Commensal microbiota affects ischemic stroke outcome by regulating intestinal γδ T cells

Abstract

Commensal gut bacteria impact the host immune system and can influence disease processes in several organs, including the brain. However, it remains unclear whether the microbiota has an impact on the outcome of acute brain injury. Here we show that antibiotic-induced alterations in the intestinal flora reduce ischemic brain injury in mice, an effect transmissible by fecal transplants. Intestinal dysbiosis alters immune homeostasis in the small intestine, leading to an increase in regulatory T cells and a reduction in interleukin (IL)-17-positive γδ T cells through altered dendritic cell activity. Dysbiosis suppresses trafficking of effector T cells from the gut to the leptomeninges after stroke. Additionally, IL-10 and IL-17 are required for the neuroprotection afforded by intestinal dysbiosis. The findings reveal a previously unrecognized gut-brain axis and an impact of the intestinal flora and meningeal IL-17(+) γδ T cells on ischemic injury.

Figure 1

Intestinal microbiota alteration protects from MCAO. (a) Experimental design of AC treatment in 7 weeks old C57BL/6 mice. AC Res mice, co-housed with AC Res seeder mice, and AC Sens flora mice received antibiotic via drinking water for 2 weeks. Stool collection time points are indicated. MCAO is induced after 2 weeks of AC and brain infarct volume is quantified 3 days later. Other groups of mice are assessed for sensorimotor function. (b) Fecal r16S DNA copy numbers in AC Res and AC Sens mice (n = 5 per group). (c) Left, family-level phylogenetic classification of fecal 16S rDNA gene frequencies from AC Res and AC Sens mice treated for 2 weeks. Each bar represents an individual animal. Right, graph depicts Shannon α-diversity index of grouped data (n = 7 per group). Only families with a frequency > 1% were included. (d) Infarct volume of AC Res and AC Sens mice 3 days post MCAO (n = 8 per group; bar, 1 mm). (e) Sensorimotor function in AC Res and AC Sens mice. Graphs show time to contact and remove the tape off the contralateral forepaw 2 days prior, 3 and 7 days after MCAO (n = 11 per group). (f) Left, fecal transplant (FT) experimental design. Mice are pulse-treated with AC for 3 days and gavaged with cecal contents of either AC Res or AC Sens donors. After 2 weeks on H₂O, FT recipient mice are subjected to MCAO and sacrificed 3 days after for infarct volume quantification. Right, graph shows infarct volumes (n = 8 per group). Columns represent mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (Student’s t-test).

Figure 2

Increased T_reg cells and reduced IL-17⁺ γδ T cells in the small intestine of AC Sens mice. (a) Representative flow cytometry plots. Left, CD4 T cells identified in side scatter (SSC)/forward scatter (FSC) plots and CD45⁺/CD4⁺ expression. Mid panel, T_reg cells (CD45⁺CD4⁺FoxP3⁺) in the LP of the small intestine of AC Res and AC Sens. Numbers represent events within the gate as percentage of CD4⁺ cells. Right, graphs represent percentages of FoxP3⁺ cells in the LP of the small intestine (n = 10 per group) and large intestine (n = 7 per group). (b) Flow cytometry analysis of IL-17 production in γδ T cells (CD45⁺TCR-γδ⁺CD4⁻) and CD4⁺ T cells (CD45⁺TCR-γδ⁻CD4⁺) in the LP of the small intestine from AC Res and AC Sens mice after 2 weeks on antibiotics. The boxes in the center dot plots identify IL-17⁺ cells and numbers represent IL-17⁺ cells as a percentage of γδ T cells (first row) and CD4⁺ cells (second row) in AC Res and AC Sens mice. The bar graphs on the right indicate percentages of IL-17-producing cells in the LP of the small and large intestine (AC Res, n = 8 and AC Sens, n = 7). Columns represent mean ± s.e.m. *P < 0.05, **P < 0.01; n.s., not significant (Student’s t-test).

Figure 3

Accumulation of IL-17⁺ γδ T cells at the meninges is associated with increased infarct size. (a) Flow cytometric gating strategy of brain microglia (CD45^intCD11b^int) and infiltrating leukocytes (CD45^high) including monocytes/macrophages (Mono/MΦ; CD11b⁺Ly6G⁻CD11c⁻), neutrophils (PMN; CD11b⁺Ly6G⁺CD11c⁻), CD4⁺ T cells (CD11b⁻CD4⁺CD8⁻), CD8⁺ T cells (CD11b⁻CD8⁺CD4⁻), B cells (CD11b⁻CD4⁻CD8⁻CD19⁺) and natural killer cells (NK; CD11b⁻NK1.1⁺) in AC Res and AC Sens naïve mice and days 1, 2 and 3 after MCAO. Graphs represent absolute number of cells in the ischemic hemisphere (n = 5–9 per group, Supplementary Table 3). Bottom right graph, percentage of leukocyte reduction 2 days after reperfusion (n = 8–9 per group, Supplementary Table 3). Values are mean ± s.e.m. **P < 0.01 versus AC Res; ^#P < 0.05 and ^##P < 0.01 versus neutrophils (Student’s t-test). (b) Cxcl1 and Cxcl2 mRNA expression in brains of AC Res and AC Sens mice 1 d after MCAO (n = 10 per group). Values are fold change over brain from naïve mice. (c) Flow cytometric gating strategy of meningeal PMN in AC Res and AC Sens mice 1 d after MCAO. Graph represents PMN cell number quantification in the meninges (n = 9 per group). (d) Flow cytometry analysis of meningeal IL-17⁺ γδ T cells (CD45^highCD11b⁻TCR-β⁻TCR-γδ⁺IL-17⁺-GFP) of AC Res and AC Sens IL-17-eGFP mice 16 h after MCAO. Left graph represents the total number of γδ T cells and right graph, total number of IL-17⁺ γδ T cells in the meninges of AC Res and AC Sens mice subjected to sham surgery (n = 7 per group) and after MCAO (AC Res, n = 7; AC Sens, n = 6). Each data point (n) is derived by pooling 2 hemispheres. (e) Representative photomicrographs of meningeal γδ T cells (green; TCR-γδ-GFP⁺), laminin (red) and cell nuclei (blue, To-Pro-3) 16 h after MCAO. Coronal section is taken at Bregma + 3 mm. Columns and line plots represent mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; n.s., not significant (Student’s t-test)

Figure 4

Migration of intestinal T cells to the meninges after ischemic brain injury. (a) Photoconversion of the distal part of the small intestine is induced in KikGR mice 7 days prior MCAO using violet light. Photoconverted cells (KikR⁺ cells) are analyzed in different tissues by flow cytometry 16 h after MCAO. (b) Flow cytometry gating strategy in photoconverted KikGR mice. T cells (CD45⁺CD11b⁻TCR⁺) and B cells (CD45⁺CD11b⁻TCR-β⁻TCR-γδ⁻) express the red variant of the protein (KikR⁺) in the meninges 16 h after MCAO. (c) Percentage of photoconverted cells (KikR⁺) of total B or T cells in superficial cervical lymph nodes (scLN; n = 4), deep cervical lymph nodes (dcLN; n = 4) and meninges (n = 4). One data point is derived from 3 pooled animals. Columns represent mean ± s.e.m. *P < 0.05 and **P < 0.01 (one way-ANOVA and Tukey’s test). (d) Proportion of KikR⁺ B and T cells in the LP of the small intestine (SI-LP; n = 8). One data point is derived from one animal. Columns represent mean ± s.e.m. *P < 0.05 (Student’s t-test). (e) Analysis of photoconverted CD45^highCD11b⁻ cells in different organs, showing the different signal from a non-photoconverted and a photoconverted mouse 16 h after ischemia. The gates in the dot plots identify KikR⁺ cells, and the numbers are the percentage of KikR⁺ cells of pan-lymphocytes (CD45^highCD11b⁻).

Figure 5

Neuroprotection conferred by intestinal dysbiosis requires a reduction in intestinal IL-17⁺ γδ T cells. (a) Infarct volume in AC Res (n = 6) and AC Sens (n = 8) IL-17 KO mice as measured by Nissl staining of coronal brain sections 3 days after MCAO (bar, 1 mm). (b) Infarct volume of AC Res (n = 7) and AC Sens (n = 9) IL-10 KO mice on day 3 post MCAO. Representative coronal sections with Nissl staining are shown (bar, 1 mm). (c) The graphs represent the percentage of IL-17⁺ γδ T, T_H17 and FoxP3⁺ cells, respectively, in the LP of the small intestine from AC Res (n = 7) and AC Sens (n = 8) IL-10 KO mice after 2 weeks on antibiotic treatment as measured by flow cytometry. Columns represent mean ± s.e.m.; n.s., not significant (Student’s t-test).

Figure 6

DCs from AC Sens originate in the intestine, induce T_reg cells and downregulate IL-17⁺ γδ T cells in vitro. (a) Flow cytometry analysis of DCs (CD45^highCD11c⁺F4/80⁻Lin⁻; Lin = B220⁺CD3ε⁺) isolated from mLN of AC Sens and AC Res mice. Graph represents percentage of CD11b⁺CD103⁺ subpopulations in CD11c⁺ cells (n = 12 per group). (b) Flow cytometry analyzing T_reg cells (CD4⁺FoxP3⁺) co-cultured with DCs from AC Res or AC Sens mice in absence (n = 7 per group, left plots) or presence of TGF-β1 (n = 5 per group, right plots). Numbers in the dot plots are percentages of T_reg cells of total CD4+ cells. Graphs represent percentage of T_reg (n = 5 per group). (c) Flow cytometric analysis of IL-17 expression in γδ T cells co-cultured with DCs from AC Res or AC Sens mice (n = 9 per group). Gates in the dot plots identify IL-17⁺ γδ T cells, and the numbers are the percentage of IL-17⁺ cells of total γδ T cells. Graph represents percentage IL-17⁺ γδ T (n = 7 per group). (d) Suppression of IL-17⁺ γδ T cells by co-culture with T_reg cells generated in vitro by incubating WT or IL-10 KO CD4+ cells with DC from AC Res or AC Sens mice. (WT n = 11, IL-10 KO n = 9 per data point), Supplementary Table 4). (e) Percentage of T_reg cells induced by DCs from AC Sens in WT and IL-10 KO mice (n = 6 per group). Columns and line plots represent mean ± s.e.m. *P < 0.05, **P < 0.01; n.s., not significant (Student’s t-test). † and #P < 0.05, †† and ##P < 0.01 (one-way ANOVA). (f) Proposed mechanism of protection from ischemic brain injury induced by intestinal microbial dysbiosis. Two weeks of AC results in microbial dysbiosis. Mesenteric lymph node DCs that originate in the small intestine induce T_reg cells. After homing to the gut, IL-10 secreting T_reg cells suppress IL-17⁺ γδ T differentiation. Effector T cells traffic from the intestine to the meninges where a reduction in IL-17⁺ γδ T cells decreases post-ischemic chemokine expression and leukocyte infiltration improving outcome after brain ischemia.