Clostridiaceae Ruminococcaceae Lachnospiraceae Unclassified Clostridiales Lactobacillaceae Streptococcaceae Turicibacteraceae S-7 Burkholderiaceae unclassified RF9 Anaeroplasmataceae ther Relative abundance (%) 8 α-diversity **** H Vancomycin H Vanco Supplementary Figure Vancomycin alters gut flora and induces protection from ischemic brain injury. (a) Experimental layout of vancomycin treatment in 7-week-old C7BL/ mice. Mice receive water alone (H ) or vancomycin (Vanco) in drinking water (. g/l) for weeks. Stool pellets are collected days after treatment for rs sequencing. Administration of antibiotic is discontinued days prior to MCA. (b) Infarct volume of H (n = 9) and vancomycin (n = ) mice as measured by Nissl staining of brain sections on day post MCA (bar, mm). (c) Left panel indicates color code for the most predominant bacterial families found in the mouse intestine. Mid panel shows phylogenetic classification of S rdna frequencies in stool samples collected from H and vancomycin mice treated for weeks. Each bar represents one mouse. Right panel depicts intestinal microbiota α-diversity (H, n = 8; vancomycin, n = ). Throughout, error bars represent mean ± s.e.m. ** <., **** <.; by Student s t-test. Nature Medicine: doi:.8/nm.8

3 Benakis et al. Supplementary Figure CBF reperfusion (% of baseline) Infarct volume (mm ) FT FT H Vancomycin Il -/- Il -/- Il7a -/- Il7a -/- Supplementary Figure Correlation plot between cerebral blood flow (CBF) reperfusion (mean s are from to minutes after reperfusion) and infarct volume 7 h after MCA in all groups tested; each data point represent one animal. Nature Medicine: doi:.8/nm.8

4 Benakis et al. Supplementary Figure EB (IH/CH) Naive MCA Supplementary Figure Measurement of blood brain barrier permeability by Evans Blue (EB) extravasation is not different in naive and mice and h after MCA. Data are expressed as ratio of the ipsilateral hemisphere (IH) and contralateral hemisphere (CH), (n = per group). Columns represent mean ± s.e.m. (Student s t-test). Nature Medicine: doi:.8/nm.8

5 a b hyla Class rder Firmicutes Clostridia Erysipelotrichi Clostridiales Erysipelotrichales Bacteroidetes roteobacteria Verrucomicrobia Bacilli Bacteroidia Betaproteobacteria Gammaproteobacteria Verrucomicrobiae Lactobacillales Bacteroidales Burkholderiales Enterobacteriales Verrucomicrobiales Tenericutes Mollicutes Anaeroplasmatales Deferribacteres Deferribacteres Deferribacterales and Family Family Clostridiaceae Ruminococcaceae Lachnospiraceae unclassified Clostridiales Erysipelotrichaceae Coprobacillaceae Lactobacillaceae Bacteroidaceae orphyromonadaceae revotellaceae unclassified Bacteroidales S-7 Alcaligenaceae Enterobacteriaceae Verrucomicrobiaceae Anaeroplasmataceae Deferribacteraceae ther Benakis et al. Supplementary Figure Relative abundance (%) 8 FT FT FT and FT Family α-diversity **** FT FT f Ruminococcaceae f Verrucomicrobiaceae f Deferribacteraceae f revotellaceae unclassified.o Clostridiales f Clostridiaceae f S.7 f Lachnospiraceae f Burkholderiaceae f Erysipelotrichaceae f Anaeroplasmataceae f orphyromonadaceae f Coprobacillaceae f Bacteroidaceae f Streptococcaceae f Alcaligenaceae f Enterobacteriaceae f Lactobacillaceae unclassified.o RF f Turicibacteraceae f Verrucomicrobiaceae f revotellaceae f orphyromonadaceae f Clostridiaceae f Lactobacillaceae f Lachnospiraceae f Anaeroplasmataceae f Bacteroidaceae f Ruminococcaceae f S.7 unclassified.o Clostridiales unclassified.o Bacteroidales f Erysipelotrichaceae f Enterobacteriaceae f Alcaligenaceae f Coprobacillaceae 8 redictive importance 8 redictive importance Supplementary Figure hylogenetic analysis of fecal transplanted (FT) mice. (a) Left panel indicates color code for the most predominant bacterial families. Mid panel shows family-level phylogenetic classification of S rdna frequencies in stool samples collected from and mice weeks after fecal transplantation. Each bar represents an individual animal. For clarity only families represented over % were included. Right, graph depicts intestinal microbiota α-diversity (n = 8 per group). Columns represent mean ± s.e.m. **** <. (Student s t-test). (b) Random forest analysis showing predictive importance of operational taxonomic units (TU) at the family level (TUs > % were included) trained for stroke outcome in and (left panel) and FT and FT mice (right panel). Nature Medicine: doi:.8/nm.8

6 Benakis et al. Supplementary Figure a SSC b SSC c 8 * CD TCR-γδ CD CD CD IL-7-GF Fox IL-7 + (% γδt cells) Fox + (%CD + ) IL-7 + (% CD + ) Small Intestine ** Small Intestine Large Intestine Large Intestine IL-7 + (% γδt cells) H Vanco Supplementary Figure T reg, but not IL-7 + γδ T cells are affected in intraepithelial lymphocytes (IELs) of mice. (a) T reg population (CD + CD + Fox + ) in the IELs of the small intestine of and analyzed by flow cytometry. Graphs represent percentage of Fox + in the epithelium of the small (, n = and, n = 7) and large intestine (, n = and, n = ). (b) Flow cytometry analyzing IL-7 production in γδ T cells (CD + TCR-γδ + CD ) and T H 7 (CD + TCR-γδ CD + ) in the IELs of the small intestine from and mice after two weeks on antibiotic. The boxes in the dot plots gate IL-7 + cells in γδ T cells (first row) and IL-7 + in T H 7 cells (second row) in and mice. The bar graphs indicate percentage of IL-7 producing cells in the small (, n = 7 and, n = 7) and large intestine (, n = 7 and, n = ). Columns represent mean ± s.e.m.; no significant changes () are observed between groups (Student s t-test). (c) Similarly to AC treatment, IL-7 + γδ T cells are suppressed in the lamina propria (L) of the small intestine in vancomycin-treated mice. Graphs show percentage of IL-7 production in γδt cells (CD + TCR-γδ + CD ) in the L of the small intestine from H (n = ) and vancomycin (n = 8) mice after weeks. Columns represent mean ± s.e.m. * <. and ** <. (Student s t-test). Nature Medicine: doi:.8/nm.8

7 Benakis et al. Supplementary Figure 7 a Lymph nodes Spleen Blood 8 SSC b CD CD Fox Fox + (%CD + ) Lymph nodes Spleen Blood SSC TCR-γδ IL-7 IL-7 + (% γδt cells) CD CD IL-7 IL-7 + (% CD + ) Supplementary Figure 7 IL-7 + γδ T cells, IL-7 + CD cells (T H 7) and T reg cell analysis in lymph nodes, spleen and blood of and mice. (a) Flow cytometry gating strategy of T reg cells (CD + CD + Fox + ) in the lymph nodes. Graphs indicate number of Fox + cells as a percentage of CD + cells in and mice in the lymph nodes (n = per group), spleen (, n = and, n = ) and blood (n = per group). (b) Flow cytometry gating strategy to identify IL-7 + γδ T cells (CD + TCR-γδ + IL-7 + ) and T H 7 cells (CD + CD + IL-7 + ) in the lymph nodes. IL-7 production is analyzed by intracellular staining of IL-7. Graphs indicate number of IL-7 + cells as a percentage of γδ T cells (first row) and IL-7 + cells as a percentage of CD cells (second row) in and mice in the lymph nodes, spleen and blood (n = per group). Throughout, error bars represent mean ± s.e.m.; no significant changes are observed between groups (Student s t-test). Nature Medicine: doi:.8/nm.8

8 Benakis et al. Supplementary Figure 8 a hyla Class rder Firmicutes Clostridia Erysipelotrichi Clostridiales Erysipelotrichales Bacteroidetes roteobacteria Verrucomicrobia Bacilli Bacteroidia Betaproteobacteria Gammaproteobacteria Verrucomicrobiae Lactobacillales Bacteroidales Burkholderiales Enterobacteriales Verrucomicrobiales Tenericutes Mollicutes Anaeroplasmatales b Family Clostridiaceae Ruminococcaceae Lachnospiraceae unclassified Clostridiales eptostreptococcaceae Erysipelotrichaceae Streptococcaceae Lactobacillaceae Enterococcaceae Bacteroidaceae orphyromonadaceae revotellaceae S-7 Burkholderiaceae Alcaligenaceae Enterobacteriaceae Verrucomicrobiaceae Anaeroplasmataceae ther Relative abundance (%) 8 Relative abundance (%) 8 α-diversity w **** w d w w c Infarct volume (mm ) 8 d Fox + (% CD + ) IL-7 + (% γδt cells) 8 IL-7 + (% CD + ) 8 Supplementary Figure 8 ne week of AC treatment does not induce protection from ischemic brain injury and changes in intestinal immune cell response. Top panel indicates color code for the most predominant bacterial families. Lower panel shows family-level phylogenetic classification of S rdna frequencies in stool samples collected (a) from mice treated for days or (b) from and after week of antibiotic. Each bar represents an individual animal. nly families represented over % were included. Right, graph depicts intestinal microbiota α-diversity (n = ). (c) Infarct volume of (n = ) and (n = ) mice treated for week as measured by Nissl staining of coronal brain sections on day post MCA (bar, mm). (d) Flow cytometry analyzing T reg (CD + CD + Fox + ), IL-7 production in γδ T cells (CD + TCR-γδ + CD ) and T H 7 cells (CD + TCR-γδ CD + ) in the L of the small intestine from and mice after week on antibiotic (n = 8 per group). Data are expressed as mean ± s.e.m. **** <.;, not significant; by Student s t-test. Nature Medicine: doi:.8/nm.8

9 Benakis et al. Supplementary Figure 9 SSC CD CDb TCR-β CD Fox Number of CD + Fox + ( ) Supplementary Figure 9 Intestinal dysbiosis does not alter meningeal T reg after brain ischemia. Flow cytometry analyzing regulatory T cells (CD high CD + TCR-β + Fox + ) in the meninges of and mice h after ischemia. The graph represents the total number of meningeal T reg h after MCA in (n = ) and (n = ) mice. ne data point represent one animal. Columns represent mean ± s.e.m.;, not significant (Student s t-test). Nature Medicine: doi:.8/nm.8

10 Benakis et al. Supplementary Figure SI-L. Blood. Non photoconverted.. KikR KikG hotoconverted Supplementary Figure hotoconversion in the small intestine does not label circulating immune cells. Distal small intestine ( cm) from KikGR mice are exposed for min to violet light ( nm) (photoconverted) or to ambient room light (non-photoconverted). Immediately after photoconversion, blood and small intestine (SI-L) are extracted and analyzed by flow cytometry for KikR + cells. The gates in the dot plots identify KikR + cells, and the numbers are the percentage of KikR + cells of total lymphocytes (CD high CDb ); gating strategy is as indicated in Figure b. Nature Medicine: doi:.8/nm.8

11 CBF MCA occlusion (%) CBF reperfusion (%) week old weight (g) body temperature ( C) 8. ±.7. ± ±.7. ±.. ±. -. ±.. ± ±.. ±..7 ±. 8 + FT 9. ±.9. ±. 9. ±.9.7 ±.**. ±. - FT 9. ±.. ± ±.. ±..8 ±. 9 + H. ±.9. ±. 9. ±.. ±.7.8 ±. 7 - Vancomycin 8. ±.7 8. ± ±..8 ±..8 ±. 7 + Il -/-.9 ±. 87. ±. 9. ±..9 ±.. ±. - Il -/- 7. ±.8. ± ±.. ±.. ±. 7 - Il7a -/- 9. ±.9 9. ±.9 9. ±.. ±.*.7 ±. + Il7a -/-. ±..9 ±.8 9. ±.. ±.7. ±. 8 + N protection (+/-) Supplementary Table ercentages of cerebral blood flow (CBF) reduction after middle cerebral artery (MCA) occlusion and CBF mean s from to min after reperfusion. Mouse age (weeks), weight (g) and body temperature ( C) at the time of MCA induction. Data are represented as mean ± s.e.m. indicate significant difference in CBF reperfusion between H and Il -/- mice, p<. (one-way ANVA), and * indicate significant difference in body weight between, and FT and Il7a -/-, * <. and ** <. (one-way ANVA). Benakis et al. Supplementary Table Nature Medicine: doi:.8/nm.8

12 N exclusion (death) exclusion (CBF) survival 7 h post MCA(%) (Fig.d) 9 88 (Fig.d) 8 (Fig.e) 9 (Fig.e) 79 FT FT 9 H Vancomycin 9 Il -/ Il -/- 9 Il7a -/- Il7a -/- 7 Supplementary Table Number of mice excluded upon death and non-satisfactory cerebral blood flow (CBF) during occlusion or min after reperfusion. The percentage of survival at 7 h after MCA is not different between groups and treatment (Fisher s exact test). Benakis et al. Supplementary Table Nature Medicine: doi:.8/nm.8

13 a microglia microglia CD CD naive naive d d d d d d mono/mph mono/mph MN MN naive naive d d d d E-9 d d CD CD CD8 CD8 naive naive d d 8.8 d d d E- d E- B cells B cells NK NK naive naive 9.9 d d 8.9 d d d d b %reduction MN of %reduction mono/mph of %reduction CD8 of %reduction B cells of %reduction NK of n mean s.e.m Supplementary Table Supportive table to Fig.a. (a) Exact n s, absolute number of cells in the ischemic hemisphere (mean ± s.e.m.) and s in and naive mice and days, and after MCA for each immune cell markers analyzed by flow cytometry. utliers were identified using the RUT method with a maximum false discovery rate of Q = %. (b) Exact n s and percentages of immune cell reduction days after MCA (mean ± s.e.m.) in mice compared to mice. Benakis et al. Supplementary Table Nature Medicine: doi:.8/nm.8

14 T reg from WT CD + cells induced with DC- T reg from WT CD + cells induced with DC- T reg from Il -/- CD + cells induced with DC- Ratio T reg /γδt mean s.e.m. n mean s.e.m. n mean s.e.m. n : 9 : : : : Supplementary Table Supportive tables to Fig.d showing exact n s, percentage of IL-7 + γδ T cells (mean ± s.e.m.) per group and ratio for each data point analyzed by flow cytometry. Benakis et al. Supplementary Table Nature Medicine: doi:.8/nm.8