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

T cells that promote memories

There is now a growing appreciation of cross-talk between the nervous and immune systems. Here, Ribeiro et al. report that interleukin-17 (IL-17)–producing γδ T cells that arise early in life and home to the meninges of neonatal mice play an essential role in the development of short-term memories. Both mice lacking γδ T cells and IL-17 show impaired performance in Y-maze tests that rely on short-term memory but did not show any deficits in other behavioral tests, including tests that gauge long-term memory. The authors found that meningeal IL-17–producing γδ T cells promote secretion of brain-derived neurotropic factor in mouse brains. Further studies are needed to understand the local and systemic effects of IL-17 on neuronal and non-neuronal cells in the brain.


The notion of “immune privilege” of the brain has been revised to accommodate its infiltration, at steady state, by immune cells that participate in normal neurophysiology. However, the immune mechanisms that regulate learning and memory remain poorly understood. Here, we show that noninflammatory interleukin-17 (IL-17) derived from a previously unknown fetal-derived meningeal-resident γδ T cell subset promotes cognition. When tested in classical spatial learning paradigms, mice lacking γδ T cells or IL-17 displayed deficient short-term memory while retaining long-term memory. The plasticity of glutamatergic synapses was reduced in the absence of IL-17, resulting in impaired long-term potentiation in the hippocampus. Conversely, IL-17 enhanced glial cell production of brain-derived neurotropic factor, whose exogenous provision rescued the synaptic and behavioral phenotypes of IL-17–deficient animals. Together, our work provides previously unknown clues on the mechanisms that regulate short-term versus long-term memory and on the evolutionary and functional link between the immune and nervous systems.


Neuroimmune interactions in the central nervous system (CNS) were until recently thought to be limited to cases of pathological insult (1). Among the important players that have been depicted to interact with the inflamed CNS, particular attention has been paid not only to conventional CD4+ αβ T cells but also to unconventional γδ T cells. In stark contrast to the former, which can take up to 5 to 7 days to clonally expand and differentiate into effector (T helper) subsets under the influence of specific “polarizing” cytokines (2), we and others have shown that murine γδ T cells are developmentally programmed in the thymus in the absence of overt inflammation, i.e., in the steady state (35). This allows them to accumulate as effector lymphocytes in peripheral tissues and respond to challenge (such as infection) much more rapidly than their αβ T cell counterparts, i.e., within a time frame that aligns with innate immunity (6).

In the murine thymus, γδ T cells are programmed into two main effector subsets that produce either interferon-γ (IFN-γ) or interleukin-17 (IL-17) and can be further distinguished on the basis of various cell surface markers, such as CD27 (3) or CCR6 (7), among others (8). Important data have highlighted a critical role for both IFN-γ–producing γδ T cells and IL-17–producing γδ T cells (γδ17 T cells hereinafter) in neuroinflammation: IFN-γ–producing γδ T cells were shown to mediate demyelination upon coronavirus infection (9), whereas γδ17 T cells were found at high frequency in the brain of mice with experimental autoimmune encephalomyelitis (EAE) and to contribute to disease development (10). This latter subset has also been shown to have a key impact in the progression of cerebral ischemia-reperfusion injury (11). In both cases, γδ17 T cells have been highlighted as critical players in disease progression by contributing to a local immune amplification loop within the brain meningeal spaces and altering the stromal microenvironment of the inflamed brain, ultimately leading to blood-brain barrier (BBB) disruption (12, 13).

In contrast with their pathogenic role in neuroinflammation, γδ17 T cells are known to constitute a major source of IL-17 in various other nonlymphoid tissues at steady state, which contributes to normal tissue physiology, as illustrated by recent works reporting their key functions in bone repair (14) and thermogenesis (15). This is an interesting nascent field that may reveal novel physiological roles for γδ17 T cells residing in other tissues.

Although the CNS has been regarded for decades as an immune-privileged organ, shielded by the BBB, current neuroimmunology now acknowledges that lymphatic vessels within the dural sinuses of the meninges establish direct communication with the draining cervical lymph nodes (cLNs) (16, 17) and that the immune system is crucial to support brain homeostasis and plasticity in a disease-free context. This stems from data establishing key roles for immune cells, particularly CD4+ αβ T cells, in physiological brain functions, including social behavior (18), sensory response (19), and spatial learning (20). Namely, previous studies have demonstrated that T cell–deficient mice display an impaired spatial memory when compared with wild-type (WT) controls, which could be restored after injection of WT splenocytes (21). Moreover, an accumulation of IL-4–producing CD4+ αβ T cells in the meningeal spaces of the murine brain upon cognitive performance has been reported (22). This would benefit the learning capacity by inducing astrocytic expression of brain-derived neurotrophic factor (BDNF) and skewing the meningeal macrophages toward an anti-inflammatory profile (22). By contrast, pro-inflammatory cytokines such as IFN-γ and tumor necrosis factor–α have been shown to exert a negative effect on cognitive behavior (23, 24). Thus, it is tempting to assume that anti-inflammatory cytokines would support physiological brain function, whereas typical pro-inflammatory signals would hinder it, but this view may well be too simplistic.

Here, inspired by their important pathophysiological roles in brain inflammation (25), we hypothesized that innate-like γδ T cells might also contribute to neurophysiology in the steady state. We show that γδ T cells are a major source of IL-17 in the brain meninges at steady state and promote short-term memory in naïve mice by increasing the glutamatergic synaptic plasticity of hippocampal neurons.


Fetal-derived γδ T cells infiltrate the meninges since birth

We started this study by analyzing the immune content of meningeal infiltrates from naïve C57BL/6 WT mice throughout ontogeny using flow cytometry. We found a sizeable population of highly proliferative and differentiated γδ T cells in the meningeal spaces already at birth and persisting throughout life, whereas their αβ T cell counterparts tended to accumulate after weaning (Fig. 1, A to C). Meningeal γδ T cells displayed a particularly activated phenotype, enriched for CD69+ CD44hiCD62Llow− cells and scored nearly 100% of proliferating cells by Ki67 staining (Fig. 1D). Their repertoire was mostly restricted to the usage of the gamma chain variable region (Vγ) 6 of the T cell receptor (TCR) (Fig. 1E), characterizing a fetal thymic-derived population reported to colonize various nonlymphoid tissues (26, 27). Consistent with their fetal origin, meningeal γδ T cells were very poorly reconstituted upon transplantation of WT adult bone marrow precursors into irradiated C57BL/6 WT mice (Fig. 1F). These data reveal a population of fetal-derived Vγ6-biased γδ T cells displaying an activated phenotype in the meninges at steady state.

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).

Meningeal γδ T cells are strongly biased toward IL-17 production

To further characterize the phenotype of meningeal γδ T cells, we focused on their effector profile. We found that they displayed signatures of bona fide γδ17 T cells (3, 7) such as the expression of the master transcription factor retinoic acid receptor–related orphan receptor γt (RORγt) and the chemokine receptor CCR6 (Fig. 2, A and B). Consistently, a large fraction (~50%) of γδ T cells from the meninges of adult mice expressed IL-17 (but not IFN-γ) ex vivo (Fig. 2C), which was notable even compared with other tissues enriched in Vγ6+ γδ T cells (fig. S1). IL-17+ cells were absent in the brain parenchyma (fig. S2) and restricted to the meninges, where they were accounted for almost exclusively by γδ T cells (Fig. 2D), whereas αβ T cells mostly provided IFN-γ (Fig. 2C). We confirmed these meningeal phenotypes using an IL-17–eYFP (enhanced yellow fluorescent protein) fate mapping reporter mouse model (Fig. 2E) (28), which allows the detection of IL-17 producers while bypassing the need for ex vivo stimulation. Most YFP+ γδ T cells coexpressed intracellular IL-17 protein, thus demonstrating their active functionality in the meninges (Fig. 2E). Meningeal γδ17 T cells peaked after the first week of life (Fig. 2F) and, consistent with their Vγ6+ phenotype (Fig. 1E) and proposed fetal origin (26), could not be efficiently generated by adult bone marrow precursor transplantation (Fig. 2G).

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.

Meningeal γδ T cell homeostasis is independent of inflammatory signals

To gain further insight into the potential mechanisms responsible for meningeal γδ T cell accumulation and maintenance, we analyzed mice deficient in specific components that could theoretically influence this process. Critically, we observed that meningeal γδ17 T cells were similarly abundant in germ-free (GF) and specific pathogen–free (SPF) mice or after treatment of SPF animals with an antibiotic cocktail (Fig. 3, A and B). Thus, meningeal-resident γδ17 T cells at steady state are independent of commensal microbiota, contrary to pathogenic γδ17 T cells that migrate from the gut to the CNS in a mouse model of ischemic brain injury (12). Furthermore, the accumulation of γδ17 T cells in the meninges was independent of the pro-inflammatory cytokines IL-1β and IL-23 (Fig. 3C), known to drive their expansion in autoimmune pathology including EAE (10). Typical pathogen-associated molecular pattern signals were also not required, as mice deficient in Toll-like receptor 2 (TLR2), TLR4, Caspase 1, or nucleotide-binding oligomerization domain-containing protein 1 (NOD1) harbored normal meningeal γδ17 T cell pools (Fig. 3D). These data collectively identify a noninflammatory γδ17 T cell subset that populates the meninges in the perinatal period and persists throughout life.

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.

γδ T cells producing IL-17 are required for short-term memory

Because they do not penetrate the brain parenchyma in the steady state (fig. S2), T cells are thought to exert their effects on cognition through the secretion of soluble factors from the meninges (1, 29). To address the role of IL-17 produced by meningeal γδ T cells in this process, we analyzed the behavior of mice deficient in IL-17 (IL-17−/−) or γδ T cells (TCRδ−/−) in classical neuroscience paradigms assessing spatial reference memory. These mice had a normal exploratory behavior and did not show any particular signs of motor deficit or anxiety (figs. S3 and S4). Strikingly, when tested on the Y-maze paradigm for short-term spatial working memory, IL-17−/− and TCRδ−/− mice displayed clear cognitive impairments as they failed to prefer the novel arm, in contrast to inbred IL-17+/+ and TCRδ+/+ littermate controls (Fig. 4, A and B, and fig. S5A) or to C57BL/6 WT mice (figs. S5B and S6). These IL-17–dependent short-term memory deficits were observed in both males (Fig. 4, A and B, and fig. S5, A and B) and females (fig. S6). We excluded a potential influence of the gut flora by showing that animals deficient in IL-17 or γδ T cells and respective WT littermate controls share similar gut microbiome compositions (fig. S7). We further validated our findings in bone marrow chimeras (BMCs) mostly devoid of meningeal γδ17 T cells (Fig. 2G), which displayed normal motor function and exploratory behavior (figs. S3C and S4C) but impaired short-term learning (Fig. 4C and fig. S5C).

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.

To test the local impact of meningeal IL-17, we neutralized IL-17 in situ by injecting specific blocking monoclonal antibodies (mAbs) through the intracerebroventricular route. Administered to WT mice 24 hours before behavioral tests, this treatment abrogated short-term memory in the Y-maze (Fig. 4D).

IL-17−/−, TCRδ−/−, and BMC mice showed normal behavior in the Morris water maze (MWM), the prototypic paradigm for long-term spatial reference memory, in which mice were tested in four trials per day over a four-day period, followed by a “probe” test at day 5 (Fig. 4E and fig. S8). To rule out different degrees of motivation and performance demand in the MWM versus the Y-maze, we adapted both paradigms to assess cognition in short-term and long-term time frames, respectively (30, 31). We confirmed in these redesigned experimental settings that both males and females deficient in IL-17 or γδ T cells suffer from short-term, but not long-term, memory deficits (Fig. 4, F to H, and figs. S9 and S10).

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

As a first and unbiased assessment of the molecular mechanisms underlying the impact of meningeal γδ T cell–derived IL-17 on learning and memory, we used a quantitative SWATH-MS proteomics approach to compare the levels of hippocampal proteins between WT and IL-17−/− animals after Y-maze training. Although changes in the expression of individual proteins were rather mild (table S1), a broader look using pathway enrichment analysis (KEGG) pointed toward changes in synaptic plasticity, such as long-term potentiation (LTP), calcium signaling, and glutamatergic synapse (figs. S11 and S12) (32). To functionally address the impact of IL-17 deletion on synaptic plasticity, we examined baseline synaptic transmission and LTP in IL-17−/− and WT animals. Consistent with our hypothesis, IL-17−/− animals displayed impaired LTP specifically after a short-term Y-maze training (but not at steady state) (Fig. 5, A and B), which was partially restored by preincubation of the hippocampal slices with IL-17 (Fig. 5B). These data demonstrate that IL-17 enhances synaptic plasticity during short-term learning. However, long-term memory trained IL-17−/− mice displayed similar LTP to WT controls (Fig. 5C), which is consistent with their normal performance in such paradigms (Fig. 4E and fig. S9 and 10) and highlights additional/compensatory mechanisms deployed in long-term cognition. This indicates a specific impact of IL-17 in these two dissociable forms of spatial information processing, both of which depend on the hippocampus (33).

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.

The fact that animals deficient in GluR1 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) display a very similar phenotype to that of IL-17−/− mice (30, 34) suggested a role of IL-17 (direct or indirect) in modulation of glutamatergic function. Consistently, we observed that IL-17−/− animals have a shifted input/output (I/O) curve after short-term Y-maze (but not at steady state) (Fig. 5, D and E) associated with a reduction in the AMPAR/N-methyl-d-aspartate receptor (NMDAR) ratio in CA1 glutamatergic neurons (Fig. 5F), whereas their paired-pulse ratio remained unaffected (Fig. 5G). The electrophysiological characterization obtained in IL-17−/− mice was phenocopied using TCRδ−/− mice (Fig. 6E and fig. S13), further validating meningeal γδ T cells as the main source of IL-17 in the steady-state CNS.

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.

IL-17 promotes glial BDNF production

On the basis of previous studies demonstrating that astrocytes and microglial cells constitutively express IL-17RA (35), we hypothesized that glial cells could be critical responders to IL-17 in our model, especially because astrocyte end feet are in close contact with the meningeal areas, and have been suggested to act as sensors of the meningeal cytokine milieu (1). To address this hypothesis, we crossed IL-17RAfl/fl mice with specific Cre lines to generate mice where IL-17RA is conditionally deleted on astrocytes [using a constitutive glial fibrillary acidic protein (GFAP)–Cre model] or on the microglia (using an inducible CX3CR1-Cre model). However, none of the single conditional knockout (KO) animals recapitulated the short-term cognitive deficits observed with mice deficient in IL-17 (fig. S14), pointing to the potential existence of compensatory mechanisms. We then crossed the two lines to generate double conditional KO mice. Although we observed a relative loss in the capacity to discriminate between the novel and other arms, this did not achieve the extent of IL-17−/− mice (fig. S14), suggesting the involvement of additional cellular populations. Neurons, pericytes, and endothelial cells are potential candidates to also respond to IL-17 (3638).

To gain further molecular insight into the link between meningeal IL-17, glial cells, and neuronal functions, we considered BDNF given that its expression is modulated by cytokines (20, 22) and it has been implicated in several learning and memory paradigms (39), especially through regulation of synaptic plasticity (40). Namely, mice deficient in BDNF display notable deficits in basal synaptic transmission characterized by an impaired LTP (41). We examined the effect of IL-17 on primary glial cultures and found increased BDNF production (Fig. 6A). Moreover, ex vivo analysis of the hippocampus showed significantly lower levels of BDNF in IL-17−/− animals compared with WT controls selectively after a short-term Y-maze (but not after a long-term MWM) paradigm (Fig. 6B). Furthermore, the incubation of hippocampal slices from IL-17−/− or TCRδ−/− mice with exogenous BDNF rescued their LTP and basal transmission deficits (Fig. 6, C to E and fig. S17), and, notably, an intracerebroventricular injection of BDNF restored short-term working memory in IL-17−/− mice (Fig. 6F). Collectively, these neurophysiology and behavioral data suggest a key role for glial BDNF as a molecular link between meningeal γδ17 T cells and hippocampal neuronal plasticity.


The immune system has been traditionally regarded as a network of interacting cells and products critical for host defense against invading microbes and tumors. This disease, notwithstanding, is a relatively rare manifestation, and maintaining such a complex structure in homeostasis represents an important biological cost. The immune system is likely to have major physiological roles extending far beyond defending the host, as suggested by recent studies showing that immune cells can sense environmental signals and regulate physiological processes, such as nervous system function, metabolic homeostasis, thermogenesis, and tissue repair (15).

In this study, we provide unexpected clues on the evolutionary and functional link between the immune and nervous systems through three major advances: (i) We reveal a previously unknown meningeal-resident γδ T cell subset that accounts for essentially all the local IL-17–producing cells at steady state, (ii) we demonstrate that this subset regulates cognitive dissociation in the hippocampus in an IL-17–dependent manner, and (iii) we show evidence that IL-17 increases glial BDNF production and modulates neuronal synaptic plasticity in the hippocampus.

BDNF has been shown to be a key molecule in various learning and memory paradigms, such as the Y-maze and MWM (39, 42). Besides our in vivo data—restoring cognitive performance in the Y-maze—we also show that BDNF rescues the LTP impairment observed in IL-17−/− and TCRδ−/− mice. LTP is the main form of synaptic plasticity reflecting the activity of synaptic information storage processes and has been often used as the gold standard cellular correlate of learning and memory in the hippocampus (4347). Although these BDNF rescue experiments may not be physiologic, as often is the case with experimental manipulation/perturbation of biological systems, we believe that they strongly support, as a proof of concept, that BDNF can bypass the downstream mechanisms affected by the lack of IL-17 in the CNS. This notwithstanding, we do not rule out that other neurotransmitters may potentially contribute to the IL-17–mediated regulation of cognitive behavior.

Previous studies have demonstrated that astrocytes and microglial cells constitutively express IL-17RA and that this receptor is functional, as glial cell cultures produced chemokines in response to IL-17 treatment in vitro (35). Astrocytes are in close contact with meningeal areas and have been suggested to act as sensors of the meningeal cytokine milieu (22), integrating the signals from the meningeal immune system and transmitting this information to the CNS. Our experiments, based on the conditional deletion of IL-17RA on astrocytes and microglia, revealed a complex system with various compensatory mechanisms still to be explored. For instance, pericytes and endothelial cells are potential candidates to also respond to IL-17 (36). A direct impact of IL-17 on neurons is also not excluded, as IL-17R expression has been observed under basal conditions in neural stem cells (48), neuronal progenitor cells (37), and dorsal root ganglion neurons (49, 50).

We show that most meningeal IL-17–producing T cells are Vγ6+ γδ T cells, which have been previously reported to infiltrate the lung, liver, tongue, dermis, fat, and genital tract (27), from where they are unlikely to act on brain cognition. Instead, we propose a local effect from γδ17 T cells that likely colonize the meninges driven by CCR6, a chemokine receptor that they express at high levels and has been recently implicated in the migration of γδ17 T cells to the dermis (51). We could abrogate short-term memory by neutralizing IL-17 in situ through intracerebroventricular injection of an anti–IL-17 specific blocking antibody, a similar approach to the one previously used to demonstrate a role for meningeal IFN-γ on social behavior (18).

In this study, we used full IL-17 and γδ T cell KO mice because the genetic ablation of γδ T cells or IL-17 does not allow tissue-specific targeting of the cells/molecule. Moreover, there are currently no suitable tools to efficiently deplete IL-17 selectively in the γδ T cell lineage. To overcome these limitations, we took advantage of the embryonic origin of the Vγ6+ γδ17 T cell subset that resides in the meninges and generated BMCs, i.e., injected adult bone marrow into irradiated adult hosts from the same genetic WT background. As previously reported (26), this procedure allows the generation of animals with a γδ T cell compartment mostly devoid of IL-17 producers. These animals recapitulated the behavior of IL-17 or TCRδ full KO animals, showing a specific alteration of their short-term, but not long-term, memory. This dichotomy also rules out a general impact of irradiation on cognitive tasks when assessed more than 8 weeks after reconstitution. Other publications in the field have used this approach to dissect the impact of radioresistant versus radiosensitive immune cells on learning (22, 52).

Intriguingly, a deleterious effect of IL-17 on synaptic plasticity has been reported in the hippocampal dentate gyrus (DG), which exhibits pronounced differences in function and signal processing from our region of interest, CA1 (37). In particular, the I/O curves and the responses to paired-pulse stimulation in DG are intrinsically distinct from CA1 (53, 54). Therefore, we believe that our report adds an unestablished role for IL-17 at steady state in the CA1 region of the hippocampus, which is distinct but not contradictory to the current literature.

On the other hand, exacerbated levels of IL-17 have been associated with neuronal death and behavioral abnormalities. This was, for example, induced by intraperitoneal injections of lipopolysaccharide, as a nonphysiological stimulus triggering systemic inflammation associated with cognitive impairments (55). Other recent work also reported a negative impact of maternal IL-17 on autism-like phenotype in offspring (56, 57). These studies concerned gut microbiota-dependent inflammatory IL-17 in pregnant mothers, likely mimicking viral infection, and its detrimental impact on neural developmental in the offspring. Furthermore, IL-17 produced by γδ T cells has also been shown to be deleterious in EAE (13) and stroke (12). We therefore propose a dual role for IL-17 in physiological versus pathological conditions. Whereas, as shown here, the steady-state (and microbiota-independent) production of IL-17 by meningeal γδ T cells promotes learning and memory, its exacerbated production (by γδ as well as recruited CD4+ T cells) upon inflammation contributes to the pathogenesis of neuroinflammation and likely also to neurodegeneration, as recently suggested for Parkinson’s disease (58, 59).

In summary, although the impact of the adaptive immune system on long-term memory has been under the spotlight (29), our work implicates innate-like γδ17 T cells, adding them to the portfolio of the immune populations infiltrating the CNS at steady state (60, 61). We thus propose a revised model that implicates resident meningeal innate-like γδ17 T cells in cognitive dissociation in the hippocampus (30, 34): They are selectively engaged in short-term memory, whereas adaptive αβ T cells seemingly control long-term memory upon recruitment to the meningeal spaces during the training period (20, 22). This provocative notion parallels the orchestration of an immune response to infection, where a rapid (within hours) engagement of innate-like (and often tissue-resident) T cells precedes the contribution of adaptive T cells, which take several days to clonally expand and migrate to the target tissues (2). Last, as IL-17 was recently described as a neuromodulator of sensory response in Caenorhabditis elegans (62) and autism-like behavior in mice (56, 57), we expect this cytokine to impact other aspects of brain functions, thus defining new avenues to explore in neuroimmunology.


Study design

The goals of this study were to characterize meningeal-resident γδ T cells and determine their impact on memory and learning under physiological conditions/steady state. Flow cytometry samples were pooled from four mice, and a minimum of four independent experiments were performed. Behavioral testing was performed using TCRδ−/− and IL-17−/−, with the respective WT littermate controls. A minimum of two independent experiments were performed for each behavior test using a minimum of eight mice per experimental group. Animals were randomized, and the experimenter was blinded to genotype for the duration of behavioral testing. The role of γδ17 T cells on glutamatergic synaptic plasticity was evaluated by performing field and patch-clamp electrophysiology recordings in hippocampal slices from IL-17−/− and TCRδ−/− mice. A minimum of three mice were used per experimental group.


Animal research was conducted at the Instituto de Medicina Molecular João Lobo Antunes (IMM). All experiments were approved by the animal ethics committee at the institute and performed according to national and European regulations. Rodents were purchased from Charles River Laboratories (Spain).

All mice were from the C57BL/6 background, either bred in-house (WT, TCRδ−/−, IL-17−/−, IL-1R−/−, IL-23R−/−, and NOD1−/−), bred at Instituto Gulbenkian de Ciência (IGC) (Oeiras, Portugal) (TLR2−/− and TLR4−/−), bred at Mill Hills (London, UK) (IL-17aCreR26ReYFP), or purchased from Charles River Laboratories (WT) or from the Jackson Laboratory (Caspase 1−/−). When possible, mice used for experiments were littermates. Mice were bred in SPF conditions and maintained under the SPF facility of Instituto de Medicina Molecular João Lobo Antunes (iMM). WT GF mice were bred and maintained in the GF facility of IGC. When indicated, an antibiotic cocktail [streptomycin (5 g/liter), ampicillin (1 g/liter), collistin (1 g/liter), and vancomycin (0.5 g/liter), all from Sigma-Aldrich] was delivered in the drinking water supplemented with 3% sucrose (Sigma-Aldrich), in utero (by treating pregnant females) or after weaning. Control mice were given 3% sucrose in drinking water. Unless specified [uterus fluorescence-activated cell sorting (FACS) characterization], all mice used were 10- to 24-week-old males.

Bone marrow chimeras

BMCs (WT ➔ WT BMC) were generated as previously described (10). Briefly, C57BL/6J WT mice were lethally irradiated (9.5 gray) and, on the next day, intravenously injected with a total of 5 × 106 to 10 × 106 whole bone marrow cells from C57BL/6J (Thy1.1+) donor mice. All BMCs were kept on antibiotics-containing water (2% Bactrim, Roche) for the first 4 weeks after irradiation. The hematopoietic compartment was allowed to reconstitute for 8 weeks before the animals were used for experiments.

Flow cytometry

Mice were euthanized with CO2 and immediately transcardially perfused with ice-cold phosphate-buffered saline (PBS). Meninges were collected and processed as previously described (7). Tongues, brains, and uterus were cut into 2-mm2 pieces and incubated for 30 min at 37°C with stirring in RPMI medium and 5% fetal bovine serum (FBS) supplemented with collagenase D (1.5 mg/ml; Roche) and deoxyribonuclease I (100 μg/ml, Roche). Supernatants were collected, and live cells were isolated on a gradient of Percoll 70 to 30% (GE Healthcare). Spleens and cLNs were homogenized and washed in RPMI medium supplemented with 10% FBS. Meninges, uterus, and tongues were pooled from three to five mice, and brains and spleens were analyzed individually. FACS stainings were performed as previously described (32) using indicated mAbs (table S2). Dead cells were excluded using LiveDead Fixable Viability dye (Invitrogen). Samples were acquired using FACSFortessa (BD Biosciences). Data were analyzed using FlowJo software (Tree Star).

Behavioral tests

Mice were handled for 5 days before behavioral tests, which were performed in the following sequence: open field (OF), elevated plus maze (EPM), Y-maze, and MWM. Mazes were cleaned with a 30% ethanol solution between each trial. Animals were randomized, and the experimenter was blinded to genotype for the duration of behavioral testing. All behavioral tests were performed during the light phase between 8 a.m. and 6 p.m., under dim light, in a sound attenuated room. Mice movements were recorded and analyzed using the video-tracking software SMART.

Open field

The OF was performed as previously described (63).

Elevated plus maze

The EPM was performed as previously described (64).


The short-term Y-maze was performed as previously described (63). Briefly, this is in a two-trial recognition test in a Y-shaped maze with three arms (each with 35 cm length by 10 cm width by 20 cm height), angled at 120°. On the first trial (learning trial), the animal explored the maze for 10 min with only two arms opened (start and other arm). After returning to his home cage for 1 hour, the same animal was re-exposed to the maze for 5 min (test trial) with the novel arm available.

In the long-term Y-maze [adapted from (30)], mice were trained for a period of 5 days, 5 min per day. On day 6, animals were allowed to explore the three arms of the maze for 5 min for the test trial. The number of transitions was used to evaluate motor performances, and the time spent exploring each arm was quantified. Discrimination ratio is calculated by dividing time in the N or O arm by the sum of the time in both arms (N + O).

Morris water maze

The long-term MWM was performed as previously described (65) for five consecutive days and consisted of a 4-day acquisition phase and a 1-day probe test. During the acquisition phase, each mouse was given four swimming trials per day (30-min intertrial interval). A trial consisted of allowing the mouse to explore and reach for the hidden platform. If the animal reached the platform before 60 s, then it was allowed to remain there for 10 s. If the animal failed to find the target before 60 s, then it was manually guided to the platform, where it was allowed to remain for 20 s. On the probe test, the platform was removed, and animals were allowed to swim freely for 60 s while recording the percentage of time spent on each quadrant. In the short-term MWM [adapted from (31)], animals were trained on the same day, seven trials per day with an intertrial interval of 30 s. One hour after the acquisition phase, animals were tested for 1 min.

Intracerebroventricular injections

WT and IL17−/− mice were administered with BDNF (0.1 mg/ml, total volume of 3 μl), saline solution (PBS, total volume of 3 μl), or anti-mouse IL-17 (0.8 mg/ml, total volume of 5 μl) (clone 17F3, BioXCell), and immunoglobulin G1 (IgG1) isotype control (0.8 mg/ml, total volume of 5 μl) (clone MOPC-21) by intracerebroventricular injection as previously described (35). Briefly, mice were anesthetized under 1.5% isoflurane in 100% oxygen in a transparent acrylic chamber. After the induction, mice were moved out of the chamber to a stereotaxic frame, and isoflurane anesthesia was maintained. A single intracerebroventricular injection was performed into the right ventricle of the brain using the stereotaxic coordinates of 0.6 mm posterior, 1.2 mm lateral, and 2.2 mm ventral to bregma. A 10-μl Hamilton syringe was used for intracerebroventricular injection. Behavioral assessment was performed 24 hours after surgery in the Y-maze.

Proteomic analysis

Sample preparation

IL-17−/− and WT controls were euthanized immediately after the Y-maze training by cervical dislocation followed by hippocampi isolation. The subproteome fractionation and protein precipitation of the hippocampi were performed as described in (66), and the protein pellets were resuspended in 2× Laemmli buffer (5% glycerol, 1.7% SDS, 100 mM dithiothreitol, and bromophenol blue in 50 mM tris buffer at pH 6.8). Before ultracentrifugation, a part of each homogenized sample (approximately 10%) was pooled per group. Total protein content was assessed with the commercial 2D Quant Kit (GE Healthcare), and 30 μg of the soluble fraction was used for SWATH-MS analysis. After denaturation at 95°C, samples were alkylated and subjected to in-gel digestion using the short-GeLC approach (67).

Information-dependent acquisition and Sequential Window Acquisition of All Theoretical - Mass Spectra (SWATH-MS) acquisition and processing

Samples were analyzed on a TripleTOF 5600 System (ABSciex) in two phases: information-dependent acquisition (IDA) of the pooled samples for protein identification and SWATH acquisition of each individual sample fraction for quantification. A specific library of precursor masses and fragment ions was created by combining all files from the IDA experiments and used for subsequent SWATH processing. Library was obtained using ProteinPilot software (v5.0, ABSciex) searching against a Mus musculus database from SwissProt (download in July 2016). SWATH data processing was performed using SWATH processing plug-in for PeakView (v2.0.01, ABSciex) as described in (67) adapted for the present samples. All the peptides that met the 1% false discovery rate (FDR) threshold in at least three replicates were retained, and the levels of the proteins were estimated by summing the respective transitions and peptides that met the criteria established [an adaptation of (68)]. For comparisons between experimental conditions, the protein levels were normalized to the total intensity of the sample. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRoteomics IDEntifications (PRIDE) partner repository (69) with the dataset identifier PXD007574.

Bioinformatic analysis

The list of total identified entities from the proteomic analysis was uploaded into the DAVID 6.8 informatic tool using the available Mus musculus database as background. Resulting clustered Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways with an enrichment score of >1.5 and a P value and Benjamini score of <0.05 were selected.

Microbiome evaluation via 16S ribosomal RNA gene sequencing

Fecal samples were collected from animals and snap-frozen and stored at −80°C until microbial DNA extraction. Before extraction, samples were homogenized in 2-ml sterile tubes containing 1-mm-diameter autoclaved zirconia/silica beads (BioSpec products), with the help of a tissue homogenizer. Microbial genomic DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen). The 16S ribosomal RNA V4 amplicons and ITS1-spanning amplicons were both generated using the following Earth Microbiome Project benchmarked protocols. Amplicons were then sequenced using a 280-multiplex approach on a 2 × 250–base pair PE MiSeq run at IGC Genomics Unit (Oeiras, Portugal). Raw sequences were first loaded into the QIIME 1.9.1 pipeline using custom analysis scripts for analysis on the UBELIX Linux cluster of the University of Bern. Operational taxonomic units were picked using UCLUST with a 97% sequence identity threshold using the default options as implemented in QIIME and followed by taxonomy assignment using the latest Greengenes database ( Calculation of the α-diversity (observed operational taxonomic units, Simpson and Shannon indices) and β-diversity (Bray-Curtis genus-level community dissimilarities, weighted and unweighted UniFrac-based PCoA) and statistical analysis of clustering using Mann-Whitney U tests for α-diversity and Adonis (PERMANOVA) for β-diversity were carried out to confirm the strength and statistical significance of groups in the same distance metrics in the QIIME pipeline and phyloseq in R. The q value package was implemented in MaAsLin to correct for multiple testing (Benjamini-Hochberg FDR correction, q value) of 0.05. After correction for an FDR, q < 0.05 was considered significant.

Enzyme-linked immunosorbent assay

After the Y-maze or MWM, mice were euthanized with CO2 and immediately transcardiacally perfused with ice-cold PBS. The meninges, hippocampi, and prefrontal cortex were dissected and frozen in liquid nitrogen. Tissues samples were then homogenized by sonication in radioimmunoprecipitation assay buffer at pH 8.0 (150 mM NaCl, 50 mM tris base, 1 mM EDTA, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, and protease inhibitors, Roche). Total protein content was quantified using the Bio-Rad DC Protein Assay Kit. Levels of BDNF were measured by enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s instructions (Promega).

Primary glial cultures

Astrocyte-enriched cultures were prepared from mice cerebral forebrain as previously reported (22). Briefly, newborn mice (0 to 3 days old) were euthanized by decapitation, and the brain was dissected in ice-cold PBS inside a laminar flow chamber. Cells were dissociated in glucose Dulbecco’s modified Eagle’s medium (4.5 g/liter; Gibco) supplemented with 10% FBS (Gibco) and 1% antibiotic/antimycotic and cultured in poly-d-lysine hydrobromide (10 μg/ml; Sigma-Aldrich). Cultures were maintained at 37°C in a humidified atmosphere (5% CO2) for 21 days, and medium was replaced every 3 to 4 days. At 14 days in vitro, cultures were supplemented with IL-17 (10 ng/ml; eBioscience). At the end of the culture, supernatants were collected for ELISAs.

Electrophysiological field excitatory postsynaptic potential recordings

IL-17−/− mice and WT controls were euthanized by cervical dislocation after Y-maze or MWM trainings. The brain was rapidly removed, and the hippocampi were dissected free in ice-cold Krebs solution (124 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 1 mM MgSO4, 2 mM CaCl2, and 10 mM d-glucose), previously gassed with 95% O2 and 5% CO2 at pH 7.4. Transverse hippocampal slices (400 μM) were obtained with a McIIwain tissue chopper, and field excitatory postsynaptic potentials (fEPSPs) were recorded in stratum radiatum of the CA1 area as previously described (70). For recovery experiments, hippocampal slices were preincubated with IL-17 (10 ng/ml; eBioscience) for 1 hour or with BDNF (30 ng/ml; Amgen Inc.) for 30 min before LTP induction and remained in circulation up to the end of the experiment. After obtaining a stable 10-min baseline, I/O curves, LTP (10c trains with four pulses at 100 Hz separated by 200 ms and induced at 0.5 mV/ms), and paired-pulse facilitation [excitatory post-synaptic current (EPSC) at 50-ms interpulse interval] were recorded. Recordings were performed at 32°C with a perfusion rate of 3 ml/min.

Patch-clamp experiments

Whole-cell patch-clamp experiments were performed in the voltage-clamp configuration (71) using a pipette solution containing 117.5 mM cesium methanesulfonate, 15 mM CsCl, 10 mM tetraethylammonium chloride, 8 mM NaCl, 10 mM Hepes, 0.25 mM EGTA, 4 mM MgATP, and 0.3 mM NaGTP. The pH was adjusted to 7.3 with CsOH. For all experiments, slices were superfused with the oxygenated artificial cerebrospinal fluid (ACSF) at 32°C in the continuous presence of 50 μM picrotoxin (dissolved in dimethylsulfoxide, Sigma-Aldrich, France) to block gamma-aminobutyric acid (GABA)–ergic transmission. The Schaffer collateral pathway was stimulated at 0.10 Hz using electrodes (glass pipettes filled with ACSF) placed in the stratum radiatum. After a tight seal (>1 GΩ) on the cell body of the selected neuron was obtained, whole-cell patch-clamp configuration was established, and cells were left to stabilize for approximately 2 min before recordings began. To calculate the AMPAR/NMDAR ratio, cells were held at −65 mV to record AMPAR excitatory postsynaptic currents (EPSCs) and at +40 mV to record NMDAR EPSCs. AMPAR EPSC amplitudes were calculated by averaging 30 consecutive EPSCs recorded at −65 mV and measuring the peak compared with the baseline. NMDAR EPSC amplitudes were calculated by averaging 30 consecutive EPSCs recorded at +40 mV and measuring the amplitude at 60 ms after EPSC onset compared with the baseline.

Statistical analysis

The values presented are mean ± SEM of n independent experiments. To test the significance of the differences between two conditions, Student’s t test, Mann-Whitney U test, and F test were used. In statistical tests between three or more conditions, one-way analysis of variance (ANOVA) or Kruskal-Wallis test followed by Bonferroni’s or Dunnett’s multiple comparison post hoc test was used as specified in the figure legends. P values of <0.05 were considered to be statistically significant.


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.


Acknowledgments: We thank the assistance of the staff of the Flow Cytometry and Rodent facilities of iMM Lisboa, IGC, and The Francis Crick Institute. We also thank H. Veiga-Fernandes, M. Veldohen, A. Almeida, A. Sebastião, V. Batalha, D. Ferreira, S. Vaz, G. Leal, P. Papotto, M. Muñoz-Ruiz, B. Di Lorenzo, M. Bordone, J. Chesné, N. Schmolka, K. Serre, J. Darrigues, G. Fiala, D. Inacio, C. Cunha, N. Gonçalves-Sousa, R. Ribeiro, B. Garcia-Cassani, T. Carvalho, R. Loureiro Gomes (iMM Lisboa, Portugal), R. Fonseca (CEDOC, Lisboa, Portugal), M. Correia-Neves, C. Miranda, J. Cerqueira (ICVS, Braga, Portugal), I. Prinz (Hannover Medical School, Hannover, Germany), H. Marie (IPMC, Nice, France), C. Beurrier (Marseille, France), D. J. Pennington (Blizard Institute, Queen Mary, London, UK), A. Iseppon, and A. Hayday (The Francis Crick Institute, London, UK) for helpful discussions and technical support. We are also grateful to M. Oukka (University of Washington, USA) and F. Powrie (Oxford University, UK) for provision of IL-23R−/− mice and S. Akira (Osaka University, Japan) for MyD88−/− mice. Funding: This work was funded by the Fundação para a Ciência e Tecnologia (IF/00013/2014 to J.C.R., IF/00105/2012 to L.V.L., PD/BD/114103/2015 to H.C.B., SFRH/BD/52228/2013 to M.T.-F., SFRH/BD/88419/2012 to C.S., and POCI-01-0145-FEDER-007440 to B.M.), the European Research Council (CoG_646701 to B.S.-S.), “COMPETE Programa Operacional Factores de Competitividade,” QREN, the European Union (FEDER–Fundo Europeu de Desenvolvimento Regional), Horizon 2020 (TwinnToInfect; grant agreement no. 692022), and Wellcome Advanced Investigator Grant (100910/Z/13/Z to B.S.). B.S. is supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001159), The UK Medical Research Council (FC001159) and the Wellcome Trust (FC001159). This publication was supported by UID/BIM/50005/2019, a project funded by Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado. Author contributions: M.R. and H.C.B. designed and performed most of the experiments and analyzed the data. M.T.-F., J.E.C., I.M.-M., C.A.V., and S.O. assisted in the experiments. P.A.P. performed the whole-cell patch clamp experiments. C.S. and B.M. performed and analyzed the proteomic quantification. T.R., A.W., B.S., L.V.L., and B.S.-S. assisted in the experimental design, provided key research tools, and contributed to the manuscript writing. J.C.R. designed the study, performed some experiments, supervised the research, and wrote the manuscript. Competing interests: The authors declare that they have no competing interest. Data and materials availability: The proteomic data for this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (69) with the dataset identifier PXD007574. All the software used in data analysis is commercially available, and the respective information is provided in each respective section. The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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