Research ArticleNEUROIMMUNOLOGY

Meningeal γδ T cell–derived IL-17 controls synaptic plasticity and short-term memory

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Science Immunology  11 Oct 2019:
Vol. 4, Issue 40, eaay5199
DOI: 10.1126/sciimmunol.aay5199
  • Fig. 1 Fetal-derived γδ T cells infiltrate the meninges from birth.

    Meningeal cell suspensions were prepared from 8- to 20-week-old C57BL/6J WT and Il17aCre R26ReYFP mice (A, B, D, and E), 0- to 52-week-old C57BL/6J WT mice (C), and 20-week-old WT and WT ➔ WT BMC mice (F). Samples were analyzed for the expression of indicated surface (CD45, CD3, TCRδ, CD4, CD8, Vγ1, Vγ4, Vγ5, and Vγ6), markers. Live cells were gated using LiveDead Fixable Viability dye as shown in (A). Dot plots represent cell populations from indicated gates. Histograms depict percentages or absolute numbers from indicated populations. Meningeal spaces were pooled from four mice. Spleens were analyzed from individual mice. Results are representative of four to seven independent experiments. Error bars, mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 as calculated by Student’s t test (parametric) or Mann-Whitney U test (nonparametric).

  • Fig. 2 Meningeal γδ T cells are biased toward IL-17 production.

    Meningeal cell suspensions were prepared from 8- to 20-week-old C57BL/6J WT and Il17aCre R26ReYFP mice (A to E), 0- to 52-week-old C57BL/6J WT mice (G), and 20-week-old WT and WT ➔ WT BMC mice (F). Samples were analyzed for the expression of indicated surface (CD45, CD3, TCRδ, CD4, CD8, CD27, and CCR6) and intracellular (RORγt, Tbet, IL-17, and IFN-γ) markers. Live cells were gated using LiveDead Fixable Viability Dye as shown in (A). Dot plots represent cell populations from indicated gates. Histograms depict percentages or absolute numbers from indicated populations. Meningeal spaces were pooled from four mice. Spleens were analyzed from individual mice. Results are representative of four to seven independent experiments. Error bars, mean ± SEM. *P < 0.05 and **P < 0.01, as calculated by Mann-Whitney U test.

  • Fig. 3 Meningeal γδ T cell homeostasis is independent of inflammatory signals.

    Cell suspensions were prepared from the meninges of 8- to 12-week-old C57BL/6J WT mice, bred in an SPF (A to D) versus GF (A) environment, treated or not with an antibiotic cocktail (Abx) (B), and compared with IL-1R−/−, IL-23R−/− (C), TLR2−/−, TLR4−/−, Caspase 1−/−, and NOD1−/− mice (D). Percentages of γδ T cells and IL-17 producers were analyzed by FACS as illustrated in Fig. 1. Results are representative of two to four independent experiments.

  • Fig. 4 γδ T cells producing IL-17 are required for short-term memory.

    (A) Representative track line from indicated animals exploring the short-term Y-maze. (B to D) Cognitive performance in the short-term Y-maze evaluated by discrimination ratio between the novel arm (N) versus the other arm (O) of IL-17−/− and TCRδ−/− compared with respective littermate controls (n = 21 to 32) (B), WT ➔ WT BMC mice (n = 23 to 27) (C), and WT after intracerebroventricular injection of isotype control (IgG) or anti–IL-17 (aIL-17) (n = 10 to 12) (D). (E) Percentages of swimming time in the test quadrant of IL-17−/− and TCRδ−/− and respective littermate controls during the probe test of the long-term MWM (n = 10 to 14). (F) Cognitive performance in the long-term Y-maze evaluated by discrimination ratio between the novel arm (N) versus the other arm (O) of IL-17−/− and TCRδ−/− compared with respective littermate controls (n = 10 to 16). (G) Representative track line from indicated animals exploring the short-term MWM during the probe test, after a training phase with a platform in the lower left quadrant of the pool. (H) Corresponding percentages of time spent in the test quadrant of IL-17−/− and TCRδ−/− and respective littermate controls (n = 8 to 16). Results are representative of two to three independent experiments in male mice. Error bars, mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. Paired Student’s t test and one-way ANOVA followed by Bonferroni’s multiple comparison test were used to analyze discrimination ratio (%) and time in quadrant (%), respectively.

  • Fig. 5 IL-17 modulates synaptic plasticity and AMPA/NMDA ratio upon a short-term memory task.

    (A to C) Time course (left panels) and magnitude (right panels) of LTP induced by theta-burst stimulation (TBS) in hippocampal slices from WT and IL-17−/− mice at steady state (A) after training in the short-term Y-maze (B) and after training in the long-term MWM (C). When indicated, hippocampal slices from IL-17−/− mice were supplemented with IL-17 (10 ng/ml) (n = 3 to 7, Kruskal-Wallis test followed by Dunn’s multiple comparisons test). (D and E) I/O curves corresponding to the fEPSP slope evoked by different stimulation intensities (0.8 to 2.8 mA) of IL-17−/− compared with WT mice at steady state (D) after training in the short-term Y-maze test (E) (n = 5 to 7, F test). (F) Representative traces of EPSCs recorded at −70 mV and +40 mV in neurons from WT and IL-17−/− mice after training in the short-term Y-maze (left panels). Arrows indicate the amplitudes considered to calculate AMPAR/NMDAR ratio, depicted in the right panel. n = 11 to 12, unpaired t test. (G) Paired-pulse facilitation, EPSCs at 50-ms interpulse intervals in WT and IL-17−/− after Y-maze (n = 5 to 7, Mann-Whitney test). Data are mean ± SEM. **P < 0.01 and ***P < 0.001.

  • Fig. 6 IL-17 promotes glial BDNF production.

    (A) BDNF concentration in mixed glial cultures supplemented with IL-17 normalized to the control condition (n = 4 to 6, Mann-Whitney U test). (B) BDNF concentration in the hippocampus at steady state after short-term Y-maze test and after long-term MWM test (n = 4 to 9, Mann-Whitney U test). (C) I/O curves corresponding to the fEPSP slope evoked by different stimulation intensities (0.8 to 3.0 mA) of WT, IL-17−/−, and L-17−/− supplemented with BDNF (30 ng/ml) after short-term Y-maze test (n = 5 to 6, F test). (D to E) Time course (left panel) and magnitude (right panel) of LTP induced by TBS in the CA1 region of hippocampal slices of (D) WT, IL-17−/−, and IL-17−/− supplemented with BDNF (30 ng/ml) and (E) WT, TCRδ−/−, and TCRδ−/− supplemented with BDNF (30 ng/ml) after short-term Y-maze test (n = 4 to 7, Kruskal-Wallis test followed by Dunn’s multiple comparisons test). Raw data from IL-17−/− in (C) and (D) are adapted from Fig. 3 (B and E). Data from WT are the same in (D) and (E). (F) Cognitive performance in Y-maze evaluated by discrimination ratio between novel arm (N) versus the other arm (O) of WT and IL-17−/− mice tested after intracerebroventricular injection of PBS (vehicle, vhc) or BDNF (n = 8 to 16, paired Student’s t test). Results are representative of two to five independent experiments. Data are mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/40/eaay5199/DC1

    Fig. S1. Tissue-resident Vγ6+ γδ T cells are enriched in IL-17 producers in the perinatal period.

    Fig. S2. γδ T cells are absent from the brain parenchyma.

    Fig. S3. Absence of IL-17 or γδ T cells does not impair mice exploratory behavior or anxiety.

    Fig. S4. Exploratory behavior in short-term Y-maze test is unaffected in IL-17−/−, TCRδ−/−, BMC, and WT mice after intracerebroventricular injection.

    Fig. S5. Short-term memory is affected in IL-17−/−, TCRδ−/−, BMC, and WT mice after intracerebroventricular injection of anti–IL-17.

    Fig. S6. IL-17−/− and TCRδ−/− females display short-term memory deficits but normal exploratory behavior in the Y-maze.

    Fig. S7. Mice deficient in IL-17 or γδ T cells share the same gut microbiota as littermate controls.

    Fig. S8. Long-term memory in the MWM is not affected by the absence of IL-17 or γδ T cells.

    Fig. S9. Long-term memory in the Y-maze is not affected in the absence of IL-17 or γδ T cells.

    Fig. S10. Short-term memory in the MWM is impaired in the absence of IL-17 or γδ T cells.

    Fig. S11. Proteomics analyses reveal mild changes in distinct signaling pathways in the IL-17−/− hippocampus.

    Fig. S12. Proteomics analyses reveal mild changes in discrete synaptic pathways in the IL-17−/− hippocampus.

    Fig. S13. TCRδ−/− mice display impaired basal transmission after short-term Y-maze.

    Fig. S14. Conditional depletion of IL-17RA in microglia, astrocytes, or both does not fully recapitulate Y-maze deficits observed in IL-17−/− mice.

    Table S1. Proteomic dataset analysis.

    Table S2. List of antibodies used for FACS analysis.

    Table S3. Raw data sets for main figures.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Tissue-resident Vγ6+ γδ T cells are enriched in IL-17 producers in the perinatal period.
    • Fig. S2. γδ T cells are absent from the brain parenchyma.
    • Fig. S3. Absence of IL-17 or γδ T cells does not impair mice exploratory behavior or anxiety.
    • Fig. S4. Exploratory behavior in short-term Y-maze test is unaffected in IL-17−/−, TCRδ−/−, BMC, and WT mice after intracerebroventricular injection.
    • Fig. S5. Short-term memory is affected in IL-17−/−, TCRδ−/−, BMC, and WT mice after intracerebroventricular injection of anti–IL-17.
    • Fig. S6. IL-17−/− and TCRδ−/− females display short-term memory deficits but normal exploratory behavior in the Y-maze.
    • Fig. S7. Mice deficient in IL-17 or γδ T cells share the same gut microbiota as littermate controls.
    • Fig. S8. Long-term memory in the MWM is not affected by the absence of IL-17 or γδ T cells.
    • Fig. S9. Long-term memory in the Y-maze is not affected in the absence of IL-17 or γδ T cells.
    • Fig. S10. Short-term memory in the MWM is impaired in the absence of IL-17 or γδ T cells.
    • Fig. S11. Proteomics analyses reveal mild changes in distinct signaling pathways in the IL-17−/− hippocampus.
    • Fig. S12. Proteomics analyses reveal mild changes in discrete synaptic pathways in the IL-17−/− hippocampus.
    • Fig. S13. TCRδ−/− mice display impaired basal transmission after short-term Y-maze.
    • Fig. S14. Conditional depletion of IL-17RA in microglia, astrocytes, or both does not fully recapitulate Y-maze deficits observed in IL-17−/− mice.
    • Table S1. Proteomic dataset analysis.
    • Table S2. List of antibodies used for FACS analysis.
    • Legend for table S3

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    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Raw data sets for main figures.

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