Arellano et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03126-3(2024) 21:144

Journal of Neuroinflammation

Open Access

RESEARCH

Therapeutic role of interferon-γ
in experimental autoimmune
encephalomyelitis is mediated througha tolerogenic subset of splenic CD11b+
myeloid cells
Gabriel Arellano1,2,3, Eric Acuña1, Eileah Loda2, Lindsay Moore2, Juan E. Tichauer1, Cristian Castillo1, Fabian Vergara1,
Paula I. Burgos4, Pablo Penaloza-MacMaster2,3, Stephen D. Miller2,3* and Rodrigo Naves1*
Abstract
Cumulative evidence has established that Interferon (IFN)-γ has both pathogenic and protective roles in Multiple
Sclerosis and the animal model, Experimental Autoimmune Encephalomyelitis (EAE). However, the underlying
mechanisms to the beneficial effects of IFN-γ are not well understood. In this study, we found that IFN-γ exerts
therapeutic effects on chronic, relapsing-remitting, and chronic progressive EAE models. The frequency ofregulatory T (Treg) cells in spinal cords from chronic EAE mice treated with IFN-γ was significantly increased withno effect on Th1 and Th17 cells. Consistently, depletion of FOXP3-expressing cells blocked the protective effectsof IFN-γ, indicating that the therapeutic effect of IFN-γ depends on the presence of Treg cells. However, IFN-γ did
not trigger direct in vitro differentiation of Treg cells. In vivo administration of blocking antibodies against either
interleukin (IL)-10, transforming growth factor (TGF)-β or program death (PD)-1, revealed that the protective effectsof IFN-γ in EAE were also dependent on TGF-β and PD-1, but not on IL-10, suggesting that IFN-γ might have anindirect role on Treg cells acting through antigen-presenting cells. Indeed, IFN-γ treatment increased the frequency
of a subset of splenic CD11b+ myeloid cells expressing TGF-β-Latency Associated Peptide (LAP) and program deathligand 1 (PD-L1) in a signal transducer and activator of transcription (STAT)-1-dependent manner. Furthermore,
splenic CD11b+ cells from EAE mice preconditioned in vitro with IFN-γ and myelin oligodendrocyte glycoprotein(MOG) peptide exhibited a tolerogenic phenotype with the capability to induce conversion of naïve CD4+ T cells
mediated by secretion of TGF-β. Remarkably, adoptive transfer of splenic CD11b+ cells from IFN-γ-treated EAE mice
into untreated recipient mice ameliorated clinical symptoms of EAE and limited central nervous system infiltrationof mononuclear cells and effector helper T cells. These results reveal a novel cellular and molecular mechanism*Correspondence:
Stephen D. Miller
s-d-miller@northwestern.edu
Rodrigo Naves
rodrigonaves@uchile.cl
Full list of author information is available at the end of the article© 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
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in this article, unless otherwise stated in a credit line to the data.

Arellano et al. Journal of Neuroinflammation(2024) 21:144

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whereby IFN-γ promotes beneficial effects in EAE by endowing splenic CD11b+ myeloid cells with tolerogenic andtherapeutic activities.
Keywords Multiple sclerosis, Experimental autoimmune encephalomyelitis, Interferon-γ, Regulatory T cells, CD11b+
cells, TGF-β

Introduction
Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), characterized by demyelination and axonal damage mediated bymyelin-specific T cells [1]. Although the etiology of MS
remains unknown, both genetic and environmental factors are involved in breaking self-tolerance [2, 3]. MS isclassified into two main clinical forms: relapsing-remitting (RR)-MS, characterized by episodes of acute neuroinflammation followed by variable recovery periods,
and progressive MS, consisting of chronic and irreversible neurological disability [4, 5]. Our understanding of
the immunopathogenesis of MS and the development oftherapies for this disease is largely based on the study ofan animal model, experimental autoimmune encephalomyelitis (EAE) [6–9]. EAE is actively induced by immunization with myelin-derived antigens associated withadjuvant and consists of an induction phase and an effector phase. During the inductive phase of EAE, peripheral myeloid antigen-presenting cells (APC) process andpresent myelin antigens to naïve CD4+ T cells inducing
differentiation of encephalitogenic interferon (IFN)-γproducing Th1 and IL-17-producing Th17 cells [10, 11].
Next, during the early effector phase, innate and adaptive immune cells, including Th1 and Th17 cells, migratefrom the periphery into the CNS generating a rapid andacute progression of disease leading to chronic demyelination and axonal damage [12]. Therefore, myeloid APC
play a critical role in MS and EAE activating encephalitogenic T cells and perpetuating the neuroinflammatory
process. However, APC also have the capability to induce
activation of Forkhead box p3 (FOXP3+) regulatory T(Treg) cells through the secretion of several molecules
such as interleukin (IL)-10 and tumor growth factor(TGF)-β, and the interaction of co-inhibitory molecules
such as those belonging to the program death-1 (PD-1)/
program-death ligands (PD-Ls) system [13–17]. In turn,
Treg cells develop a key protective role in EAE and MS,
exerting immunosuppressive activities on effector T cells
and regulating disease onset and progression [18–23].
Indeed, a functional impairment of Treg cells has been
related with MS pathogenesis [24–27].
IFN-γ is an essential cytokine that plays an importantrole in both innate and adaptive immune response. Its
receptor (IFN-γR) is ubiquitously expressed on all nucleated cells in the body [28], primarily signaling throughthe transcription factor signal transducers and activators of transcription (STAT)-1 [29], to induce expressionof multiple interferon-stimulated genes (ISGs) [30, 31].
Historically, IFN-γ is thought to have a pro-inflammatory role in MS and EAE [32–34]. However, several
studies have paradoxically shown that IFN-γ can also
mediate protective functions in EAE and MS (reviewed
in [35, 36]). In MS, induction of endogenous IFN production in progressive-MS patients showed that somepatients with improving symptoms had high levels of
serum IFN-γ, while worsening clinical symptoms was
related to low serum IFN-γ levels [37]. In EAE, cumulative evidence has demonstrated that IFN-γ signaling isnecessary to suppress EAE, and that IFN-γ treatment
has anti-inflammatory and tolerogenic effects on diseaseprogression [38–40]. Moreover, it has been reported thatthe therapeutic activity of IFN-β, an extensively usedtherapy in RRMS patients, depends on IFN-γ signaling
because IFN-β treatment has no effect on the development of disease in IFN-γR-deficient mice [41]. Also, we
have shown that IFN-γ has a dual role depending on
the phase of EAE: playing a pathogenic role during theinductive phase and a protective during the acute effector phase of disease [42]. Similar results have been foundusing neutralizing antibodies against IFN-γ in EAE mice[43]. Recently, we have reported that IFN-γ administered at the peak of EAE results in amelioration of clinical symptoms and attenuation of neuroinflammation,
in a STAT-1-dependent manner. Histological analyses
showed that spinal cord sections from IFN-γ-treated EAE
mice exhibit significantly less infiltration of inflammatory
cells and fewer demyelinated areas compared to controltreated EAE mice. Besides, IFN-γ treatment promoted ashift from activated microglia (MG) to resting MG and
the induction of a subset of CX3CR1highPD-L1low MG
characterized by a homeostatic and anti-inflammatorytranscriptional signature [44]. Therefore, accumulative
evidence has demonstrated a beneficial role of IFN-γ
in EAE and MS. However, after 30 years of study, themechanisms underlying the protective effects of IFN-γ
in these pathologies remain largely unknown. Because
EAE and MS are diseases mainly mediated by myelinspecific T cells, in this study we addressed the impact
of IFN-γ treatment on peripheral and CNS infiltrating
CD4+ T cell subpopulations, including Th1, Th17, andTreg cells, in EAE. We found that the therapeutic effectof IFN-γ requires the presence of functional FOXP3+
Treg cells, although IFN-γ does not directly regulate
Treg cell differentiation. Instead, IFN-γ acts directly on
splenic CD11b+ cells inducing a tolerogenic phenotype

Arellano et al. Journal of Neuroinflammation(2024) 21:144

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in a STAT-1-dependent manner. Interestingly, IFN-γstimulated splenic CD11b+ cells induce in vitro conversion of naïve CD4+ T cells to Treg cells mediated bysecretion of tumor growth factor (TGF)-β. Furthermore,
adoptive transfer of splenic CD11b+ cells from IFN-γtreated EAE mice to untreated EAE mice induces diseaseamelioration by reducing CNS infiltration of effector
helper T cells.

diphtheria toxin (Sigma-Aldrich, Saint Louis, Missouri,
USA) at day − 2 and 0 before starting IFN-γ treatment.
EAE symptoms were scored using a 0–5 scale as follows:
0, no symptoms; 1, loss of tail tone; 2. flaccid tail; 3, partial paralysis of lower extremities; 4, complete paralysis
of lower extremities; 5, moribund/death (animals are
humanely euthanized).

Materials and methods

Mice were perfused intracardially with PBS and brains
and spinal cords were isolated and incubated with
DNAse (10 units/mL) (New England Biolabs, Ipswich,US) and collagenase D (0.5 mg/mL) (Roche, Manheim,
Germany) for 1 h at 37 °C. Then, tissues were disaggregated and centrifuged. Mononuclear cells were obtainedusing a discontinuous Percoll gradient (Amersham, Piscataway, US). Draining cervical/axillary lymph nodes(LN) and spleens were screened to yield single cell suspensions and splenocytes were treated with red blood
lysis buffer (155mM NH4Cl, 10mM KHCO3, y 1mM
EDTA, pH 7.3). For T cell flow cytometry analysis, cells
were first stimulated with PMA (50 ng/mL)/ionomycin (500 ng/mL) (Sigma-Aldrich, Saint Louis, US) and
Brefeldin A (5 µg/mL) (Sigma-Aldrich, Saint Louis, US)for 4 h. Then, cells were stained with LIVE/DEAD™ Fixable dye-Dead Cell Stain Kit (Thermofisher, Carlsbad,US) to determine viability, blocked with Fc Block (Biolegend, San Diego, US) and fixed/permeabilized with
FOXP3/transcription factor staining buffer set (eBioscience, San Diego, US) for intracellular staining. A T-cellstaining panel consisting of the following markers wasused: CD45, CD3, CD4, CD25, FOXP3, IFN-y andIL-17 A. The APC staining panel included the following
markers: CD45, CD11b, TGF-β-Latency Associated Peptide (LAP), PD-L1, PD-L2, CD80, CD86 and MHC-II. All
antibodies were purchased from Biolegend (San Diego,
California, US). Samples were acquired with FACS LSR
Fortessa at University of Chile or FACS Celesta (BD bioscience, New Jersey, US) at Northwestern University andanalyzed with Flowjo X v10.7 software (Tree Star, Ashland, Oregon, US). Gating flow cytometry strategies are
represented in Supplementary Fig. 1.

Mice

C57BL/6 (CD45.1, CD45.2, and Stat-1−/−) mice, nonobese diabetic (NOD) mice, and SJL/J mice werepurchased from The Jackson Laboratory (US). The
C57BL/6-FOXP3GFP − DTR (FOXP3-DTR) mice werekindly provided by Dr. Pablo Penaloza-MacMaster(Northwestern University, US). Mice were maintained
and bred at the animal core facility of Faculty of Medicine-Universidad de Chile or Center for Comparative Medicine-Northwestern University and treated inaccordance with the Institutional Animal Care and Use
guidelines (IACUC) of the Universidad de Chile andNorthwestern University.
EAE induction, clinical scoring, and treatments

Mice 8 to 12-weeks-old were induced with different types
of EAE. Chronic EAE was induced by subcutaneous(s.c.) immunization of C57BL/6 male mice with 150 µg
MOG33 − 55 peptide (CPC Scientific, California, US) and500 µg Mycobacterium tuberculosis (MT) H37Ra (BDDifco, Detroit, US) in incomplete Freund’s adjuvant (BDDifco, Detroit, US) followed by an intraperitoneal (i.p.)
injection with 200 ng pertussis toxin (PTx) (Calbiochem,
Campbell, US) on days 0 and 2 post-immunization (p.i.).
RR-EAE was induced by s.c. immunization of SJL female
mice with 150 µg PLP139 − 151 peptide. Chronic progressive EAE was induced in NOD female mice by s.c. immunization with 150 µg MOG33 − 55 peptide, 400 µg MT, anda single i.v. injection of 200 ng PTx on day 0. For treatment with IFN-γ, 1 µg/day of recombinant murine IFN-γ(Biolegend, San Diego, US) was administered i.p. for 5days in chronic and RR-EAE or for 10 days in progressive
EAE. For in vivo neutralization of PD-1, mice received
two i.p. doses of 500 µg of anti-PD-1 (clone RMP1-14) or
isotype Ig control (rat IgG2a, clone 2 A) at day − 2 and
0 of IFN-γ treatment. For in vivo cytokine neutralization, three doses of 100 µg anti-TGF-β (clone 1D11.16.18)
and anti-IL-10 (clone JES5-2A5), or their isotype Ig control (mouse anti-IgG1, clone MOPC-2, or rat anti-IgG1,
clone HRPN, respectively), were administered i.p. at days
0, 2, and 4 after IFN-γ treatment. All neutralizing antibodies and isotype controls were purchased to BioXcell(Lebanon, US). To deplete FOXP3+ Treg cells, FOXP3DTR mice were i.p. treated with one dose of 2.5 µg of

CD4+ T cell and APC phenotyping

Cell purification

CD4+CD25− and CD4+CD25+ T cells were isolated byimmunomagnetic selection using the EasySep™ mouse
CD4+CD25+ regulatory T cell isolation kit II (> 90%
purity). CD4+CD62L+CD44low naïve CD4+ T cells wereisolated using the EasySep™ mouse CD4+ T cell isolation
kit (> 95% purity). CD11b+ cells were purified using the
EasySep™ mouse CD11b positive selection kit II (> 87%
purity). All purification kits were from Stem Cell (Vancouver, Canada).

Arellano et al. Journal of Neuroinflammation

Treg cell induction by IFN-γ+−+(2024) 21:144+

CD4 CD25 and CD4 CD25 T cells isolated from thespleen and LN of mice at the peak of the disease (day12–15 p.i.) were cultured with serum-free X-VIVO 20
medium (Lonza, Basel, Switzerland) in the presence of
2 µg/mL of plate-bound anti-CD3 antibody and 1 µg/mL
of soluble anti-CD28 antibody (both from Biolegend, San
Diego, California, US), with or without IFN-γ, at 37 °C
for 3 days. Different concentrations of IFN-γ (1, 10, 25,
50, and 100 ng/mL) were tested (Supplementary Fig. 3B)
to select the final concentration of 25 ng/mL, which waspreviously described to induce Treg cells in similar conditions [45]. Controls with 5 µg/mL anti-IFN-γ antibody
or isotype control antibody were used. Similar experiments were also performed in the presence of TGF-β (2ng/mL) and IL-2 (10 ng/mL) (both from Thermofisher,
Waltham, Massachusetts, US) to induce differentiationof Treg cells (iTreg conditions). Cell culture supernatants were collected and stored frozen for further cytokine analysis. The frequency of FOXP3+ Treg cells andthe expression of CTLA-4, LAG-3, HELIOS, and TGF-βLAP were determined by flow cytometry.
+

Treg cell induction by IFN-γ-stimulated CD11b cells

CD11b+ cells isolated from the spleen of EAE mice atthe peak of the disease (day 12–15 p.i.) were cultured in
the presence of 10 µg/mL MOG33 − 55 peptide and stimulated with 25 ng/mL IFN-γ for 24–72 h. The expressionof TGF-β-LAP, PD-L1, PD-L2, CD80, CD86, and MHC-II
was determined by flow cytometry. Cell culture supernatants were collected to determine TGF-β levels. In otherassays, CD11b+ purified cells from the spleen of EAE
mice were preconditioned with 10 µg/mL MOG33 − 55
peptide and 25 ng/mL IFN-γ for 24 h, washed, and cocultured with naïve CD4+ T cells (ratio 1:1) from nonimmunized mice in the presence of 1 µg/mL soluble
anti-CD3 antibody (Biolegend, San Diego, California, US)
and with either 5 µg/mL anti-TGF-β antibody (BioXcell,
Lebanon, US) or anti-PD-L1 antibody (Thermofisher,Carlsbad, US), and their isotype control antibodies. After
72 h, the frequency of Treg (CD4+CD25+FOXP3+) cells
was determined by flow cytometry.
Adoptive transfer of splenic CD11b+ cells from IFN-γtreated EAE mice into recipient EAE miceCD11b+ cells (1 × 106) were purified from spleens ofCD45.1+ EAE mice treated with IFN-γ or PBS for 5 days
and i.v. transferred into untreated CD45.2+ EAE mice atthe peak of disease. After 3 days, splenocytes and mononuclear cells from spinal cords of recipient EAE mice
were isolated and absolute cell number and frequencies
of mononuclear cells, CD45.2+ cells, CD45.1+CD11b+
cells, CD3+ T cells, CD3+CD4+ T cells, CD3+CD4− Tcells, CD3+CD4+CD25+FOXP3− T cells, and Treg cells

Page 4 of 17(CD3+CD4+CD25+FOXP3+) were determined by flow
cytometry.
Cytokine measurements

Cell culture supernatants were used to determine thelevels of TGF-β1, IL-10, IL-27, Granzyme B, and sFASL
by MILLIPLEX® Multiplex Assay kits (Millipore-Sigma,
Saint Louis, US) and quantitated using the Luminex®
200™ System (Northbrook, Illinois, US).
Statistical analysis

Results were analyzed using a Mann–Whitney U testor Kruskal-Wallis test. For comparison of EAE scores
between two or multiple groups, we conducted two-way
ANOVA or one-way ANOVA followed by multiple comparisons with Bonferroni post-hoc test. Statistical analyses were performed with GraphPad Prism v.9.0 software(San Diego, California, US). All data are presented asmean ± SEM. Values were considered statistically significant when p < 0.05.

Results

Therapeutic effect of IFN-γ is dependent on FOXP3+ Treg
cells in EAEFirst, we confirmed that treatment of C57BL/6 micedeveloping chronic EAE with IFN-γ for 5 days startingat peak of disease induced a significant suppression ofclinical symptoms. After cessation of treatment, disease
severity slowly returned to levels similar to control EAE
mice (Fig. 1A). Next, we asked whether IFN-γ might havebeneficial effects in two other clinically distinct EAE
models mimicking RRMS (SJL/J mice) and chronic progressive MS (NOD mice). The results showed that IFN-γ
administered at the peak of the first flare of RR-EAE (SJL
mice) induced a significant attenuation of clinical symptoms and prevention of further relapses compared to
PBS-treated mice (Fig. 1B). In addition, IFN-γ treatment
promoted significant suppression of disease in chronic
progressive EAE mice (NOD mice) after 10 days of treatment (Fig. 1C).
Next, we investigated the impact of IFN-γ treatment on
peripheral and CNS-infiltrating CD4+ T cell subpopulations in chronic MOG35 − 55 peptide EAE in B6 mice. Spinal cords isolated from EAE mice treated with IFN-γ for
5 days (Fig. 2A) exhibited a significant reduction in thenumber of mononuclear cells (MNC) and infiltrating
CD45+ cells compared with those from PBS-treated EAE
mice (Fig. 2B). There was no statistical difference in theabsolute number of MNC and CD45+ cells in brains from
IFN-γ and PBS-treated EAE mice and similar numbersof cells were found in spleen and LN from both groups
of EAE mice (Supplementary Fig. 2A). Lower absolute
numbers of CD3+ T cells were observed in IFN-γ-treated
EAE mice compared to PBS-treated EAE mice, but this

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Fig. 1 Therapeutic administration of IFN-γ ameliorates clinical severity in chronic, relapsing-remitting, and chronic progressive-EAE. (A) Two groups of
C57BL/6 mice were induced with chronic EAE (n = 9/group) and daily treated with 1 µg IFN-γ (empty black circles) or PBS (solid black circles) at peak of
disease for 5 days. (B) SJL mice were induced with relapsing-remitting (RR)-EAE (n = 6/group) and daily treated with 1 µg IFN-γ (empty black circles) or
PBS (solid black circles) at peak of the first flare for 5 days. (C) Chronic progressive-EAE was induced in NOD mice (n = 13/group) and treated with 1 µg
IFN-γ (empty black circles) or PBS (solid black circles) during the chronic phase of the disease (starting at 45 days post-immunization) for 10 days. Clinicalsymptoms were monitored daily using the standard scoring scale. Results are shown as the mean ± SEM. Data were analyzed using two-way ANOVA followed by Bonferroni post-hoc test. *p < 0.05, **p < 0.01

difference did not reach statistical significance (Fig. 2B).
Analysis of the impact of IFN-γ on different subpopulations of CD4+ T lymphocytes (Supplementary Fig. 1A
and B) showed a lower absolute number of CD4+ T cells
and Th1 cells in spinal cords from IFN-γ-treated EAE
mice than in control EAE mice, but these differenceswere not significant. The number of Th17 and Treg cells
was similar between both groups of mice (Fig. 2C). The
absence of impact on Th17 and Treg cell number led us
to determine if the frequencies might be modulated by
IFN-γ treatment. Similar frequencies of CD4+ T cells
and Th1 cells and a slight but not significant increase of
Th17 cells were determined in spinal cords from IFN-γ
and PBS-treated EAE mice. Notably, a significantly
higher frequency of Treg cells was found in spinal cords
from IFN-γ-treated EAE mice (Fig. 2D). No significant
differences in numbers or frequencies of CD4+ T cell
subpopulations were observed in the brain and secondary lymphoid organs (cervical/axillary draining LN
and spleen); although the absolute number of Treg cells
tended to be higher in brains and spleens of IFN-γtreated EAE mice (Supplementary Fig. 2B). These results
led us to examine the functional requirement of Treg cells
in mediating the protective effect of IFN-γ in EAE. To dothat, we used the transgenic FOXP3-DTR mouse model,
which allows the conditional depletion of FOXP3+ Treg
cells after systemic administration of diphtheria toxin(Supplementary Fig. 2C). Consistent with a previous
study [21], depletion of FOXP3+ Treg cells at the peak of
EAE induced a more severe disease over time. Interestingly, EAE mice depleted of Treg cells were refractory to
the protective effects of IFN-γ, suggesting that functional

Treg cells are required at the time of IFN-γ treatment foreffective therapeutic regulation of EAE (Fig. 2E).
IFN-γ does not exert a direct effect on Treg celldifferentiation

In order to determine whether IFN-γ might directly trigger the conversion of CD4+CD25− T cells into Treg cells,
CD4+CD25− T cells were purified from spleen and draining LN of EAE mice at the peak of the disease (> 90%
purity, Supplementary Fig. 3A) and in vitro activatedwith anti-CD3 and anti-CD28 antibodies with IFN-γ oranti-IFN-γ antibody for 72 h. Different concentrations of
IFN-γ (range between 1 and 100 ng/ml) did not induce
conversion of CD4+CD25− T cells isolated from spleen or
LN of EAE mice into Treg cells (Fig. 3A and Supplementary Fig. 3B). Next, we analyzed whether IFN-γ might
have a direct effect on the generation of inducible Treg
cells (iTreg) promoted by TGF-β and IL-2. The results
showed that addition of IFN-γ did not change the frequency of iTreg cells starting from CD4+CD25− T cells
isolated from spleens or LN (Fig. 3B). Moreover, addition of blocking antibodies to IFN-γ led to significantly
higher generation of iTreg cells compared to untreatedand IFN-γ-stimulated CD4+CD25− T cells (Fig. 3B). We
also determined whether IFN-γ might have a direct effecton the fraction of CD4+CD25+ T cells obtained during
the purification of CD4+CD25− T cells (> 89% of total
FOXP3+ cells - Supplementary Fig. 3A). IFN-γ treatment
did not affect the expression of regulatory markers associated with Treg cells such as FOXP3, CTLA-4, HELIOS,
LAG-3, and TGF-β-LAP (Fig. 3C) or the secretion of
IL-10, Granzyme B, and soluble FAS ligand (Fig. 3D) in
CD4+CD25+ T cells in vitro activated with anti-CD3 andArellano et al. Journal of Neuroinflammation(2024) 21:144

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Fig. 2 Therapeutic effect of IFN-γ depends on the presence of FOXP3+ Treg cells in EAE. (A) Mice induced with chronic EAE were treated daily with 1 µg
IFN-γ (empty black circles) or PBS (solid black circles) at the peak of disease for five days (n = 10/group, mean of three independent experiment). (B-D) At
the end of treatment, spinal cords were isolated and the absolute number of (B) mononuclear cells (MNC), CD45+ cells, and CD3+ cells, and (C) the absolute number and (D) frequency of CD4+ T cells, Th1, Th17 and Treg cells were determined by flow cytometry. (E) Wild type (n = 13/group) and FOXP3-DTR
mice (n = 5/group) were induced with EAE and treated with 1 µg IFN-γ (empty black and red circles) or PBS (solid black and red circles) at the peak of disease for five days. To deplete FOXP3+ Treg cells, FOXP3-DTR mice were i.p. treated with one dose 2.5 µg of diphtheria toxin (DTx) two days before starting
IFN-γ treatment and with another similar dose two days later. Three independent experiments were pooled. Results are shown as the mean ± SEM. Data
were analyzed using two-way ANOVA followed by Bonferroni post-hoc test (A), Mann–Whitney U test (B-D), or one-way ANOVA followed by Bonferroni
post-hoc test (E). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

anti-CD28 antibodies. Taken together, these results indicate that IFN-γ does not directly promote the generationof Treg cells, but does, in fact, inhibits the differentiationof iTreg cells.
Therapeutic effect of IFN-γ is dependent on TGF-β andPD-1

Because IFN-γ has no direct effect on the induction ofTreg cells, we asked if this cytokine might modulate
regulatory molecules involved in the differentiation andactivity of FOXP3+ Treg cells, such as TGF-β [46], IL-10[47], and PD-1 [16]. As expected, in vivo blockade ofeither TGF-β or IL-10 in untreated (without IFN-γ) EAE
mice slightly exacerbated disease severity compared tocontrol groups. Interestingly, neutralization of TGF-β,
but not IL-10, interfered with the protective effects of
IFN-γ on EAE (Fig. 4A and B). Similarly, disease amelioration induced by IFN-γ was prevented when EAE mice
were administered blocking antibodies to PD-1 (Fig. 4C).
Based on these results, we hypothesized that IFN-γ might
have an indirect role on FOXP3+ Treg cells regulating theexpression of TGF-β and ligands of PD-1 in APC.
IFN-γ/STAT1 axis induces a subset of splenic CD11b+
myeloid cells expressing PD-L1 and TGF-β in EAE mice

To address the above hypothesis, we determined theimpact of IFN-γ on myeloid APC in EAE by analyzing
the frequency of CD11b+ myeloid cells expressing PD-L1

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Fig. 3 IFN-γ does not exert a direct effect on Treg cell differentiation. (A) CD4+CD25− T cells isolated (purity > 90%) from spleen or draining lymph
nodes (LN) of EAE mice at the peak of disease were stimulated for 3 days with 2 µg/mL plate-bound anti-CD3 and 1 µg/mL soluble anti-CD28 in theabsence (control) or presence of either 25 ng/mL IFN-γ, 5 µg/mL anti-IFN-γ antibody or 5 µg/mL isotype control antibody. (B) Similar assay as described in (A) in the presence of 2 ng/mL TGF-β and 10 ng/mL IL-2 to promote differentiation of inducible Treg cells (iTregs). The frequency of Treg cells(CD3+CD4+CD25+FOXP3+) was determined by flow cytometry. (C) Purified fraction of CD4+CD25+ cells (containing > 82% of Treg cells) obtained during
the purification of CD4+CD25− T cells were stimulated with 2 µg/mL plate-bound anti-CD3 and 1 µg/mL soluble anti-CD28 for 3 days in the absence (control, solid gray bars) or presence of 25 ng/mL IFN-γ (empty gray bars). The expression of regulatory markers FOXP3, HELIOS, CTLA-4, LAG-3 and TGF-β-LAP
was determined as median fluorescence intensity (MFI) by flow cytometry. (D) Secretion of IL-10, Granzyme B and sFASL in cell culture supernatants wasassessed by multiplex assay. Results are shown as the mean ± SEM (n = 4). Data were analyzed using Mann–Whitney U test. *p < 0.05

Arellano et al. Journal of Neuroinflammation(2024) 21:144

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and TGF-β-LAP in brain, spinal cord, spleen, and LN
from EAE mice. The results showed that the frequency
of CD11b+ cells expressing PD-L1 and TGF-β-LAP was
significantly higher in the spleen from IFN-γ-treated EAE
mice than in those from PBS-treated EAE mice. There
was no difference in other organs (Fig. 5A).
The biological activity of IFN-γ is mainly mediatedthrough STAT-1, thus we determined whether the in vivo
modulation of splenic CD11b+ myeloid cells by IFN-γ isalso dependent on STAT-1. We found that the enhanced
frequency of CD11b+ cells expressing PD-L1 and TGF-βLAP induced by IFN-γ was significantly reversed in EAE
mice lacking STAT-1 (Fig. 5B and C). Therefore, theseresults indicate that the IFN-γ-STAT-1 axis mediates
protective effects in EAE via inducing a subset of splenic
CD11b+ myeloid cells expressing PD-L1 and TGF-β-LAP.
IFN-γ acts directly on splenic CD11b+ cells inducing atolerogenic phenotype and function

Fig. 4 Therapeutic effect of IFN-γ is dependent on TGF-β and PD-1. EAE
mice were treated at the peak of disease for 5 days with 1 µg/day IFN-γ
alone (empty black circles) or along with (A) 100 µg of neutralizing antibody against TGF-β (solid blue circles, n = 6) or (B) IL-10 (solid blue circles,n = 7) administered at days 0, 2 and 4 after IFN-γ treatment, or with (C)500 µg neutralizing anti-PD-1 antibody (solid blue circles, n = 8) administered two days before starting IFN-γ treatment and at the same time of
IFN-γ treatment. Control EAE mice were treated with PBS (vehicle, solid
black circles) or corresponding isotype antibody alone (empty red circles)or along with IFN-y (empty blue circles). Two independent experiments
were pooled. Results are shown as the mean ± SEM. Data were analyzed
using one-way ANOVA followed by the Bonferroni post-hoc test. *p < 0.05,**p < 0.01

Next, we wanted to determine whether IFN-γ might
directly regulate the expression of PD-L1, TGF-βLAP, and other molecules involved in T cell activation
on CD11b+ myeloid cells. CD11b+ cells isolated fromspleen (> 87% purity, Supplementary Fig. 4) at peak EAE
were stimulated with IFN-γ and MOG35 − 55 peptide for
24–72 h and the expression of TGF-β-LAP, co-inhibitory
molecules (PD-L1 and PD-L2), co-stimulatory molecules(CD80 and CD86), and MHC-II molecules was evaluated by flow cytometry. The levels of secreted TGF-β
were assessed in the cell culture supernatant by multiplex
assay. The results showed that CD11b+ cells treated with
IFN-γ for 24 h presented a significantly lower expression of TGF-β-LAP on the cell surface, as measured bymean fluorescence intensity (MFI), which was associatedwith a higher secretion of TGF-β1 compared to unstimulated cells (Fig. 6A and C); suggesting an enhanced processing and secretion of TGF-β in response to IFN-γ
in these cells. After 72 h, IFN-γ-treated CD11b+ cells
showed a significantly higher expression of TGF-β-LAP+on the cell surface but a similar secretion of TGF-β thanuntreated CD11b+ cells (Fig. 6B and C). Given that it has
been reported that IL-27, a cytokine induced by IFN-γ, isinvolved in antigen-specific immune tolerance in EAE by
inducing PD-L1 expression in dendritic cells and production of IL-10 in CD4+ T cells [48, 49], we also determined
the levels of IL-27 and IL-10 in the cell culture supernatant. The results showed that splenic CD11b+ cells fromEAE mice secreted low levels of IL-27 and IL-10 andthat they were not induced by IFN-γ after 24 and 72 h oftreatment (Supplementary Fig. 5A). Remarkably, stimulation with IFN-γ for 24 h and 72 h induced a significantly
higher frequency of CD11b+ cells expressing PD-L1
as well as a higher expression of PD-L1 compared tountreated CD11b+ cells (Fig. 6A and B). The expression

Arellano et al. Journal of Neuroinflammation(2024) 21:144

Page 9 of 17

Fig. 5 IFN-γ/STAT-1 axis induces splenic CD11b+ myeloid cells expressing PD-L1 and TGF-β-LAP. (A) Frequency of PD-L1+ and TGF-β-LAP+ CD11b+ cells inbrain, spinal cord (SC), draining lymph nodes (LN) and spleen from EAE mice treated at the peak of disease with IFN-γ (empty gray bars, n = 4) or PBS (solid
gray bars, n = 5) for 5 days was determined by flow cytometry. (B) Frequency of CD11b+PD-L1+ cells and (C) CD11b+TGF-β-LAP+ cells were determined byflow cytometry in spleen from wild-type mice (solid and empty gray bars, n = 6) and STAT-1 deficient mice (solid and empty red bars, n = 6) induced with
EAE and treated at the peak of disease with IFN-γ (empty bars) or PBS (vehicle, solid bars) for 5 days. Three independent experiments were pooled. Results
are shown as the mean ± SEM. Data were analyzed using Mann–Whitney U or Kruskal-Wallis test. *p < 0.05, **p < 0.01

Arellano et al. Journal of Neuroinflammation(2024) 21:144

Page 10 of 17

Fig. 6 IFN-γ directly induces a tolerogenic phenotype and function in splenic CD11b+ cells from EAE mice. Splenic CD11b+ cells isolated from EAE mice
were in vitro stimulated with 25 ng/mL IFN-γ (empty gray bars) or left untreated (UN, solid gray bars) for (A) 24 and (B) 72 h in the presence of 10 µg/
mL MOG33 − 55 peptide. Frequency and median fluorescence intensity (MFI) of TGF-β-LAP, PD-L1, PD-L2, CD80, CD86 and MHC-II were analyzed by flow
cytometry (n = 4). (C) Cell culture supernatants were collected at 24 and 72 h and concentration of TGF-β1 was measured by multiplex assay (n = 8). (D)Splenic CD11b+ cells from EAE mice were preconditioned with 10 µg/mL MOG33 − 55 peptide and 25 ng/mL IFN-γ for 24 h, washed, and co-cultured
with naïve CD4+ T cells (ratio 1:1) from healthy mice in the presence of 1 µg/mL soluble anti-CD3 antibody and with either anti-TGF-β antibody (solid
blue bar), anti-PD-L1 antibody (solid red bar), or corresponding isotype control antibodies (empty blue and red bars). After 72 h, the frequency of Treg
cells (CD4+CD25+FOXP3+) was determined by flow cytometry. Results are shown as the mean ± SEM. Data were analyzed using Mann–Whitney U test.
*p < 0.05, **p < 0.01

Arellano et al. Journal of Neuroinflammation

Fig. 7 (See legend on next page.)(2024) 21:144

Page 11 of 17

Arellano et al. Journal of Neuroinflammation(2024) 21:144

Page 12 of 17(See figure on previous page.)
Fig. 7 Adoptive transfer of splenic CD11b+ cells from IFN-γ-treated EAE mice into recipient EAE mice suppresses disease progression. (A) Splenic CD11b+
cells (106) from CD45.1 EAE mice treated for 5 days with IFN-γ (empty black circles) or PBS (solid black circles) were i.v. transferred into CD45.2 EAE miceat the peak of disease (n = 15). Clinical symptoms were monitored daily. Four independent experiments were pooled. (B-C) After three days of transfer,mononuclear cells from spinal cords of recipient EAE mice receiving CD45.1+CD11b+ cells from EAE mice treated with IFN-γ (empty gray bars, n = 8) or PBS(solid gray bars, n = 8) were isolated and absolute number and frequencies of (B) mononuclear cells (MNC), CD45.2+ cells, and CD45.1+CD11b+ cells, and(C) CD3+ T cells, CD3+CD4+ T cells, CD3+CD4− T cells, CD3+CD4+CD25+FOXP3− T cells, and Treg cells (CD3+CD4+CD25+FOXP3+), were determined by flow
cytometry (n = 8, two different experiments were pooled). Results are shown as the mean ± SEM. Data were analyzed using two-way ANOVA followed by
the Bonferroni post-hoc test (A) or Mann–Whitney U test (B-C). *p < 0.05, **p < 0.01, ***p < 0.001

of PD-L2 on CD11b+ cells was significantly lower in IFNγ-treated CD11b+ cells than untreated cells at 24 h, andthe frequency of CD11b+ cells expressing PD-L2 was alsosignificantly reduced after 72 h.
The frequency of CD11b+ cells expressing CD80
and CD86 was initially increased at 24 h by IFN-γ but
decreased to control levels after 72 h. The expression of
CD80 was significantly lower in response to IFN-γ after
72 h (Fig. 6A and B). Although there was no differencein the frequency of CD11b+ cells expressing MHC-II
molecules, CD11b+ cells stimulated with IFN-γ showedsignificantly lower expression of MHC-II molecules at
24 h compared to untreated CD11b+ cells (Fig. 6A). After
72 h, IFN-γ induced a significantly higher frequency of
CD11b+MHC-II+ cells but with a similar level of expression of MHC-II molecules than control cells (Fig. 6B).
Taken together, these results suggest that IFN-γ acts
directly on CD11b+ cells regulating the expression ofmolecules involved in immune tolerance resulting in an
early secretion of TGF-β1, enhanced expression of PD-L1
and CD86, and decreased expression of MHC-II molecules, followed by an increased expression of TGF-β-LAP
and PD-L1, and a reduced expression of CD80.
Therefore, we next investigated the capability of IFNγ-treated CD11b+ cells to induce differentiation of Treg
cells. CD11b+ cells isolated from spleen at peak EAE were
cultured in the presence of IFN-γ and MOG35 − 55 peptide for 24 h, washed, and then co-cultured with naïve
CD4+ T cells from healthy mice (1:1 ratio) and soluble
anti-CD3 antibody for 72 h. The expression of FOXP3+(MFI) in CD4+ T cells and the frequency of Treg cells(CD4+CD25+FOXP3+) was significantly higher in naïve
CD4+ T cells co-cultured with IFN-γ-stimulated CD11b+
cells than with untreated CD11b+ cells (Fig. 6D and Supplementary Fig. 5B). Interestingly, neutralization of TGFβ, but not of PD-L1, significantly decreased the expressionof FOXP3+ in CD4+ T cells and the frequency of Treg cells
to a similar level of untreated cells. These results indicatethat IFN-γ induces tolerogenic splenic CD11b+ cells withthe capability to convert naïve CD4+ T cells into Treg cells.
Transfer of CD11b+ myeloid cells from IFN-γ-treated EAE
mice to untreated EAE mice induces disease amelioration
by reducing CNS-infiltrating helper T cells

To demonstrate the therapeutic and tolerogenic potential of splenic CD11b+ myeloid cells induced by IFN-γ,

these cells were isolated from IFN-γ or PBS-treated
EAE mice (CD45.1) and transferred to untreated EAE
mice (CD45.2) at the peak of EAE. EAE mice receiving a single injection of splenic CD11b+ myeloid cells
from IFN-γ-treated EAE mice exhibited a significant
and rapid amelioration of clinical symptoms followed
by a relapse of disease compared to control EAE micereceiving CD11b+ cells from PBS-treated EAE mice
(Fig. 7A). After three days of transfer, the total number of mononuclear cells and CD45.2+ cells were significantly lower in spinal cord from EAE mice receiving
splenic CD11b+ cells from IFN-γ-treated EAE micethan those receiving splenic CD11b+ myeloid cells from
PBS-treated EAE mice (Fig. 7B). Instead, the absolute
cell number and frequency of CD45.1+CD11b+ cells
in spinal cord were not significantly different between
both groups of recipients EAE mice (Fig. 7B). There
was also no difference in the absolute cell number andfrequency of CD45.2+ cells and CD45.1+CD11b+ cells
in the spleen from both groups of recipients EAE mice
(Supplementary Fig. 6A). Interestingly, spinal cords
from EAE mice receiving CD11b+ myeloid cells from
IFN-γ-treated EAE mice exhibited a significantly lower
absolute number of CD3+ T cells, CD3+CD4+ T helper
cells, CD3+CD4− cells, and CD25+FOXP3− cells compared to spinal cords from EAE mice receiving CD11b+
myeloid cells from PBS-treated EAE mice. Likewise, theabsolute number of Treg cells was lower in EAE micereceiving CD11b+ myeloid cells from IFN-γ-treated
EAE mice; but it did not reach statistical significance(Fig. 7C). Although reduction in all these cell populations might be influenced by the lower absolute numbers of total mononuclear cells observed in spinal cordfrom EAE mice receiving CD11b+ myeloid cells from
IFN-γ-treated EAE mice (Fig. 7B), the frequency of
CD3+ T cells and CD3+CD4+ helper T cells was also significantly lower in recipients of CD11b+ myeloid cells
from IFN-γ-treated EAE mice (Fig. 7C). There was nodifference in the absolute cell number and frequency of
all T cell subpopulations analyzed in spleen from bothgroups of recipients EAE mice (Supplementary Fig. 6B).
Taken together, these results indicate that adoptive
transfer of splenic CD11b+ myeloid cells from IFN-γtreated EAE mice to recipient (untreated) EAE mice
suppresses disease progression by limiting CNS-infiltrating effector helper T cells.

Arellano et al. Journal of Neuroinflammation(2024) 21:144

Discussion
IFN-γ is primarily considered a pro-inflammatory cytokine based on its key role against infections and tumors
as well as the promotion of Th1 cell-driven inflammation.
However, cumulative evidence has revealed that IFN-γ
also exerts protective effects in EAE (reviewed in [35,36, 42, 44]). Here, we provide further support for thesefindings, demonstrating that IFN-γ limits the magnitudeof new relapses in RR-EAE and slows clinical course inchronic progressive EAE. Also, beneficial effects of IFN-γ
have been reported in other murine models of transplantation, asthma, and autoimmune diseases such as collagen-induced arthritis (CIA), experimental autoimmune
uveitis (EAU), autoimmune nephritis, and autoimmune
myocarditis [50–58]. More recently, we have shown that
IFN-γ is essential for maintaining immune tolerance toaquaporin-4 (AQP4) antigen and that its absence leads
to autoimmune inflammation and severe clinical disease
resembling AQP4-IgG+ neuromyelitis optica spectrum
disorders (NMOSD) [59].
Treg cells play a critical role in maintaining immune
tolerance in EAE and MS [60]. Indeed, FOXP3+-depleted
EAE mice exhibit worsening of disease associated witha significant increase in CNS-infiltrating CD4+ T effector cells [21]. In addition, several reports have shownthat there is a dysregulation in the Treg cell populationin both MS and EAE due to either a reduction in theirfrequency [61, 62], or in their functionality [18, 25, 63].
In this study, we found that although the therapeutic
activity of IFN-γ in EAE is dependent on the presence offunctional Treg cells, IFN-γ does not trigger direct differentiation of FOPX3+ Treg cells and; on the contrary,in vitro neutralization of IFN-γ induces an increase in
their cell number. Consistent with our findings, several
studies have shown that IFN-γ inhibits the in vitro andin vivo development of Treg cells and that the neutralization of IFN-γ markedly promotes the differentiationand suppressive function of iTregs [64–67]. Furthermore, some reports have included antibodies against
IFN-γ to improve in vitro differentiation of iTreg cells[65, 68]. In contrast, Wang et al., reported that IFN-γ
can directly induce in vitro FOXP3 expression in murine
TCR-stimulated CD4+CD25− T cells and that their transfer to EAE recipient mice reduced clinical symptoms.
Furthermore, IFN-γ induced, in a dose-dependent manner, increased expression of FOXP3 in CD4+CD25− Tcells from healthy individuals and the resulting Treg cells
inhibited autologous T cell proliferation [45]. Similarly,
IFN-γ promoted a significant increase in the conversion
of TCR-stimulated CD4+CD25− T cells to suppressive
CD4+CD25+FOXP3+ T cells in patients with GuillainBarré syndrome or myasthenia gravis [69, 70]. Anotherstudy showed that differentiation of iTreg cells starting from healthy human CD4+CD45RA+ T cells in the

Page 13 of 17

presence of IFN-γ exhibited higher expression of FOXP3
than iTreg cells differentiated only with TGF-β and both
TGF-β-induced iTreg cells and IFN-γ-boosted iTreg cells
had similar suppressive activity [71]. Therefore, the roleof IFN-γ in the conversion of CD4+CD25− T cells to Treg
cells is controversial. The difference between these results
might be explained by the microenvironment from which
CD4+ T cells were isolated. Wang et al. [45], generated
FOXP3+ Treg cells from CD4+CD25− T cells obtained
from the spleen of unprimed (without EAE) mice. In thisstudy, we used CD4+CD25− T cells obtained from LN andspleens of EAE mice. In addition, the conversion rate of
CD4+CD25− T cells into CD4+CD25+FOXP3+ T cells was
significantly higher in healthy individuals than in patientswith Guillain-Barré syndrome or myasthenia gravis [69,70]. Therefore, the response of CD4+CD25− T cells to
IFN-γ may differ between patients and healthy subjects
and between patients in different stages of disease. In thesame way, the inflammatory microenvironment mightalso affect the responsiveness of Treg cells to signals promoting suppressive activity, such as IFN-γ. Supporting
this notion, it has been reported that FOXP3+ Treg cells
readily accumulate in the CNS of EAE mice, but they areunable to inhibit MOG35 − 55 peptide-specific effector T
cells derived from the CNS at the peak of inflammation
due to their production of IL-6 and TNF [18, 72].
It has been reported that RRMS patients exhibit asignificantly higher percentage of Treg cells producing
IFN-γ than healthy individuals. Furthermore, the in vitro
suppressive activity of IFN-γ+FOXP3+ Treg cells fromRRMS patients was significantly lower than that fromhealthy controls. Besides, blocking IFN-γ significantly
increased the suppressive activity of IFN-γ+FOXP3+ Treg
cells from RRMS patients, but not to the same level ofsuppressive activity as IFN-γ+FOXP3+ Treg cells fromhealthy subjects [66]. Therefore, these results suggest thatthe dual role of IFN-γ on the differentiation and tolerogenic function of Treg cells might be determined by theconditions of the particular inflammatory microenvironment. Indeed, we and other investigators have found thatthe opposite roles of IFN-γ in EAE are dependent on thestage of disease: pathogenic during the induction phase
of EAE, but beneficial during the early and chronic effector phases [39, 42–44, 73].
Our results show that the therapeutic activity of IFN-γ
in EAE also depends on TGF-β and PD-1. PD-1 is a coinhibitory surface receptor that plays a pivotal role in regulating T cell activation. The interaction between PD-1
and its ligands PD-L1 and PD-L2 regulates peripheral
CD4+ and CD8+ T cell tolerance attenuating self-reactive
T cell response and promoting Treg cell differentiationand function [16]. In particular, expression of PD-L1 isregulated by IFN-γ and this ligand has a critical regulatory role in EAE [16, 74–78]. In turn, TGF-β has been

Arellano et al. Journal of Neuroinflammation(2024) 21:144

described as one of the most essential cytokines involvedin Treg cell development and function as well as a key
factor regulating EAE development [46, 79–81]. Interestingly, Treg cell conversion induced by splenic DCs
in a tumor microenvironment is dependent on TGF-βand PD-L1 signaling [82]. In addition, Casella et al. [49],have described that antigen-specific tolerance induction
by intravenous injection of high doses of autoantigen
halts EAE progression by inducing PD-L1 expression in
CNS monocyte-derived DCs via IFN-γ/IL-27-dependent mechanism. Based on this evidence, we hypothesized that IFN-γ might have an indirect role on FOXP3+
Treg cells by inducing expression of TGF-β and ligandsof PD-1 in APC. In line with our hypothesis, we foundthat IFN-γ induces an increased frequency of a subset of
splenic CD11b+ cells expressing PD-L1 and TGF-β-LAP+
in EAE. Furthermore, our ex vivo assays show that IFN-γ
acts directly on CD11b+ cells inducing a tolerogenic phenotype characterized by an early secretion of TGF-β1,
enhanced expression of PD-L1, and decreased expressionof MHC-II molecules, followed by an increased expression of TGF-β-LAP and PD-L1, and a reduced expressionof CD80. IL-27 and IL-10 are not induced by IFN-γ andthey would not be involved in the tolerogenic mechanism
mediated by IFN-γ. Consistently, in vivo neutralizationof IL-10 does not interfere with the protective effectsof IFN-γ on EAE. Notably, IFN-γ-treated CD11b+ cells
promote antigen-specific conversion of CD4+ T cells
into Treg cells through the secretion of TGF-β. Because
PD-L1 was not involved in this process, it is not ruled outthat the role of PD-1 in EAE is partially independent ofTreg induction. In this case, it is plausible that the roleof PD-1 is rather related with attenuation of self-reactive
T cell response [16]. Supporting our results showing thatthe beneficial activity of IFN-γ is dependent on TGFβ, we have recently found that primary myeloid cells/
microglia cultures obtained from spinal cord of IFN-γtreated EAE mice that were ex vivo re-stimulated withlow dose (1 ng/ml) IFN-γ and MOG35 − 55 peptide showedsignificantly enhanced tolerogenic activity, promoting
induction of CD4+ Treg cells associated with increased
TGF-β secretion [44]. Additionally, it has been reported
that IFN-γ signaling is required for tolerogenic functionof DCs mediated by TGF-β in EAE [83], and that IFN-γ
along with TNF-α produced by T cells in combination
with TGF-β and B7 molecules expressed by neurons are
critical in the generation of neuron-induced Treg cells
in the CNS of EAE mice [84]. Collectively, this evidence
supports the ability of IFN-γ to endow different cell types
with tolerogenic properties.
Remarkably, splenic CD11b+ myeloid cells from IFNγ-treated EAE mice showed a significant therapeuticpotential in that intravenous injection of these cells in
untreated EAE mice induced a rapid and significant

Page 14 of 17

amelioration of clinical symptoms. Disease attenuationinduced by adoptive transfer of splenic CD11b+ myeloid
cells from IFN-γ-treated EAE mice was associated withlower CNS infiltration of mononuclear cells and effector
CD3+CD4+ helper T cells. Although there was no significant difference in Treg cells at the time of evaluation (3days after injection), it does not rule out the possibility
that Treg cells are induced by splenic CD11b+ myeloid
cells earlier and/or transitorily. Further experiments will
be necessary to identify which type of APC mediates thetherapeutic effects of splenic CD11b+ myeloid cells. A
possible cellular target of IFN-γ might be DCs because
transfer of healthy splenic DC ex vivo stimulated with
IFN-γ significantly suppressed clinical symptoms of EAE
and prevented diabetes onset in NOD mice [49, 85, 86].
We found that that the induction of splenic CD11b+
myeloid cells expressing PD-L1 and LAP-TGF-β depends
on STAT-1, an essential transcriptional factor involvedin the maintenance of immunological self-tolerance andfunction of Treg cells in EAE [87, 88]. Interestingly, we
have recently reported that amelioration of EAE symptoms and induction of homeostatic microglia by IFN-γ isalso dependent on STAT-1. Therefore, our previous andcurrent results reveal a novel role of the IFN-γ/STAT-1
axis in the induction of tolerogenic microglia and splenic
CD11b+ myeloid cells involved in the suppression of EAE
development and shed light on the protective mechanismof IFN-γ showing that it induces tolerogenic activity in
EAE targeting both peripheral and CNS-resident APC.

Conclusions
Collectively, our results show that the protective mechanisms of IFN-γ in EAE depend on the presence of
FOXP3+ Treg cells. However, IFN-γ does not exert a
direct effect on Treg cells but rather induces a subset of
splenic CD11b+ myeloid cells with tolerogenic phenotype
and function able to induce conversion of naïve CD4+ T
cells into Treg cells signaling through STAT-1 and promoting TGF-β secretion. Furthermore, IFN-γ-induced
splenic CD11b+ myeloid cells exert therapeutic activity
in recipient EAE mice limiting CNS infiltration of mononuclear cells and helper T cells. Therefore, our findings
reveal a previously unknown protective mechanism ofIFN-γ in a neuroinflammatory context and contribute toclarifying its paradoxical role in EAE and MS.
Abbreviations
APC	antigen presenting cellsCD	cluster of differentiation
CNS	central nervous system
CTLA	Cytotoxic T-lymphocyte antigen
DNAse	deoxyribonuclease
DTR	diphtheria toxin receptor
DTx	diphtheria toxin
EAE	experimental autoimmune encephalomyelitis
FOXP3	Forkhead box p3
IFN	interferon

Arellano et al. Journal of Neuroinflammation(2024) 21:144

IFN-γR	interferon-γ receptorIL	interleukin
i.p.	intra peritoneal
LAG	Lymphocyte-activation gene
LAP	Latency associated peptide: LN, lymph node
MFI	mean fluorescence intensity
MHC	major histocompatibility complex
MNC	mononuclear cells
MOG	myelin oligodendrocyte glycoprotein
MS	Multiple SclerosisMT	
mycobacterium tuberculosis
PBS	phosphate-buffered saline
PD	Program-death
PD-L	Program-death Ligand
p.i.	post-immunization
PLP	proteolipid protein
PMA	phorbol myristate acetatePTx	pertussis toxin
RR	relapsing-remittings.c.	subcutaneousSC	spinal cord
STAT	Signal Transducers and Activators of Transcription
Th	T helper cells
TGF	Tumor growth factor
Treg	regulatory T
WT	wild type

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03126-3.
Supplementary Material 1
Acknowledgements
We thank the members of Naves and Miller labs for their helpful suggestions
and discussions.
Author contributions
R.N. and G.A. conceived the study, designed experiments, analyzed data, andwrote the manuscript. E.A., E.L., L.M., J.E.T., C.C., and F.V. performed experiments
and analyzed data. P.I.B., P.P.M., and S.D.M. provided resources, analyzed anddiscussed results, and revised the manuscript. All the authors have read andapproved the final manuscript.
Funding
This work was supported by the National Doctoral scholarship CONICYT-CHILE
21130452 and MECESUP UCH 1304 (GA). FONDECYT/ANID 1140049 (RN),1141211 (PB), 1191874 (RN), 1231672 (RN), and FONDECYT/ANID postdoc
3150133 (JT). MED.UCHILE-FACS Laboratory is supported by CONICYT-CHILE
through grants FONDEQUIP140032 (BD LSR Fortessa X-20, Special Order)
and AIC-08 (BD FACSAria III) and by the Institute of Biomedical Sciences(ICBM), School of Medicine, Universidad de Chile, Chile. Some of this work
was supported with grant from the National Institute on Drug Abuse (NIDA,
DP2DA051912) (PPM). Supported by gifts from the Johnnie Walkers MS
Foundation, the Amy and David Fulton Foundation, the Crammer Family
Foundation, the Thomas and Deige McLaughlin Foundation, and the Rottering
Family Foundation (SDM).
Data availability
The data supporting the findings of this work are available upon reasonable
request.

Declarations
Ethical approval
All animal experiments and procedures were conducted in accordance with
the Institutional Animal Care and Use guidelines (IACUC) of the Universidad
de Chile and Northwestern University.

Page 15 of 17

Competing interests
SDM is a co-founder, paid consultant, scientific advisory board member, andgrantee of COUR Pharmaceuticals. SDM is also a consultant for NextCure andTakeda Pharmaceuticals. The other authors have declared that no conflicts ofinterest exist.
Author details
1
Program of Immunology, Institute of Biomedical Sciences, Faculty of
Medicine, Universidad de Chile, Santiago, Chile2
Department of Microbiology-Immunology, Feinberg School of Medicine,Northwestern University, Chicago, IL, US
3
Center for Human Immunology, Feinberg School of Medicine,Northwestern University, Chicago, IL, US
4
Department of Clinical Immunology and Rheumatology, School of
Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
Received: 26 March 2024 / Accepted: 5 May 2024

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