Meningeal γδ T cells regulate anxiety-like behavior via IL-17a signaling in neurons

Abstract

Interleukin (IL)-17a has been highly conserved during evolution of the vertebrate immune system and widely studied in contexts of infection and autoimmunity. Studies suggest that IL-17a promotes behavioral changes in experimental models of autism and aggregation behavior in worms. Here, through a cellular and molecular characterization of meningeal γδ17 T cells, we defined the nearest central nervous system–associated source of IL-17a under homeostasis. Meningeal γδ T cells express high levels of the chemokine receptor CXCR6 and seed meninges shortly after birth. Physiological release of IL-17a by these cells was correlated with anxiety-like behavior in mice and was partially dependent on T cell receptor engagement and commensal-derived signals. IL-17a receptor was expressed in cortical glutamatergic neurons under steady state and its genetic deletion decreased anxiety-like behavior in mice. Our findings suggest that IL-17a production by meningeal γδ17 T cells represents an evolutionary bridge between this conserved anti-pathogen molecule and survival behavioral traits in vertebrates.

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Fig. 1: Dural meninges harbor γδ17 T cells.
Fig. 2: Functional and molecular characterization of meningeal γδ17 T cells.
Fig. 3: Meningeal γδ17 T cells regulate anxiety-like behavior.
Fig. 4: Steady-state expression of IL-17a by meningeal γδ T cells is partially regulated by TCR engagement and commensal-derived signals.
Fig. 5: IL-17a signaling through cortical neurons regulates anxiety-like behavior.
Fig. 6: Neuronal IL-17a signaling shapes the transcriptional landscape of mPFC neurons.

Data availability

FASTQ files and quantified gene counts for single-cell sequencing are available from the Gene Expression Omnibus under accession number GSE147262.

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Acknowledgements

We thank S. Smith for editing the manuscript, J. Sokolowski for help with the collection of human samples and M. Beenhakker for help with interpreting the electrophysiology studies. We thank all members of the Kipnis laboratory and members of the Center for Brain Immunology and Glia (BIG) for valuable comments during multiple discussions of this work. We also thank the UVA Flow Cytometry Core for help with cell sorting, the Genome Analysis and Technology Core for help with library preparation and sequencing, and J. Graham from the Virginia Commonwealth University School of Nursing for performing the Meso Scale Discovery assay. This work was supported by grants from the National Institutes of Health (MH108156, AT010416 and AG034113) to J.K.

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Authors

Contributions

K.A.d.L. designed and performed the experiments, analyzed and interpreted the data and wrote the manuscript. M.W. analyzed the scRNA-Seq raw data and provided intellectual contributions. J.R. helped with the immunohistochemistry studies and provided intellectual contributions. S.D.M. assisted with the behavior experiments and provided intellectual contributions. A.F.S. helped with the sNuc-Seq experiments and carried out the TRAP2 experiments. I.S. performed the surgeries and animal injections. G.C.M. helped with the flow cytometry experiments. T.M. helped with the immunohistochemistry studies. W.B. assisted with the experimental procedures. Z.P. helped with the foraging behavior experiments. M.B.L. provided the human tissue samples. X.S.X. and W.S.C. performed the electrophysiology studies. J.H. helped with the CyTOF experiments. J.K. provided intellectual contribution, oversaw data analysis and interpretation and wrote the manuscript.

Corresponding authors

Correspondence to Kalil Alves de Lima or Jonathan Kipnis.

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Competing interests

J.K. is a member of a scientific advisory group for PureTech Health.

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Editor recognition statement Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Meningeal γδ17 T cells are long-lived cells with low proliferative capacity in steady state.

a, Representative contour plot and histogram of the frequency of meningeal TCR β− or TCR γδ− expressing cells on the double negative (DN, CD4negCD8neg) T cell population under steady state by flow cytometry. b, Representative histograms of CD62L, CD44, IL-1R and RORγt expression in meningeal and splenic γδ T cells from naïve mice by flow cytometry. c, Representative contour plots of the intracellular staining for IL-17a, IFNγ and IL-22 in γδ T cells (TCR γδ+), conventional αβ T cells (TCR β+NK1.1neg) and NK T lymphocytes (TCR β+NK1.1+) isolated from spleen. d, Graph bars for the frequency of IL-17a, IFNγ and IL-22 for each isolated meningeal T cell subset. di, Meningeal γδ T cells; dii, conventional αβ T cells and diii, NK T lymphocytes (n = 5 per group). One-way ANOVA followed by Bonferroni’s multiple comparisons test. e, Absolute number of γδ T cells isolated from dura, enriched subdural meninges (eSDM), choroid plexus or brain parenchyma under homeostasis (n = 5 per group). One-way ANOVA followed by Bonferroni’s multiple comparisons test. fi, Gating strategy for the flow analysis of fresh dura sample isolated from humans. Single live cells were gated as CD45+ and the γδ T cell frequency was determined in the CD3+. fii, Contour plots of the frequency of γδ T cells isolated from arachnoid or dura mater by flow cytometry (n = 2). gi, Representative dot plots of the percentage of TCR γδ− or αβ− expressing cells obtained from meninges during prenatal stages (around E18.5) or at 8-weeks of age (Adult). gii, Graph bars of the percentage and giii, absolute number of γδ T cells. Each pre-natal sample represents a pool of three embryos. Unpaired two-tailed t test. Data are from one single experiment. hi, Representative flow cytometry analysis of γδ T lymphocytes in the blood (top), and meninges (bottom) of CD45.1+ and CD45.2+ congenic parabiotic pairs joined for 4 weeks (n = 4 per group). hii, Mean frequency of either CD45.1+ or CD45.2+ γδ T cells in each parabiotic pair in the blood and meninges. i, WT mice at post-natal day 14 (P14) or j, adult (8 week-old) were injected daily with EdU (i.p., 10 mg/kg) and three days later the proliferating EdU+ cells were determined by flow cytometry. ii, Histograms showing the EdU frequency in γδ T cells and CD11bhi myeloid cells isolated from meninges and spleen of P14 mice. iii, Graph bars of the percentage of γδ T cells or iiii, CD11bhi myeloid cells co−expressing EdU. P14 (n = 5 per group). ji, Representative histograms of γδ T lymphocytes and CD11bhi myeloid cells expressing EdU in meninges and spleen of adult mice. jii, Graph bars of the percentage of γδ T cells and jiii, CD11bhi myeloid cells co-expressing EdU. Adult (n = 5 per group). ki, Scheme of the experimental details used to assess the proliferation capacity of meningeal γδ T cells by immunohistochemistry. P14 or adult TcrdCreERT2:Ai6 mice were injected daily with EdU and tamoxifen (both i.p., 10 mg/kg) and three days later the proliferation of γδ T cells was determined in the whole-mount meninges by confocal microscopy. kii, Representative images of meningeal whole-mounts isolated from P14 (left) and Adult (right) mice and stained for CD31 (blue) and EdU (red). Arrows indicate γδ T cells proliferating in P14 meninges (TCR δ (ZsGreen)+EdU+). SSS, superior sagittal sinus. Scale bar, 100 μm. Representative image of one single experiment (n = 5). Data are shown as mean ± s.e.m. and each dot symbol represents individual mouse.

Extended Data Fig. 2 Meningeal γδ T cells expression profile and additional behavior paradigms using γδ T cell-deficient mice.

a, Normalized expression of Tcrgv6 transcripts (Vγ4 chain, Garman nomenclature) in meningeal and splenic γδ T cells obtained by single cell RNA-Seq. b, Graph bars of the TCR Vγ-usage in γδ T cells. bi, Meningeal and bii, splenic γδ T cells. biii, Overview of Vγ-chain usage (Vγ1, Vγ2, Vγ3 or others (Vγ4 or Vγ5)). ci, Representative contour plots of the intracellular staining for IL-17a and IFNγ in αβ T cells (TCR β+NK1.1neg) and γδ T lymphocytes (Vγ1, Vγ2, Vγ3−negative or positive fractions) isolated from steady state meninges. cii-v, Graph bars of the percentage of IL-17a− or IFNγ− producing cells in these different meningeal T cell subsets. One-way ANOVA followed by Bonferroni’s multiple comparisons test. d, UMAP projection of γδ T cells colored by the scaled expression of Slc6a13 (GAT2). e, Average expression of chemokines in CD45+ cells isolated from steady state dura mater obtained from public database21. f, Representative histogram of the expression of CXCR6-GFP in meningeal T cell subsets. g, Absolute numbers of conventional αβ T cells isolated from CXCR6−sufficient and −deficient mice. Unpaired two-tailed t test. Data represent one independent experiment. hi, Adult Ccr2CreERT2:Ai6 mice were injected daily with tamoxifen (i.p., 10 mg/kg) and three days later meninges, spleen and blood were harvested for FACS analysis. Representative histograms of CCR2-ZsGreen expression in γδ T cells and conventional αβ T lymphocytes by flow cytometry. hii, Graph bars of the percentage of γδ T cell− and hiii, αβ T cell− expressing CCR2-ZsGreen in spleen, blood and meninges. hiv, Histograms of the expression of CCR2-ZsGreen in meningeal γδ and αβ T lymphocytes. hv, Frequency of CCR2−expressing cells. One-way ANOVA followed by Bonferroni’s multiple comparisons test. ii, Representative dot plots of the percentage of meningeal TCR γδ− and TCR β− expressing cells isolated from WT or Ccr2−/− (Ccr2RFP/ RFP) mice. iii, Percentage and iiii, absolute number of meningeal γδ T cells. iiv, Percentage and iv absolute number of meningeal αβ T cells. Unpaired two-tailed t test. ji, Dot plots of the frequency of inflammatory monocytes (CD11bhiLy6Chi) isolated from WT or CCR2-deficient mice. jii, Percentage and jiii, absolute number of meningeal inflammatory monocytes. Data are pooled from two independent experiments with similar results. Unpaired two-tailed t test. k, Social interaction of wild-type and Tcrd−/− mice was assessed in the three-chamber test and the percentage of time investigating either a mouse (social) or an object (inanimate) was calculated. WT (n = 9); Tcrd−/− (n = 11). Two-way ANOVA followed by Bonferroni’s multiple comparisons test. li, For the spatial memory task, WT and γδ T cell-deficient mice were assessed in the training and lii, novel location tasks of the novel location recognition (NLR) test and the time spent with either a familiar or novel object was determined. WT (n = 8); Tcrd−/− (n = 8). Two-way ANOVA followed by Bonferroni’s multiple comparisons test. m, Foraging behavior of wild-type and Tcrd−/− mice assessed in the exploratory and n, foraging phase of the foraging paradigm. Data is pooled from three independent cohorts. WT (n = 18); Tcrd−/− (n = 18); Unpaired two-tailed t test. Data are shown as mean ± s.e.m. and each dot symbol represents individual mouse.

Extended Data Fig. 3 γδ17 T cells are localized in the meningeal stroma and actively transcribe IL-17a in vivo.

a, Flow cytometry analysis of meninges three days after treatment with 25 μg of isotype control or anti-TCR γδ (i.c.m.). ai, Percentage of CD45+ live cells. aii, Frequency of αβ T lymphocytes gated on the CD45+ fraction. aiii, Absolute number of CD45+ leukocytes, aiv, αβ T cells and av, γδ T cells in the meninges. Isotype control (n = 3); anti-TCR γδ (n = 3). b, Absolute number of γδ T cells of mice treated as described in a. To bypass their TCR staining, γδ T cells were gated as Thy1.2+TCR βnegRORγt+ cells. Isotype control (n = 4); anti-TCR γδ (n = 4). c, Levels of TCR γδ expression in splenic γδ T cells three days after treatment with 2.5 μg of isotype control or anti-TCR γδ (i.c.m.). Isotype control (n = 4); anti-TCR γδ (n = 4). d, Expression per cell of IL-17a in Thy1.2+TCR βnegRORγt+IL-17a+ T cells (“visible” and “invisible” γδ T cells). Isotype control (n = 4); anti-TCR γδ (n = 4). ei, WT mice were injected with 2.5 μg of anti-TCR γδ or isotype control (i.c.m.) and seven days later were assessed in the elevated plus maze. Representative track plots of the cumulated movement. eii, Percentage time spent in the open arms of the maze. Isotype control (n = 10); anti-TCR γδ (n = 10). fi, Representative histogram of the levels of TCR γδ expression in meningeal γδ17 T cells seven days after injection with isotype control or anti-TCR γδ. fii, Contour plots of the percentage of IL-17a− and IFNγ−producing meningeal T lymphocytes. fiii, Absolute number of meningeal γδ T cells. fiv, Analysis of the TCR γδ MFI (gated on γδ T cells). fv, Graph bars for the percentage of IL-17a−producing meningeal γδ T cells or fvi, αβ T cells. Isotype control (n = 3); anti-TCR γδ (n = 3). g, Homeostatic IL-17a-eGFP expression in meningeal T cell by immunohistochemistry using the IL-17a reporter mice (Il17aeGFP/eGFP). Insets of the superior sagittal sinus (SSS) of meningeal dural whole-mounts showing CD3 (red), IL-17a (eGFP, green) and CD31 (blue) staining in adult mice. White arrows highlight γδ T cells (defined by the CD3 and IL-17a double-staining). Scale bar, 100 μm. Representative image of at least three independent experiments with similar results (n = 3 per experiment). hi, Representative FACS plot of the IL-17a-eGFP expression of meningeal and splenic γδ T cells under steady state. hii, Graph bars of IL-17a−expressing cells in the meninges or hiii, spleen (n = 3 per group). ii, Representative dot plots of IL-17a-eGFP+ meningeal T cells three days after treatment with 2.5 μg of anti-TCR γδ treatment (i.c.m.). To bypass their TCR staining, “visible” and “invisible” γδ T cells were gated as Thy1.2+RORγt+ cells. iii, Frequency of IL-17a-eGFP+ meningeal γδ17 T cells (defined as Thy1.2+RORγt+TCR βneg cells). iiii, IL-17a expression per a cell basis on IL-17a-eGFP−expressing γδ T cells. Isotype control (n = 5); anti-TCR γδ (n = 5). ji, Scheme of the experimental approach used to label intravascular and tissue-localized cells in steady state meninges. WT mice were given 0.25 μg of anti-CD45 (i.v.) and 3 min later blood was collected. Animals were perfused, and meninges were harvested for flow cytometry analysis. jii, Representative dot plots and jiii, Graph bars of either intravascular (i.v. CD45+) or tissue-localized (i.v. CD45neg) γδ T cells (n = 4 per group). k, Representative meningeal coronal section of IL-17a-eGFP reporter mice stained for DAPI (blue), CD31 (white), and CD3 (red). Arrows indicate a T cell expressing IL-17a-eGFP in meningeal stroma. Scale bar, 100 μm. Representative image of one single experiment (n = 5). l, MSD assay of CSF obtained from WT and Tcrd−/− mice. Technical replicate of meninges pooled from WT or Tcrd−/− mice (n = 10 per group). ND, not detected. m, Steady state concentration of plasmatic IL-17a obtained from WT and Tcrd−/− mice by MSD assay (n = 6 per group). Data are shown as mean ± s.e.m. and each dot symbol represents individual mouse. a–f, h, i, and m, Unpaired two-tailed t test.

Extended Data Fig. 4 Pathways and stimuli involved in the activation of meningeal γδ17 T cells.

a, Stress was induced in IL-17a-eGFP mice by the exposure to electronical foot shock (ES) for seven consecutive days. ai, Representative dot plots of the IL-17a-eGFP expression by meningeal T cells by flow cytometry. aii, Frequency of IL-17a-eGFP+ γδ17 T cells or aiii, conventional αβ T cells. Data are from one single experiment. Naive (n = 5); ES (n = 5). b, Unpredictable chronic mild stress (UCMS) was induced for over eight weeks in WT mice. bi, Representative expression of IL-17a and IFNγ assessed by the intracellular staining following ex vivo stimulation. bii, Percentage of IL-17a− or biii, IFNγ− producing γδ T cells. Naive (n = 4); UCMS (n = 5). Data are from one single experiment. ci, Percentage of conventional αβ and γδ T cells isolated from meninges of specific pathogen free (SPF) and germ-free mice. cii, Representative contour plots of the percentage of IL-17a−positive meningeal γδ cells. ciii, Graph bars of the frequency and civ, absolute number of meningeal γδ T lymphocytes in SPF versus germ-free animals. cv, Analysis of the frequency and cvi, absolute number of meningeal γδ T cell-producing IL-17a. SPF (n = 5); Germ-free (n = 5). di, Contour plots of the percentage of IL-17a−positive meningeal γδ cells obtained from meninges of untreated (No Abx) or broad-spectrum antibiotics-treated (Abx) animals and assessed by ex vivo stimulation. dii, Graph bars of the frequency and diii, absolute number of IL-17a−producing meningeal γδ T lymphocytes. No Abx (n = 3); Abx (n = 4). ei, Representative contour plots of IL-17a and IFNγ expression by meningeal γδ T cells 24 h after aryl hydrocarbon receptor (AHR) activation by the endogenous ligand 6-Formylindolo[3,2-b]carbazole (FICZ, 10 ng, i.c.m.). eii, Analysis of the frequency of IL-17a− and eiii, IFNγ− producing cells assessed by ex vivo stimulation. N = 3 per group. fi, Representative dot plots of the percentage of TCR γδ- or αβ-expressing cells obtained from WT or Il1r1−/− mice. fii, Analysis of the percentage and fiii, absolute number of meningeal γδ T cells. fiv, Graph bars of the frequency and fv, absolute number of meningeal αβ T lymphocytes. WT (n = 5); Il1r1−/− (n = 5). gi, Contour plots of the IL-17a expression by meningeal γδ T cells following the ex vivo stimulation by PMA/Ionomycin. gii, Analysis of the percentage and giii, absolute number of IL-17a−producing γδ T cells. WT (n = 5); Il1r1−/− (n = 5). hi, Representative dot plots of the percentage of TCR γδ- or αβ-expressing cells obtained from WT or Il23a−/− mice. hii, Analysis of the frequency and hiii, absolute number of meningeal γδ T cells. hiv, Graph bars of the percentage and hv, absolute number of meningeal αβ T lymphocytes. WT (n = 3); Il23ra−/− (n = 3). ii, Contour plots of the IL-17a and IFNγ expression by meningeal γδ T cells following ex vivo stimulation. iii, Analysis of the percentage and iiii, absolute number of IL-17a−producing γδ T cells. WT (n = 3); Il23ra−/− (n = 3). ji, Representative contour plots of the percentage of IL-17a−eGFP+ meningeal Τ cells seven days after active EAE induction (EAE 7dpi). jii, Graph bars of the frequency of meningeal γδ T lymphocytes (total TCR γδ+ cells), jiii, conventional αβ T cells (TCR β+ cells) and jiv, Vγ2+ γδ T cells (gated on total γδ T cells). jv, Percentage of IL-17a−eGFP+ cells gated on innate-like γδ T cells (Vγ2neg) and jvi, expression per cell of IL-17a. jvii, Frequency of IL-17a−eGFP+ αβ T cells. Naive (n = 4); EAE (n = 5). ki, Representative contour plots of the percentage of IL-17a−eGFP+ meningeal Τ cells 14 days post EAE induction (EAE 14dpi). kii, Graph bars of the frequency of meningeal γδ T lymphocytes (TCR γδ+ cells), kiii, αβ T cells and kiv, Vγ2+ γδ T cells (gated on γδ T cells). kv, Percentage of IL-17a−eGFP+ cells gated on innate-like γδ T cells (Vγ2neg) and kvi, expression per cell of IL-17a−eGFP. kvii, Frequency of IL-17a−eGFP+ cells in αβ T cells. Naive (n = 4); EAE (n = 4). Data are shown as mean ± s.e.m. and each dot symbol represents individual mouse. a–k, Unpaired two-tailed t test.

Extended Data Fig. 5 Lack IL-17a signaling does not affect social or learning behaviors.

ai, Representative track plots of the cumulative total distance of Il17a+/− and Il17a−/− mice in the elevated plus maze test. aii, Percentage of total time spent in the open arms. Il17a+/− (n = 11) and Il17a−/− (n = 10). Unpaired two-tailed t test. b, Representative images of IL-17Ra (green), NeuN (red) and DAPI (blue) staining in the PFC, c, hippocampus and d, cerebellum of WT mice. b–d, Representative image of at least three independent experiments with similar results (n = 3 per experiment). Scale bar, 100 μm. e, Expression of Il17ra (Cyan) at anterior–posterior (AP) + 1.32 mm, f, AP: −2.75 and g, in the cerebellum by in situ hybridization. e, f, Scale bar, 500 μm. g, Scale bar, 100 μm. Il17ra (Cyan) was co-labelled with GABAergic (Gad2, green), glutamatergic markers (Slc17a7, magenta), and DAPI (blue). e–g, Representative image of 5 independent experiments with similar results (n = 3 per experiment).hi, AAV Syn1Cre was bilaterally injected into the mPFC of WT or Il17rafl/fl mice. Four weeks later, mPFC slices were prepared for immunohistochemistry analysis. hii Representative images of IL-17Ra expression in the mPFC of WT or Il17rafl/fl. hii Analysis of the efficiency transfection. hiii Coverage of IL-17Ra staining and hiv Quantification of IL-17Ra-positive cells as a fraction of total NeuN+ cells. Scale bar, 30 μm. WT (n = 5) and Il17rafl/fl (n = 5), with 20 slices per mouse. Unpaired two-tailed t test. i, Sociability assay was performed in the three-chamber test and the percentage of time investigating either a mouse (social) or an object (inanimate) was calculated. WT (n = 9) and Il17rafl/fl (n = 11). Two-way ANOVA followed by Bonferroni’s multiple comparisons test. ji, In the fear conditioning paradigm, the percentage of time freezing was evaluated in the context and jii, cued trial. WT: AAV Syn1Cre (n = 10) and Il17rafl/fl: AAV Syn1Cre (n = 9). k, AAV Syn1Cre was bilaterally injected into the S1DZ region of WT or Il17rafl/fl mice. Mice were tested in the elevated plus maze four weeks later. Percentage of total time spent in the open arms of the maze is presented. WT (n = 8) and Il17rafl/fl (n = 10). Unpaired two-tailed t test. Data represents one single experiment. Data is presented as mean ± s.e.m. and each dot symbol represents individual mouse.

Extended Data Fig. 6 Neuronal loss of IL-17a signaling changes the transcriptional landscape of mPFC neurons.

ai, Representative track plots of the cumulative total distance of Syn1Cre, Il17rafl/fl and Syn1Cre:Il17rafl/fl tested in the elevated plus maze test just before nuclei isolation for the scRNA-Seq experiment. aii, Percentage of total time spent in the open arms. Il17rafl/fl (n = 5); Syn1Cre (n = 5); and Syn1Cre:Il17rafl/fl (n = 4). Data represents one single experiment. Il17rafl/fl vs. Syn1Cre:Il17rafl/fl, P = 0.049; Syn1Cre vs. Syn1Cre:Il17rafl/fl; P = 0.011; One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test. b, Heatmap showing cluster signatures across all mPFC nuclei. Each line represents the scaled expression of the corresponding gene within each nucleus, with more highly expressed genes shown in black. The top five most highly expressed genes from each cluster (compared against all other clusters) are shown. c, Dot plot showing the expression of canonical neuronal subclass markers across each of our Seurat identified clusters. Color represents the scaled average expression and size indicates the percentage of cells within each cluster expressing the gene. di, Dot plot showing the expression of Slc17a7, Gad2 and Il17ra among 2,590 nuclei isolated from the mouse hippocampus (dentate gyrus). Color represents the scaled average expression and size indicates the percentage of cells within each cluster expressing the gene. dii, violins show the normalized expression of Il17ra within each cell across clusters. e, Bar graph showing the proportion of cells from each sample belonging to each cluster across all mPFC nuclei. f, Violins show the normalized expression of Il17ra within each cell across clusters. g, Dot plot showing information about each cluster. In the top row, dot size indicates the total number of cells belonging to the cluster and color indicates the number of differentially expressed genes (DEG) overlapping between Syn1Cre:Il17rafl/fl vs. Syn1Cre and Syn1Cre:Il17rafl/fl vs. Il17rafl/fl (adjusted p value < 0.05, absolute log fold change > 0.1). In the second row, the size of the dot shows the percentage of cells within the cluster expressing Il17ra while the color represents the scaled average expression of Il17ra across cells in the cluster. h, Heatmaps showing overlapping differentially expressed genes between Syn1Cre:Il17rafl/fl vs. Syn1Cre and Syn1Cre:Il17rafl/fl vs. Il17rafl/fl that are significantly changed in all three layer subsets (far left), the top 50 (based on absolute average log fold change) differentially expressed genes that changed uniquely in Layer II/III (left), and the significantly differentially expressed genes that were uniquely changed in Layer V (right) and Layer VI (far right) with the loss of functional Il17ra. Above each heatmap is a pie chart showing the percentage of significantly differentially expressed genes combined across all layers, that belong to each category described (shared, unique to Layer II/III, unique to Layer V, or unique to Layer VI). i, Plots showing the most significantly enriched KEGG pathways based on the set of significantly up- (top) or down- (bottom) regulated genes shared between Syn1Cre:Il17rafl/fl vs. Syn1Cre and Syn1Cre:Il17rafl/fl vs. Il17rafl/fl with colored squares indicating the contribution of each gene to the corresponding enriched pathway.

Extended Data Fig. 7 IL-17a effects on electrophysiological parameters of mPFC neurons.

a–f, Spontaneous excitatory postsynaptic action potentials (sEPSCs) in the mouse mPFC before and after IL-17a application via bath-application for 20 to 25 min (n = 8 neurons). The data points represent sEPSCs frequency or amplitude on a given neuron pre- and post- IL-17a treatment (blue and orange dots respectively – plotted on the right y-axis). Light grey bars represent the frequency or peak amplitude (pA) as % of control in the given neuron (mean and s.e.m., plotted on the left y-axis). ai, Recordings of spontaneous action potentials before (pre) and after (post) IL-17a application (1 ng/mL). aii, Frequency of spontaneous action potentials pre and post treatment of 1 ng/mL of IL-17a. bi, Representative amplitude of the spontaneous excitatory postsynaptic action potentials in the mouse mPFC before and after application of IL-17a (1 ng/ml) as described before. bii, Analysis of the peak amplitude. ci, Recordings of spontaneous action potentials before and after IL-17a application (10 ng/mL). cii, Frequency of spontaneous action potentials pre and post treatment of 10 ng/mL of IL-17a. di, Representative amplitude of the spontaneous excitatory postsynaptic action potentials before and after application of IL-17a (10 ng/ml). dii, Analysis of the peak amplitude. ei, Recordings of spontaneous action potentials before and after IL-17a application (100 ng/mL). eii, Frequency of spontaneous action potentials pre and post treatment of 100 ng/mL of IL-17a. fi, Representative amplitude of the spontaneous excitatory postsynaptic action potentials before and after application of IL-17a (100 ng/ml). fii, Analysis of the peak amplitude. g–l, Spontaneous inhibitory postsynaptic action potentials (sIPSCs) in the mouse mPFC before and after IL-17a application via bath-application for 20 to 25 min (n = 8 neurons). gi, Recordings of spontaneous action potentials before and after IL-17a application (1 ng/mL). gii, Frequency of spontaneous action potentials pre and post treatment of 1 ng/mL of IL-17a. hi, Representative amplitude of the spontaneous inhibitory postsynaptic action potentials before and after application of IL-17a (1 ng/ml). hii, Analysis of the peak amplitude. ii, Recordings of spontaneous action potentials before and after IL-17a application (10 ng/mL). iii, Frequency of spontaneous action potentials pre and post treatment of 10 ng/mL of IL-17a. ji, Representative amplitude of the spontaneous inhibitory postsynaptic action potentials before and after application of IL-17a (10 ng/ml). jii, Analysis of the peak amplitude. ki, Recordings of spontaneous action potentials before and after IL-17a application (100 ng/mL). kii, Frequency of spontaneous action potentials pre and post treatment of 100 ng/mL of IL-17a. li, Representative amplitude of the spontaneous inhibitory postsynaptic action potentials before and after application of IL-17a (100 ng/ml). lii, Analysis of the peak amplitude. a–l, Wilcoxon matched-pairs signed rank test.

Extended Data Fig. 8 IL-17a effects on the evoked action potentials before and after IL-17a treatment.

ai-iii, Recordings of evoked action potentials in a neuron in the mouse mPFC before (pre) and after (post) IL-17a application (n = 8 neurons). The current injection protocol (−40, 0, 40, 80, 120, 160, 200 pA; 500 ms) is shown in gray underneath the traces. bi-iii, Graphs show the frequency of action potentials (y-axis) in response to different levels of current injection (x-axis) before and after IL-17a bath-application for 20 to 25 min. biv, Graph summarizes the change in frequency pre and post IL-17a treatment. Two-way ANOVA followed by Sidak’s multiple comparisons test. Data points and error bars represent the mean and s.e.m., respectively, of n = 8 sets (Pre vs. Post IL-17a treatment).ci, Scheme of the experimental details to target IL-17a−responding cells. FosCreERT2:Ai14 mice were treated with 25 ng of IL-17a or saline (i.c.m.) and then given an injection of 4-hydroxytamoxifen (4-OHT, i.p.) one hour later. After seven days, animals were perfused and the mPFC was collected for immunohistochemistry staining and analysis. cii, Representative photomicrographs from the mPFC of mice injected with IL-17a or saline and stained for a neuronal marker, NeuN (blue), DAPI (red) and showing c-Fos expression (green). ciii, Quantification of c-Fos+ cells in the mPFC of stimulated mice. Scale bar, 200 μm. aCSF (n = 5); IL-17a (n = 5). Unpaired two-tailed t test. Representative image of two independent experiments with similar results (n = 5 per experiment). Data is presented as mean ± s.e.m. and each dot symbol represents individual mouse.

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Alves de Lima, K., Rustenhoven, J., Da Mesquita, S. et al. Meningeal γδ T cells regulate anxiety-like behavior via IL-17a signaling in neurons. Nat Immunol (2020). https://doi.org/10.1038/s41590-020-0776-4

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