Guadalupi et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03120-9

Journal of Neuroinflammation(2024) 21:128

Open Access

RESEARCH

Interleukin-9 protects from microgliaand TNF-mediated synaptotoxicity
in experimental multiple sclerosis
Livia Guadalupi1,2†, Valentina Vanni2†, Sara Balletta3, Silvia Caioli3, Francesca De Vito3, Diego Fresegna1,2, Krizia Sanna1,
Monica Nencini2, Gloria Donninelli4, Elisabetta Volpe4, Fabrizio Mariani1, Luca Battistini5, Mario Stampanoni Bassi3,
Luana Gilio3, Antonio Bruno3,6, Ettore Dolcetti3,6, Fabio Buttari1,3, Georgia Mandolesi2,7, Diego Centonze1,3* andAlessandra Musella2,7
Abstract
Background Multiple sclerosis (MS) is a progressive neurodegenerative disease of the central nervous systemcharacterized by inflammation-driven synaptic abnormalities. Interleukin-9 (IL-9) is emerging as a pleiotropic cytokine
involved in MS pathophysiology.
Methods Through biochemical, immunohistochemical, and electrophysiological experiments, we investigated theeffects of both peripheral and central administration of IL-9 on C57/BL6 female mice with experimental autoimmune
encephalomyelitis (EAE), a model of MS.
Results We demonstrated that both systemic and local administration of IL-9 significantly improved clinical disability,
reduced neuroinflammation, and mitigated synaptic damage in EAE. The results unveil an unrecognized central
effect of IL-9 against microglia- and TNF-mediated neuronal excitotoxicity. Two main mechanisms emerged: first, IL-9
modulated microglial inflammatory activity by enhancing the expression of the triggering receptor expressed on
myeloid cells-2 (TREM2) and reducing TNF release. Second, IL-9 suppressed neuronal TNF signaling, thereby blocking
its synaptotoxic effects.
Conclusions The data presented in this work highlight IL-9 as a critical neuroprotective molecule capable ofinterfering with inflammatory synaptopathy in EAE. These findings open new avenues for treatments targeting theneurodegenerative damage associated with MS, as well as other inflammatory and neurodegenerative disorders of
the central nervous system.
Keywords Experimental multiple sclerosis, Neuroinflammation, Synaptopathy, Microglia activation, Inflammatory
cytokines, Glutamate transmission†

Livia Guadalupi, Valentina Vanni contributed equally to this work.

*Correspondence:
Diego Centonze
centonze@uniroma2.it
1
Department of Systems Medicine, University of Rome Tor Vergata,
Rome 00133, Italy
2
Synaptic Immunopathology Lab, IRCCS San Raffaele Roma, Rome
00166, Italy

3

Unit of Neurology, IRCCS Neuromed, Pozzilli (Is) 86077, Italy
Molecular Neuroimmunology Unit, IRCCS Fondazione Santa Lucia, Viadel Fosso di Fiorano 64, Rome 00143, Italy
5
Neuroimmunology Unit, IRCCS Fondazione Santa Lucia, Via del Fosso diFiorano 64, Rome 00143, Italy
6
Ph.D. Program in Neuroscience, Department of Systems Medicine,
University of Rome Tor Vergata, Rome 00133, Italy
7
Department of Human Sciences and Quality of Life Promotion,
University of Rome San Raffaele, Rome 00166, Italy
4© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Guadalupi et al. Journal of Neuroinflammation(2024) 21:128

Introduction
Multiple sclerosis (MS) is an inflammatory and neurodegenerative autoimmune disease resulting in a diffuse
demyelination, neuroinflammation, neuroaxonal loss anddysfunction of the central nervous system (CNS) [1, 2]. In
the last decade, both clinical and preclinical studies in MS
and in its animal model, the experimental autoimmune
encephalomyelitis (EAE), have highlighted degenerativechanges of the grey 4matter as crucial pathological events
independent of demyelination [3, 4].One of the mostprominent features of this neuronal damage is a synaptic
dysfunction and loss, named synaptopathy, characterized
by a marked imbalance between glutamatergic and GABAergic transmissions, caused by the neuroinflammatory
milieu that characterizes MS and EAE brains [3, 5, 6].
Microglia and astrocytes extensively contribute to MS
neuroinflammation and greatly modulate synaptic transmission by releasing proinflammatory cytokines. In particular, in the striatum of EAE mice, the proinflammatory
cytokine TNF, mainly released by activated microglia andmacrophages, enhance excitatory transmission acting atpostsynaptic glutamate receptor sites [7–9]. The longlasting perturbation of synaptic homeostasis ultimately
results in excitotoxic neurodegeneration. Since primaryalterations of the synaptic compartments are potentiallyreversible, synaptopathy represents an attractive therapeutic target in MS [10].
Interleukin-9 (IL-9) is a pleiotropic cytokine which
affects both immune and non-immune cells, acting on
both peripheral and CNS compartments [12]. IL-9 has
emerged as an important signature within the cytokine
microenvironment that characterize MS pathophysiology [11]. Accordingly, levels of IL-9 in the cerebrospinal
fluid (CSF) of relapsing-remitting (RR) MS patients wereshown to inversely correlate with indexes of inflammation, neurodegeneration, disability progression, andresponse to first line therapies [12]. Furthermore, IL-9
was shown to be expressed in the CNS of progressive MS
patients and its expression was mainly associated withmacrophage and CD4+ T cell infiltration and microglial
activation [13].
A large number of different cell types express IL-9R,
such as CD4+ and CD8+ T cells, B cells, macrophages,
mast cells, and eosinophils [11].Importantly, IL-9 wasshown to regulate and decrease the activation phenotype
of human macrophages [13] and to inhibit TNF releasefrom LPS stimulated- human monocytes [14]. Moreover,a growing body of evidence has shown the expression of
IL9R in CNS-resident cells, including microglia cells [11,15, 16].
Despite the encouraging data related to IL-9 in MS
patients, studies in animal models of MS aimed to better characterize its role during the pathology, did notreach conclusive results. Indeed, studies performed using

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systemic administration of recombinant IL-9, IL-9 blockers, or IL-9 receptor (IL-9R) knockout mice have yielded
conflicting results [17–20]. Moreover, stimulation of
IL-9 production from T cells has been shown to either
ameliorate or worsen EAE disease in mice, dependingon whether treatment occurred before or concurrently
with EAE induction, respectively [21]. The mRNA andprotein level of IL-9 increase in EAE spinal cords, andsuch increase correlates with a worse clinical score [22].
Finally, IL-9R expression was shown to be upregulated in
the brain and spinal cord during EAE development [15].
Building upon promising findings concerning the roleof IL-9 in MS, this study aimed to elucidate its controversial role in the mouse model of the disease and shed lighton its effects during neuroinflammatory conditions. To
achieve this goal, we conducted three different pharmacological treatments with IL-9. Through a combinationof clinical score analysis, electrophysiological recordings, immunofluorescence, and biochemical studies, we
uncovered the peculiar neuroprotective properties of
IL-9, demonstrating its efficacy in counteracting inflammatory synaptopathy and clinical deficits during EAE.

Materials and methods
Mice

Animals employed in this study were 6–8-week-old
C57BL/6 female mice, obtained from Charles-River(Milan, Italy). Mice were housed under constant conditions in an animal facility with a regular 12 h light/darkcycle. Food and water were supplied ad libitum. All theefforts were made to minimize the number of animals
used and their suffering. In particular, when animals
experienced hindlimb weakness, moistened food andwater were made easily accessible to the animals on thecage floor. Mice with hindlimb paresis received glucose
solution by subcutaneous injection or food by gavage
during the entire procedure. In the rare presence of atetraparalyzed animal, mice were sacrificed. Minipumpimplanted mice were housed in individual cages endowedwith special bedding (TEK-Fresch, Envigo, Casatenovo(LC), Italy) in order to avoid skin infections aroundthe surgical scar. Animal experiments were performed
according to the Internal Institutional Review Committee, the European Directive 2010/63/EU and the European Recommendations 526/2007, and the Italian D.Lgs
26/2014.
EAE model

Chronic-progressive EAE was induced as previously
described [23]. Six-eight weeks old C57BL/6 female
mice were active immunized with an emulsion of mouse
myelin oligodendrocyte glycoprotein peptide 35–55(MOG35–55, 85% purity; 0,66 mg/ml; Espikem, Prato,
Italy) in Complete Freund’s Adjuvant (CFA; Difco, Los

Guadalupi et al. Journal of Neuroinflammation(2024) 21:128

Altos, CA, USA), followed by intravenous administrationof pertussis toxin (500 ng; Merck, Milan, Italy) on the dayof immunization and two days post immunization (dpi).
Control mice, hereafter referred to as CFA, received thesame treatment as EAE mice without the MOG peptide,
including complete CFA and Pertussis toxin. Animals
were daily scored for clinical symptoms of EAE according
to the following scale: 0 no clinical signs; 1 flaccid tail; 2hindlimb weakness; 3 hindlimb paresis; 4 tetraparalysis;
and 5 death due to EAE; intermediate clinical signs werescored by adding 0.5. The body weight (g) of each mouse
was measured over the course of the disease course andcompared with body weight value at 0 dpi.
Grip strength test

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sonication, the homogenates were centrifuged at 13,000
×g for 20 min and the supernatant was collected. Protein
content was quantified according to the Bradford Assay
method (Thermofisher). 20 µg of striatal extract were
loaded onto a sodium dodecyl-sulfate polyacrylamidegel. Gel was blotted onto Nitrocellulose membrane (Millipore). WB experiments were performed as previously
described [24] and the following primary antibodies
were used: o/n mouse anti-TREM2 (1:1000,R&D system); mouse anti- β-actin (1:20000, Sigma) for 1 h room
temperature (RT). Membranes were incubated with asecondary HRP-conjugated IgG anti-mouse (1:10000 for
β-actin and 1:2000 for TREM2, 1 h RT, Abcam). Results
were presented as data normalized to β-actin and EAEVHL or CTRL values. N = 4–5 per group for EAE mice.

All mice were tested for grip strength performance using
the Grip Strength Meter (Ugo Basile, Italy), as in [24] at
2,7,14 and 21 dpi. The Grip Strength Meter consisted of asteel wire grid (8 × 8 cm) connected to an isometric force
transducer. Mice were lifted by their tail so that theygrasp the grid with their paws. Mice from the two experimental groups were then gently pulled backward until
they released the grid and the maximal force in newtons(N) exerted by the mouse before losing the grip was measured. The mean of three consecutive measurements foreach animal was calculated and values were normalizedby mouse body weight.

Conditioned medium from BV2 cells non-activated(control), Th1 activated and Th1 activated in the presence of IL-9 (see above) was harvested and centrifugedat 1,200 rpm in microfuge in order to remove any dead or
detached cells. TNF ELISA was performed according to
manifacturer’s guide (Biotechne). The medium (n = 4–5
samples for each condition) were run in the same assay.
Quantification of cytokine content was made according
to standard curve in the linear range (from 10 to 700 pg/
ml).

BV2 immortalized murine microglial cell line

Electrophysiology

The BV2 immortalized murine microglial cell line wasconstructed by infecting primary microglia with a v-raf/v-myc oncogene-carrying retrovirus (J2). The murine
BV2 microglia were cultured in DMEM supplemented
with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, and were maintained in a humidified incubatorwith 5% CO2. BV2 cells were in vitro activated for 6 h,
18 h and 24 h with a Mix of Th1-specific proinflammatory cytokines: 100 U/mL IL-1β (Euroclone, Milan, Italy),
200 U/mL tumor necrosis factor (TNF, Miltenyi Biotec,
Bologna, Italy), and 500 U/mL interferon γ (IFNγ, Becton Dickinson, Milan, Italy). Only the 24 h activated BV2cells were afterwards treated with IL-9 (100 μm) for 6 h.
Then, 5 × 10^5 to 1 × 10^6 cells were put on single striatal
slices (for 60 min) and whole-cell patch-clamp recordings
were made as above. For biochemical evaluation, following IL-9 treatment, activated cells were cultured for 3 h
in a non-activated medium. Cell medium was used for
ELISA experiment (see after).

Mice were killed by cervical dislocation, and corticostriatal coronal slices (200 μm) were prepared from fresh
tissue blocks of the brain with the use of a vibratome. A
single slice was then transferred to a recording chamberand submerged in a continuously flowing ACSF (34 °C,
2–3 mL/min) gassed with 95% O2–5% CO2. The composition of the control ACSF was (in mM): 126 NaCl, 2.5KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 11 glucose,
25 NaHCO3. To study spontaneous glutamate-mediatedexcitatory postsynaptic currents (sEPSCs), the recording pipettes were filled with internal solution of the following composition (mM): K+-gluconate (125), NaCl(10), CaCl2 (1.0), MgCl2 (2.0), 1,2-bis (2-aminophenoxy)ethane-N, N,N, N-tetra acetic acid (BAPTA; 0.5), HEPES(19), GTP; (0.3), Mg-ATP; (1.0), adjusted to pH 7.3 with
KOH. Bicuculline (10 µM) was added to the external
solution to block GABAA-mediated transmission.
To study GABA-mediated spontaneous inhibitory
postsynaptic currents (sIPSCs), the recording pipettes
were filled with internal solution of the following composition (mM): 110 CsCl, 30 K + − gluconate, 1.1 EGTA,
10 HEPES, 0.1 CaCl2, 4 Mg-ATP, 0.3 Na-GTP. MK-801
and CNQX were added to the external solution toblock, respectively, NMDA and non-NMDA glutamate
receptors.

Western blot

Whole striata dissected from EAE mice (i.p. treatment
starting from 0 dpi) in the acute phase of the disease(24 dpi) were homogenized in RIPA buffer plus protease inhibitor mixture (Sigma) and sonicated. After

TNF ELISA measurement

Guadalupi et al. Journal of Neuroinflammation(2024) 21:128

The detection threshold of spontaneous currents was
set at twice the baseline noise. Offline analysis was performed on spontaneous synaptic events recorded during fixed time epochs (1–2 min, three to five samplings),
sampled every 5–10 min. Only cells that exhibited stablefrequencies in control (less than 20% changes during thecontrol samplings) were used for analysis. For kinetic
analysis, events with peak amplitude between 10 and 50
pA were grouped, aligned by half-rise time, normalized
by peak amplitude, and averaged to obtain rise times anddecay times.
Real time PCR (qPCR)

Total RNA was extracted from treated BV2 cells according to the standard miRNeasy Micro kit protocol (Qiagen). The RNA quantity and purity were analyzed with
the Nanodrop 1000 spectrophotometer (Thermo Scientific). The quality of RNA was assessed by visual inspection of the agarose gel electrophoresis images. Next, 900
ng of total RNA was reverse-transcribed using highcapacity cDNA reverse transcription kit (Applied Biosystem) according to the manufacturer’s instructions
and 27 ng of cDNA were amplified and each reaction of
amplification was performed in triplicates with SensiMix II Probe Kit in triplicate using the Applied Biosystem 7900HT Fast Real Time PCR system. The expressionof IL-9R (Mm00434313_m1) and TNF (Mm00443258_
m1) mRNAs were evaluated by using TaqMan technology and data were represented as 2-ΔΔCt. β-actin(Mm00607939_s1) was used as endogenous control formRNA normalization.
Immunofluorescence and confocal microscopy

Mice were deeply anesthetized and intracardially perfused with ice-cold 4% paraformaldehyde (PFA) at 21–25
dpi (N = 3 per group). Collected brains were post-fixed
in 4% PFA for 2 h and equilibrated with 30% sucrose forat least one night. Thirty-micrometer-thick coronal sections were serially cut on a frozen microtome including the whole striatum to perform immunofluorescence
experiments as previously described (20). Brain regions
were identified using a mouse brain atlas, and for eachanimal, at least five serial sections were used for immunofluorescence as described above. The following primary antibodies were used overnight at 4 °C in Triton
X-100 0.25%: rabbit anti-Iba1 (1:750, Wako), rabbit
anti-GFAP (1:500, Dako), goat anti-DARPP-32 (1:1000,
R&D systems), mouse anti-IL-9 receptor (1:200, Santa
Cruz Biotechnology) and rat anti-CD3 (1:300, Biorad).
After being washed, sections were incubated with thesecondary antibody: Alexa-488 conjugated donkey antiRabbit, anti-Gt or anti-Rat (1:200, Invitrogen); Alexa647 conjugated donkey anti-Rabbit (1:200, Invitrogen);Cy3-conjugated donkey anti-mouse (1:200, Jackson) for

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2 h at RT. Nuclei were counterstained with DAPI (1 µl/
ml; Sigma Aldrich). Images were acquired using a Nikon
Eclipse TI2 confocal laser-scanner microscope with 20x
and 40x objectives and were processed using ImageJ software. All images acquired by the confocal laser-scanner
microscope had a pixel resolution of 1024 × 1024. Z-stackacquisitions (20x objective, zoom 1x with 2 μm interval for a total of 17 steps) were made applying the same
intensity and exposure time. A large image function thatgenerates a single high-magnification image (capturing
2 images) was made to detect IBA1+ and GFAP+ cells.
In the z-projections, to evaluate microglia and astroglia
density the number of IBA1+ and GFAP+ cells, respectively, were automatically determined and divided by thearea covered by the Roi. Data were expressed as the number of cells per mm2. All images were processed using
ImageJ software and were adjusted for reducing noise by
applying smooth and background subtraction as requiredby the NIH ImageJ. ImageJ software was used to generate intensity threshold images (binary images), setting
equal intensity thresholds between groups. A 40x objective (zoom 2x with 1 μm interval for a total of 30 steps)
was used to detect IL-9 receptor expression in DARPP32+, IBA1+, GFAP+ or CD3+ cells. A 40x objective zoom
1x with 2 μm interval for a total of 21 steps (with a large
image function that generates a single high-magnification
image capturing 4 images) was used to detect IBA1 and
TNF. Binary images of IBA1 ImageJ software were used
to quantify the microglial total area (IBA1+ surface %) in
z-projected images and the measure of microglial surface
was calculated by the software. Binary images on single
steps of IBA1 and TNF were used to generate a colocalization mask to visualize IBA1 and TNF colocalization.
Then single images were z-projected (Mk, TNF/IBA1)
and merged on the binary images of IBA-1. Mk total area
(TNF/IBA-1 surface %) was calculated by the software.
IL-9 treatment

As schematized in experimental design (Fig. 1A-A’) threedifferent in vivo treatments were performed at different
times of EAE disease. The following intraperitoneal (ip)treatments were performed: peripheral treatment with
IL-9 (200ng/mouse/day; Peproteck) and PBS as vehicle
starting at 0 dpi (preventative treatment) and at onset
of EAE (around 12–14 dpi, therapeutic treatment). Central treatment was performed by implanting, one week
before immunization, a subcutaneous osmotic minipumps allowing continuous intracerebroventricular (icv)
infusion of either IL-9 (30ng/mouse/day) or vehicle, for
4 weeks. Alzet osmotic minipumps (model 1004; Durect)
connected via catheter tube to an intracranial cannula(Brain Infusion Kit 3; Alzet), delivered vehicle or IL-9 into
the right lateral ventricle at a continuous rate of 0.11 µl/h.
The coordinates used for icv minipump implantation

Guadalupi et al. Journal of Neuroinflammation

Fig. 1 (See legend on next page.)(2024) 21:128

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Guadalupi et al. Journal of Neuroinflammation(2024) 21:128

Page 6 of 16(See figure on previous page.)
Fig. 1 IL-9 effects on EAE disease and striatal synaptic transmission. A-A’) Schematic representation of the experimental design. A)In vivo experiments.
Three in vivo treatments were performed at different times of EAE disease: peripheral intraperitoneal (i.p.) injections of IL-9 or vehicle (VHL) was carriedout starting both from the day of EAE induction (preventative treatment, in yellow) and from the day of disease onset (therapeutic treatment, in purple).
Central intracerebroventricular infusion of IL-9 or VHL was performed starting one week before immunization for four weeks (in blue). A’)In vitro experiments. BV2 immortalized murine microglial cells were activated with a Th1 Mix and treated with IL-9. B) The graph shows the clinical course of EAE micetreated with IL-9 (200 ng/mouse/day) or VHL (PBS) starting from the day of EAE induction (one of two different experimental sets). IL-9 treatment significantly ameliorated EAE disease progression. EAE VHL N = 9, EAE IL-9 N = 8; Mann-Whitney test from day 18 to 30 dpi, ***p < 0.001. c) Plasma levels of IL-9
in EAE mice following i.p. injections of IL-9 or VHL. EAE VHL N = 3, EAE IL-9 N = 3 unpaired T-test *p < 0.05. D-F) Electrophysiological properties recordedby whole-cell patch clamp technics from MSNs of EAE mice after ip injections from 0 dpi. Rise time, decay time and half width of glutamatergic currents,
were increased in EAE striatum and were completely rescued in EAE IL-9 mice (D), CTR n = 7, EAE VHL n = 15, EAE IL-9 n = 18; one-way ANOVA followed
by Tukey’s post hoc, *p < 0.05, ***p < 0.001; The frequency of the glutamatergic currents was unaffected by IL-9 treatment in EAE mice (D) CTR n = 9, EAE
VHL n = 12, EAE IL-9 n = 12. Frequency and amplitude of GABAergic transmission were comparable between IL-9 and vehicle EAE-treated mice (E) EAE
VHL n = 15, EAE IL-9 n = 9; Unpaired T-test p > 0.05. Ex vivo treatment of corticostriatal slices from control mice with IL-9 (100 μm, 1 h) didn’t impact on
glutamatergic synaptic transmission (F), Frequency: CTR n = 14, CTR IL-9 n = 17; kinetic parameters: CTR n = 10, CTR IL-9 n = 24; unpaired T-test p > 0.05.
Example of electrophysiological traces on the bottom

were as follows: anteroposterior = − 0.4 mm from bregma;lateral = − 1 mm; depth: 2.5 mm from the skull.
For the ex vivo electrophysiological experiments, fresh
striatal slices were incubated with IL-9 (100 μm), for
1 h. For BV2 experiments, 24 h activated BV2 cells weretreated with IL-9 (100 μm) for 6 h.
Statistical analysis

Statistical analysis was performed with Prism GraphPad
version 9.0. Data distribution was tested for normality byusing Kolmogorov–Smirnov test. Non-normally distributed data were analyzed through non-parametric tests.
Differences between two groups were analysed using
two-tailed Unpaired Student’s t test or Mann Whitney
test, as appropriate. Multiple comparisons were performed by ANOVA followed by Tukey’s HSD. The twoway ANOVA for repeated measures was used to assess
the interaction between time and treatment. Data were
presented as the mean ± S.E.M, unless otherwise specified. The significance level was established at p < 0.05.
Throughout the text “N” refers to the number of animals.
For electrophysiological experiments, quantitative immunofluorescence, and biochemical analysis “n” refers to thenumber of slices or BV2/BV2 medium samples.

Results
Peripheral in vivo administration of IL-9 ameliorates EAE
motor disability and striatal inflammatory synaptopathy

In order to clarify the pharmacological role of IL-9 in
EAE pathology, we treated EAE mice by ip injections of
IL-9 or VHL, starting from the day of EAE induction.
We observed that daily systemic administration of thiscytokine, which significantly increases IL-9 blood levels(Fig. 1C), ameliorated EAE disease promoting a beneficial impact on motor disability during the acute phase of
the disease (Fig. 1B).
Then, we evaluated the impact of daily treatment with
IL-9 on synaptic damage. Indeed, the striatum of EAE
mice is characterized by an unbalance between glutamatergic and GABAergic transmission [7, 25]. Whole-cellvoltage-clamp recordings from medium spiny neurons(MSNs) revealed that IL-9 completely rescued EAEinduced kinetic alterations of spontaneous postsynaptic
excitatory currents (sEPSCs), in terms of rise time, decay
time and half width (Fig. 1D). Conversely, the frequencyof glutamatergic (Fig. 1D) and GABAergic (Fig. 1E) EAE
transmissions were unaffected by the treatment.
To better characterize this synaptic effect of IL-9,
we performed patch-clamp recordings in corticostriatal slices of healthy mice incubated with IL-9 or vehicle.
Kinetic and frequency parameters of spontaneous glutamatergic currents were similar between IL-9-treated anduntreated slices (Fig. 1F), suggesting a peculiar synaptic
effect of IL-9, only in the context of neuroinflammation.
Brain resident immune cells, like microglia and astrocytes, are the main mediators of EAE inflammatory
synaptopathy. Thus, we performed a detailed immunofluorescence analysis of microgliosis and astrogliosis extent
in the striatum of CFA and EAE mice. Quantificationof microglia and astroglia density showed a significant
reduction of both IBA1+ and GFAP+ cells in EAE-IL-9
mice compared to EAE-VHL mice, as highlighted in thebinary images (Fig. 2).
Overall, these results reveal a beneficial effect of IL-9 ip
treatment on EAE clinical disability, striatal synaptopathy
and neuroinflammation.
IL-9 mediated neuroprotection is independent of its
peripheral immune effect

The amelioration of EAE motor disability, coupledwith the reductions of striatal neuroinflammation andimprovements in glutamatergic transmission defects,
highlights the beneficial influence of IL-9 within the
CNS. To rule out a secondary cytokine effect into thebrain promoted by the involvement of peripheral compartment, we treated for 4 weeks EAE mice with icv
injections of IL-9 or vehicle. We observed that daily central treatment with this cytokine ameliorated the clinical score of the disease (Fig. 3A), resembling the effectobtained after ip treatment. Data on clinical score wasGuadalupi et al. Journal of Neuroinflammation(2024) 21:128

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Fig. 2 IL-9 effects on EAE striatal microgliosis and astrogliosis. A-B) Representative confocal microscopy images of coronal corticostriatal slices of CFA andEAE mice treated with ip injections from 0 dpi, stained with IBA1 (green fluorescence, A) and GFAP (red fluorescence, B) antibodies. Scale bar: 40 µm. Blackand white binary images of IBA-1 (A) and GFAP (B) clearly highlight IL-9 effect on microgliosis and astrogliosis, respectively. A’-B’) Both IBA1+ cell density
and GFAP+ cell density are reduced in the striatum of EAE-IL9 mice (N = 3, n = 11) compared to EAE-VHL mice (N = 3, n = 10), reaching values comparable
to CFA mice (N = 2, n = 6). One-way ANOVA followed by Tukey’s post hoc, ** p < 0.01, **** p < 0.0001

corroborated by other typical clinical parameters of the
EAE condition, such as body weight and neuromuscularstrength. Specifically, both body weight change (Fig. 3A’)
and 4 limbs strength (Fig. 3A’’) were significantly ameliorated by IL-9 treatment. Moreover, icv infusion with IL-9
replicated the anti-synaptotoxic effect observed following systemic treatment. Indeed, whole-cell patch clamp
recordings showed that the increase of glutamatergic
kinetics induced by EAE was rescued in EAE-IL9 (icv)
mice, while sEPSC frequency was unaffected (data notshown). Importantly, IL-9 blood levels were comparable
between groups (Fig. 3C), indicating a negligible IL-9 systemic involvement.
Overall, these results suggest that IL-9 ameliorates EAE
clinical disability and neuronal toxicity independently of
its peripheral immune effect.

Characterization of IL-9 receptor in EAE striatum

Based on the central effect promoted by IL-9 administration, we investigated the expression of IL-9 receptor(IL-9R) in the striatum of EAE mice. Firstly, by performing immunostaining for IL-9R, we observed an overexpression of this receptor in EAE compared to CFA
mice (Fig. 4A), in line with previous data obtained in
EAE brain and spinal cord [15]. Moreover, we observedthat IL-9 treatment reduced IL-9R staining in EAE mice
(Fig. 4A and B’), and images (Fig. 4B-B’) confirmed the
reduction of microgliosis following IL-9 treatment. Then,
immunostaining for IL-9R combined with neuronal
DARPP-32, microglia IBA-1, astroglia GFAP and infiltrating lymphocytes CD3 markers showed that microglia
clearly express IL-9R (Fig. 4B-B’). Conversely, this receptor was undetectable in GFAP + cells and CD3 infiltrating
lymphocytes (Supplementary Fig. 1), as well as into thecell body of medium spiny neurons (Fig. 4B), although its

Guadalupi et al. Journal of Neuroinflammation(2024) 21:128

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Fig. 3 IL-9 icv treatment impact on EAE clinical disability and striatal synaptopathy. A) The graph shows the clinical course of EAE mice treated for fourweeks with IL-9 (30ng/mouse/day) or VHL, delivered by icv infusion. IL-9 icv treatment significantly ameliorated EAE disease progression. EAE VHL N = 10,
EAE IL-9 N = 11; Mann-Whitney test from 19 to 24 dpi, *** p < 0.001). A’) The graph shows the beneficial impact of IL-9 on body weight change obtainedby comparing the body weight measured at 7,10,14,17 and 21 dpi with respect to the 0 dpi value. EAE VHL N = 10, EAE IL-9 N = 11, 2way ANOVA followed
by Bonferroni’s multiple comparisons, 17 dpi **p < 0.01 and 21 dpi *p < 0.05. A’’) The grip strength performance was significantly ameliorated by IL-9 treatment in EAE mice after 14 and 21 dpi. EAE VHL N = 10, EAE IL-9 N = 11, 2way ANOVA followed by Bonferroni’s multiple comparisons, 14 dpi **p < 0.01 and21 dpi ***p < 0.001. B) The increased kinetic properties (rise time, decay time, and half width) of EAE sEPSCs were rescued in EAE-IL9 mice. CTR n = 17, EAE
VHL n = 12, EAE IL-9 n = 21; one-way ANOVA followed by Tukey’s post hoc, *p < 0.05, **p < 0.01, ***p < 0.001. On the bottom electrophysiological traces
representative of glutamatergic currents recorded from MSNs of EAE VHL and EAE IL-9 mice. C) Plasma levels of IL-9 in EAE mice following icv injectionsof IL-9 or VHL. EAE VHL N = 4, EAE IL-9 N = 3, unpaired T-test p > 0.05

expression on dendrites and axons could not be excluded.
These results indicate that activated microglia represent
the main cellular population expressing IL-9R in the
EAE striatum and that in vivo treatment with the cytokine decreases the EAE-mediated enhancement of IL-9R
expression.
IL-9 counteracts TNF mediated excitotoxicity

Based on the evidence that TNF is a critical player in theexacerbation of striatal glutamate transmission induced
by EAE [7], we explored the link between IL-9 and TNF
to understand if the recovery of synaptic defects mediated by IL-9 was correlated to the reduction of TNF
expression in EAE brain.
Microglia cells are the main source of TNF in the
inflamed EAE brain [26], and selectively express IL-9R(present study). Thus, we performed co-immunolabelingof the microglia marker IBA1 and of TNF on EAE striatal slices. As expected, we observed a strong expressionof TNF by IBA-1+ cells and the analysis of the total area
covered by microglia cells confirmed the impact of IL-9
on microglia density reduction (Fig. 5A). IL-9 treatment
significantly reduced TNF expression from microglia, as
highlighted by colocalization mask (mk-magenta) analysis (Fig. 5A).
Next, to better define the implication of microglia cells
on IL-9-mediated neuroprotective effects, we investigated the impact of IL-9 on BV2 immortalized murine
microglial cells. Indeed, BV2 stimulation with a proinflammatory Th1 cytokine mix (IL-1β, IFNγ, and TNFα)
well resembles EAE cytokine microenvironment andpromotes inflammatory synaptopathy typical of EAEGuadalupi et al. Journal of Neuroinflammation(2024) 21:128

Page 9 of 16

Fig. 4 Characterization of IL-9 receptor in EAE striatum. A) Representative confocal microscopy images of coronal corticostriatal slices stained with IL-9R
antibody. The expression of IL-9R is markedly increased in the striatum and corpus callosum of EAE mice compared to CFA condition and is reduced
following IL-9 treatment. B) Triple immunostaining of coronal striatal sections (in gray DAPI nuclei) showing the expression of IL-9R (red fluorescence)in IBA1-positive microglia/macrophage cells (blue fluorescence) and DARPP32-positive neurons (green fluorescence) in EAE VHL and EAE IL-9 mice. As
highlighted by the white asterisks IL-9R expression seems to be undetectable on neuron body cells while white arrows show the high expression of IL-9R
by microglia. B’) Double immunostaining for IBA1 and IL-9R showing the marked reduction of IL-9R expression in IL-9 treated mice and the strong expression of IL-9R in microglia cells. Scale bar: 20 μm[7, 27]. We stimulated BV2 cells for 6 h, 18 h and 24 h.
qPCR analysis revealed a significant increase of IL-9R
mRNA levels at 24 h in BV2 treated cells compared tocontrol conditions (Fig. 5B), suggesting the suitability of
BV2 experimental model to study IL-9 effects. Therefore,

following 24 h of activation, BV2 cells were treated with
IL-9 for 6 h. IL-9R mRNA levels in activated BV2 cells
were strongly reduced by IL-9 treatment, resembling theeffect observed in EAE condition (Fig. 5B). Then, we evaluated the impact of IL-9 on TNF release by measuring

Guadalupi et al. Journal of Neuroinflammation

Fig. 5 (See legend on next page.)(2024) 21:128

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Guadalupi et al. Journal of Neuroinflammation(2024) 21:128

Page 11 of 16(See figure on previous page.)
Fig. 5 IL-9 prevents TNF detrimental effects on striatal synaptic transmission. (A) Representative confocal microscopy images of coronal striatal slices
stained with IBA1 (green fluorescence) and TNF-ɑ (red fluorescence) showing that IL-9 treatment decreased microgliosis and TNF-ɑ expression in the
striatum of EAE mice. Percentage of the area occupied by microglia is reduced in the striatum of EAE IL-9 mice compared to EAE VHL mice. The overlapping area, mask (mk TNF/IBA1, magenta), is clearly reduced in microglia from EAE IL-9 striatum compared to EAE VHL group, indicating less production of
TNF-ɑ from these cells. EAE VHL N = 3,n = 9, EAE IL-9 N = 3,n = 11, Unpaired two-tailed t-test, * p < 0.05,** p < 0.01. Scale bar: 20 μm. (B) qPCR experiments
performed on BV2 microglial cells activated by Th1 Mix for 6 h, 18 h and 24 h show an overexpression of IL-9R only following 24 h of cytokine activation.
n = 6 per condition, Unpaired t-test, * p < 0.05. Incubation of 24 h activated BV2 cells with IL-9 (6 h, 1 μm) significantly reduced IL-9R mRNA level. n = 6 percondition, one-way ANOVA followed by Tukey’s post hoc CTR *** p < 0.001, ** p < 0.01. All data are expressed as mean ± SEM and as fold change of untreated controls. (C) qPCR quantification of TNF-ɑ transcriptional levels reveals that IL-9 treatment of BV2 activated cells do not modulate TNF mRNA. n = 6
per condition, one-way ANOVA Tukey’s post hoc analysis ***p < 0.001. (D) TNF concentration (pg/ml) in the medium of BV2 cells non-activated, activatedand activated in the presence of IL-9. Incubation with IL-9 caused the reduction of TNF protein release by activated microglia. CTRL n = 4, MIX (activated
BV2 cells) n = 5, MIX + IL-9 (activated BV2 cells + IL-9) n = 5; one-way ANOVA Tukey’s post hoc analysis *** p < 0.001. E)In vitro activated microglia stimulated with IL-9 (MIX + IL-9) significantly ameliorated the altered rise time, half-width and decay time of sEPSC of striatal slices. Control slices (CTRL) n = 17,
activated BV2 (MIX) n = 18, activated BV2 + IL-9 (MIX + IL-9) n = 18; unpaired T-test *p < 0.05, **p < 0.01. F) The increased kinetic parameters induced by
TNF in striatal slices were rescued by concomitant incubation of IL-9. VHL n = 17, TNF + VHL n = 19, TNF + IL-9 n = 17; One-way ANOVA * p < 0.05, **p < 0.01,***p < 0.001; Unpaired T-test #p < 0.05

TNF protein level in the conditioned medium of BV2
cells. As indicated by the immunofluorescence analysis
performed in EAE striatal slices, ELISA immunoassay
confirmed that IL-9 treatment significantly reduced BV2
TNF release induced by Th1 cytokines mix (Fig. 5D).
Conversely, TNF mRNA levels were similar between IL-9
and vehicle BV2-trated cells (Fig. 5C), suggesting a posttranscriptional effect.
As previously demonstrated, activated BV2 cells promote the increase of sEPSC duration (inducing thesame alterations seen in EAE), through a mechanism
dependent on TNF release. Thus, we recorded corticostriatal slices incubated with activated BV2 cells treatedwith IL-9 or vehicle. IL-9 prevented neuronal toxicity induced by activated BV2 cells, rescuing both decay
time and half width parameters (Fig. 5E), demonstrating
the direct impact of IL-9 on microglia-induced synaptictransmission.
The dual role of IL-9 in the modulation of TNF mediatedexcitotoxicity

To better characterize the biological interconnection
between IL-9 and TNF, the downstream impact of IL-9
on TNF synaptic toxicity was also investigated. To thisaim, striatal slices from healthy mice were incubated with
TNF in the presence of IL-9 or vehicle. Electrophysiological alterations induced by exogenous TNF in control
striatal slices were rescued by concomitant incubation
with IL-9 (Fig. 5F). This effect was particularly evident
in terms of decay time, as slices treated with both TNF
and IL-9 reached values similar to the control condition(vehicle) and significantly lower compared to the TNF
condition. Although less pronounced, a similar effect
was also observed for the half-width kinetic parameter(Fig. 5F).These results suggest a neuronal role for IL-9,
unveiling a functional interplay between TNF and IL-9
signaling.
In summary, our findings indicate that IL-9 contrasts
TNF-dependent synaptotoxicity via multiple actions:influencing microglial activation, reducing TNF expression and release, and interfering with the neurotoxic
effects of TNF.
The impact of IL-9 treatment on TREM2 expression

Clinical and preclinical data pointed out triggering receptor expressed on myeloid cells-2 (TREM2) as a signatureof alternative microglia/macrophage M2 activated profilethat promotes the resolution of inflammation and clearance of myelin debris in MS disease [28–30].
To investigate the impact of IL-9 on the microglia activated profile, we evaluated TREM2 levels in both ex vivo
and in vivo conditions. Specifically, Western blot experiments were performed on BV2 cells activated as previously described (24 h of activation) and treated with IL-9
or VHL (6 h). The analysis revealed that IL-9 treatment
significantly increased TREM2 expression compared tocontrol and stimulated conditions (Fig. 6A). A similar
result was obtained also in EAE mice. Indeed, TREM2
expression in the striatum of EAE mice systematicallytreated with IL-9 and sacrificed at 24 dpi was significantly
higher compared to EAE-VHL animals (Fig. 6B). Notably,
TREM2 was shown to inhibit inflammatory response in
microglia cells by reducing the production of inflammatory mediators such as TNF [31, 32]. Altogether thesefindings support the beneficial role of IL-9 on microgliamediated toxicity in MS disease, suggesting a mechanism
dependent on modulation of TREM2 expression.
Effectiveness of IL-9 therapeutic treatment in EAE

To investigate the translational relevance of IL-9 treatment in MS disease, we performed a therapeutic treatment using daily ip injections of IL-9 in EAE mice,starting from the onset of clinical disease. Similar to
the results observed in other experimental conditions,this therapeutic approach significantly ameliorated EAE
clinical disability as well (Fig. 7A). In parallel, IL-9 waseffective in mitigating EAE-related striatal synaptopathy,
leading to a significant reduction in the decay time andGuadalupi et al. Journal of Neuroinflammation(2024) 21:128

Page 12 of 16

Fig. 6 IL-9 modulates TREM2 expression in BV2 and in EAE slices. Western blot comparing the probe for anti-TREM2 antibody in BV2 cells (CTRL, 24 h of
MIX activation and 24 h of MIX activation + 6 h of IL-9 treatment) and in EAE VHL and EAE-IL9 striatal extracts (i.p. treatment from day 0). Densitometricanalysis of the bands shows an increased TREM2 content, revealing that IL-9 induces an upregulation of TREM2 level in both BV2 cells (A) and EAE striatum(B). One-way ANOVA and Unpaired T-test; * p < 0.05, **p < 0.01. n = 3 per condition for in vitro experiment and N = 4–5 per group in EAE micehalf-width of sEPSC (Fig. 7B). This experiment unequivocally demonstrates the beneficial effects of IL-9 in EAE
pathology and underscores its therapeutic potential in
the disease.

Discussion
In the present study, we investigated the role of IL-9 in
the mouse model of MS, uncovering for the first time a
relevant neuroprotective role of this cytokine. The beneficial effect of IL-9 in MS disease was previously suggested
in a clinical study where CSF levels of IL-9 have been negatively correlated with neurodegeneration, inflammatory
activity and disease severity in RRMS patients [12], while
information on progressive patients is missing. Most of
the preclinical experiments performed to understand theIL-9 role in MS disease focused on its peripheral immunomodulatory effect and reached controversial results.
Genetic deletion of IL-9R in EAE mice was shown toboth increase [17, 18] and attenuate [21] the severity of
EAE. The reduction of physiological IL-9 levels by antiIL-9 monoclonal antibody (ɑIL-9) reduced EAE severity,

in both active [19, 20] and passive [20] EAE. Noteworthy,
the anti-IL9 treatment promoted a feedback enhancement of IL-9 production from T cell [19]. Furthermore,stimulation of IL-9 production from T cells ameliorated
or worsened EAE clinical course in mice treated before
or concomitantly with EAE induction, respectively [21].
To clarify the role of IL-9 in EAE disease and to discriminate peripheral and central functions of this cytokine, we
performed different in vivo pharmacological treatments,
combined with ex vivo and in vitro data.
Besides demyelination, the complex alterations of
excitatory and inhibitory transmission induced by neuroinflammation is increasingly considered as a relevant pathogenic and neurodegenerative mechanism in
EAE and MS brains [3]. Synaptic damage occurs in theabsence of overt demyelinating lesions, and even before
the appearance of the EAE-associated neurological deficits. Among proinflammatory cytokines, TNF plays acrucial role in this inflammatory synaptopathy, mediating
the increase of glutamatergic postsynaptic duration in
the striatum of EAE mice [7]. Microglia cells are the mainGuadalupi et al. Journal of Neuroinflammation(2024) 21:128

Page 13 of 16

Fig. 7 Therapeutic treatment with IL-9 ameliorates EAE disease. (A) The graph shows representative clinical courses of EAE IL-9 and EAE VHL mice treatedstarting from the day of disease onset. IL-9 therapeutic treatment significantly ameliorated EAE disease progression. EAE VHL N = 8, EAE IL-9 N = 8; MannWhitney test from 19 to 28 dpi, *** p < 0.001). (B) MSNs from mice therapeutically treated with IL-9 (grey) showed reduced half width and decay timecompared to EAE VHL (black) condition. EAE VHL n = 7, EAE IL-9 n = 18; Unpaired T-test *p < 0.05. **p < 0.01

source of inflammatory mediators in the EAE/MS brains
and are convincingly recognized to exert a crucial role in
the pathogenic mechanisms leading to CNS inflammation and damage in MS [7, 33]. Activated microglia were
found in brain lesions of MS patients, in all stages of thedisease, and were thought to contribute in recruitment ofadaptive immune cells to the CNS [34]. Diffuse microglial
and astroglial reactions indicate that the innate immune
system is chronically activated in the gray matter andfeatures of degeneration-associated/pro-inflammatory
states of microglia increase with chronicity of the disease [35]. Importantly, persistent inflammation triggers
uncontrolled synaptic activation and excitotoxicity [36].
We have previously demonstrated that activated microglia incubated on brain slices of healthy mice mimicked

the aberrant glutamatergic transmission observed in thebrain of EAE mice [7, 37–39]. Specifically, TNF released
by reactive microglia led to enhanced AMPAR localization at the postsynaptic surface causing an increase ofglutamatergic transmission [8].
Current disease modifying therapies (DMTs) exerts
a limited impact on microglia cells through indirect mechanisms [40]. Understanding how to restore
microglia to homeostasis and shift pro-inflammatory
microglia toward an anti-inflammatory phenotype hassignificant implications for MS therapies, leading to new
approaches to counter microglia-driven neurodegeneration in MS [35, 41]. In the present study, we demonstratethat IL-9 was able to mitigate EAE clinical disability andto reduce neuroinflammation and synaptic damage byGuadalupi et al. Journal of Neuroinflammation(2024) 21:128

a preferential interaction with microglial cells and TNF
release and synaptic effects.
The CNS role of IL-9 is supported by the identification of IL-9 receptor in the brain. Astrocytes, microglia,
oligodendrocytes and neurons were shown to express
IL-9R and to be modulated after IL-9 in vitro stimulation [15, 16, 22, 42]. In this study we examined for the
first time the expression of IL-9R in EAE striatum. By
immunofluorescence analysis we observed that microglia/macrophage cells represent the main cellular population expressing IL-9R indicating that microglia may
strongly respond to IL-9 pharmacological treatment during EAE disease. In line with this, studies on post-mortem
brain tissues showed that IL-9R is expressed by macrophages/microglia in the CNS of MS patients [13]. Moreover, among human blood cells, infiltrating monocytes
expressed the highest level of this receptor [13].
In vitro experiments showed that IL-9 reduced inflammatory properties and inhibited TNF release in LPS
stimulated- human monocytes/macrophages [13, 14].
Quantitative immunofluorescence analysis performedin this study clearly demonstrates that, in the striatum
of EAE mice, TNF production in microglia cells was
reduced by IL-9 treatment. Experiments on immortalized
murine microglial cell line BV-2 confirmed the directinterplay between IL-9 and microglia cells. Indeed, IL-9
in vitro treatment promoted both the reduction of TNF
release and the rescue of synaptic modulation induced by
activated BV2 cells.
In multiple sclerosis (MS), the resolution of inflammation is facilitated by the alternative M2-activated microglia/macrophage profile [28–30]. TREM2 is a reliable
marker of the M2 microglia profile and plays a pivotal
role in limiting central nervous system (CNS) damage.
Notably, a recent clinical study uncovered a significant
positive correlation between IL-9 and TREM2 levels in
the cerebrospinal fluid (CSF) of relapsing-remitting MS
patients [43]. Within the CNS, the TREM2 receptors
appear to be exclusively expressed by microglial [44]. In
EAE mice, TREM2 was shown to play an important rolein limiting the extent of CNS damage, since its blockade
exacerbates EAE disease.
Moreover, TREM2 expression is up-regulated during EAE, and its blockade has been shown to exacerbate
the severity of the disease [29]. Furthermore, intravenous administration of TREM2-transduced myeloid precursors in EAE mice led to an improvement in clinicalsymptoms, reduced axonal damage, enhanced removalof degenerated myelin, and decreased gene transcription of proinflammatory cytokines, including TNF [30].
The promotion of remyelination and myelin debris clearance through TREM-2 activation on microglia was also
observed in a different mouse model of MS, the cuprizone model [28].

Page 14 of 16

Finally, to investigate a putative interplay between IL-9
and TNF synaptic function and not only TNF release,
we explored the anti-synaptopathic activity of IL-9 in
healthy corticostriatal slices incubated with TNF. Suchan ex-vivo experiment showed that IL-9 blocked TNF
synaptic toxicity, suggesting a further mechanism independent of TNF release and probably due to a neuronalaction. Indeed, although we did not detect IL-9R signal
in MSN soma, we cannot rule out an expression of thisreceptor in neuronal terminations.
The evidence that IL-9 significantly attenuated excitotoxicity ascribed to TNF mediated synaptic damage[7, 45, 46] leads us to propose that the beneficial effectof IL-9 observed in the EAE striatum can be, at least inpart, related to its anti-TNF action. In vitro experiments
on BV2 cells and ex vivo investigation on brain slices
reveal two mechanisms by which IL-9 can promote thisprotective role: first, IL-9 reduces the release of TNF by
microglia cells; second, IL-9 directly blocks the neuronal
TNF effect. This synergistic effect of IL-9 on TNF mediated excitotoxicity could explain the efficacy of in vivo
treatment.
The increase of IL-9R expression in EAE brain [15] andpresent study], indicates the high susceptibility of CNS
to IL-9 pharmacological treatment during the disease.
Moreover, BV2 experiments confirm the link between
IL-9R expression and inflammatory microenvironment,
since only a sustained inflammatory stimulus (at least24 h) allows the increase of IL-9R. Of note, an important
negative regulatory pathway has been described between
IL-9 and its receptors. Indeed, IL-9 stimulation promotes
the down-regulation of cell surface receptor [47]. Suchnegative feedback could explain our experimental data on
both striatal slices and BV2 cells showing a strong reduction of IL-9R levels following its stimulation.
The clinical relevance of IL-9 treatment was finallyinvestigated by assessing its impact when administered
starting from the onset of clinical manifestation. In linewith the other in vivo treatment, the cytokine significantly ameliorated the EAE disease, highlighting its therapeutic potential in MS pathology.

Conclusions
This study reveals a central action of IL-9 in ameliorating EAE disease. Our data indicate that IL-9 attenuates
neuroinflammation and synaptic damage, counteracting
TNF-mediated neuronal toxicity.
The recognition that not only myelin but also synapses are early and privileged sites of damage in MS andinvolved in tissue loss could have profound therapeutic
implications; mitigation of inflammatory synaptopathy
can cause in parallel the correction of the synaptic alterations and the attenuation of motor deficits of EAE mice.

Guadalupi et al. Journal of Neuroinflammation(2024) 21:128

Altogether the data strengthen the relevance of an
immunomodulatory and neuroprotective action of IL-9
in MS, and shed light on IL-9 modulation as an effective disease modifying therapeutic strategy to counteract
neuronal and synaptic loss, as well as irreversible disability in MS.
Abbreviations
ACSF	Artificial CSF
BBB	Blood-brain barrier
CNS	Central nervous system
CFA	Complete Freund’s Adjuvant
dpi	Day post-immunization
EAE	Experimental autoimmune encephalomyelitis
IL	Interleukin MSNs: medium spiny projection neurons
MS	Multiple sclerosis
MOG35-55	Myelin oligodendrocyte glycoprotein peptide 35–55
PFA	Paraformaldehyde
PGs	Prostaglandins
sEPSC	Spontaneous postsynaptic glutamatergic current
sIPSC	Spontaneous postsynaptic inhibitory current
VHL	Vehicle
TREM2	Triggering receptor expressed on myeloid cells-2

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03120-9.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Acknowledgements
The authors thank Stefano Vellucci and Massimo Tolu for helpful technicalassistance. The experimental design in Fig. 1 was created with BioRender.com.
Author contributions
LG, VV, DC, and AM conceived and designed the study. LG, VV, SB, SC, FDV, DF,
KS, MN, EV, FM, MSB, LG, AB, ED, FB acquired and analyzed the data. LG, VV, DC,
and AM drafted the manuscript. All the authors critically revised the article
for important intellectual content and approved the version to be published.
LG and VV contributed equally to this work, as first authors. DC and AM
contributed equally to this work, as senior authors.
Funding
The study was supported by: Italian Ministry of Health (Ricerca Corrente
IRCCS Neuromed to DC; Ricerca Corrente IRCCS San Raffaele to GM; projectGR-2016-02361163 to AM and EV); FISM-Fondazione Italiana Sclerosi Multipla(cod. FISM2016/R/31 to EV; cod. FISM2020/R-Single/007 to EV); FISM andco-financed with the ‘5 per mille’ public funding, research fellowship 2020/
BS/003 to FDV; Ministry of University and Research and the European Union- Next Generation EU - National Recovery and Resilience Plan (NRRP), project
MNESYS (PE0000006) – A Multiscale integrated approach to the study ofthe nervous system in health and disease (DN. 1553 11.10.2022) to DC, GM,
AM and FB; CNR FOE 2020 and 2021 project ‘Nuovi Biomarker Diagnosticie Terapeutici delle Malattie Neurodegenerative’ to DC and GM; private
donations in memory of Veronica Tozzi to DC.
Data availability
All the research data are available from the corresponding author on request.

Declarations
Ethics approval and consent to participate
Animals experiments followed the guidelines of Animal Research Reporting
In Vivo Experiments (ARRIVE) and were carried out according to the Internal

Page 15 of 16

Institutional Review Committee, the European Directive 2010/63/EU, theEuropean Recommendations 526/2007 and the Italian D.Lgs 26/2014 (Prot.
n°337/2021-PR).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 9 February 2024 / Accepted: 1 May 2024

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