Report

A Diet Mimicking Fasting Promotes Regeneration

and Reduces Autoimmunity and Multiple Sclerosis
Symptoms
Graphical Abstract Authors
In Young Choi, Laura Piccio,
Patra Childress, ..., Friedemann Paul,
Markus Bock, Valter D. Longo

Correspondence
[email protected]

In Brief
Choi et al. show that cycles of a fasting
mimicking diet (FMD) ameliorate disease
severity by suppressing autoimmunity
and stimulating remyelination via
oligodendrocyte regeneration in multiple
sclerosis (MS) mouse models. They also
show that a similar FMD is a safe, feasible,
and possibly a potentially effective
treatment for patients with relapsing-
remitting MS.

Highlights
d FMD reduces pro-inflammatory cytokines and increases
corticosterone levels

d FMD suppresses autoimmunity by inducing lymphocyte

apoptosis

d FMD promotes regeneration of oligodendrocyte in multiple

MS models

d FMD is a safe, feasible, and potentially effective treatment for

MS patients

Choi et al., 2016, Cell Reports 15, 111

June 7, 2016 2016 The Author(s)
http://dx.doi.org/10.1016/j.celrep.2016.05.009
 Please cite this article in press as: Choi et al., A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple Sclerosis
Symptoms, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.05.009

Cell Reports

Report

A Diet Mimicking Fasting Promotes

Regeneration and Reduces Autoimmunity
and Multiple Sclerosis Symptoms
In Young Choi,1,10 Laura Piccio,2,10 Patra Childress,3 Bryan Bollman,2 Arko Ghosh,4 Sebastian Brandhorst,1
Jorge Suarez,1 Andreas Michalsen,5 Anne H. Cross,2 Todd E. Morgan,1 Min Wei,1 Friedemann Paul,6,7 Markus Bock,6,7,11
and Valter D. Longo1,4,8,9,11,*
1Longevity Institute, School of Gerontology, and Department of Biological Sciences, University of Southern California, Los Angeles,

CA 90089, USA
2Department of Neurology and Neurosurgery and Hope Center for Neurological Disorders, Washington University School of Medicine,

St. Louis, MO 63110, USA

3Global Medicine Program, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
4Department of Neuroscience, Dana and David Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles,

CA 90089, USA
5Institute of Social Medicine, Epidemiology and Health Economics, Charite University Medicine Berlin, 10117 Berlin, Germany
6NeuroCure Clinical Research Center and Clinical and Experimental Multiple Sclerosis Research Center, Department of Neurology, Charite

University Medicine Berlin, 10117 Berlin, Germany

7Experimental and Clinical Research Center, a joint cooperation between the Charite Medical Faculty and the Max-Delbrueck Center for

Molecular Medicine, 10117 Berlin, Germany

8Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern

California, Los Angeles, CA 90089, USA

9IFOM, FIRC Institute of Molecular Oncology, 20139 Milan, Italy
10Co-first author
11Co-senior author

*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.celrep.2016.05.009

SUMMARY INTRODUCTION

Dietary interventions have not been effective in the Multiple sclerosis (MS) is an autoimmune disorder characterized
treatment of multiple sclerosis (MS). Here, we show by T cell-mediated demyelination and neurodegeneration in the
that periodic 3-day cycles of a fasting mimicking CNS (Friese and Fugger, 2005; Pender and Greer, 2007; Sospedra
diet (FMD) are effective in ameliorating demyelin- and Martin, 2005). In experimental autoimmune encephalomyelitis
ation and symptoms in a murine experimental auto- (EAE), an animal model for MS, activated myelin-specific TH1 and
TH17 cells cross the blood-brain barrier and migrate into the CNS,
immune encephalomyelitis (EAE) model. The FMD
where they are activated by local antigen-presenting cells (APCs)
reduced clinical severity in all mice and completely
and promote inflammation (Dhib-Jalbut, 2007; Fletcher et al.,
reversed symptoms in 20% of animals. These 2010; Goverman, 2009; Hemmer et al., 2002). This inflammatory
improvements were associated with increased process leads to oligodendrocyte death, demyelination, and
corticosterone levels and regulatory T (Treg) cell axonal damage, which eventually cause neurologic damage (Luc-
numbers and reduced levels of pro-inflammatory chinetti et al., 1999; Raine and Wu, 1993). Although oligodendro-
cytokines, TH1 and TH17 cells, and antigen-present- cyte precursor cells (OPCs) can migrate to the sites of MS lesions,
ing cells (APCs). Moreover, the FMD promoted they often fail to differentiate into functional oligodendrocytes
oligodendrocyte precursor cell regeneration and (Chang et al., 2002; Wolswijk, 1998). Several MS treatment drugs
remyelination in axons in both EAE and cuprizone have been effective in reducing immune responses, but their
MS models, supporting its effects on both sup- impact on long-term disease progression, accrual of irreversible
neurological disability, and immune system function remains
pression of autoimmunity and remyelination. We
largely unclear, underlining the need for novel therapeutic strate-
also report preliminary data suggesting that an
gies (Wingerchuk and Carter, 2014). Therefore, effective treat-
FMD or a chronic ketogenic diet are safe, feasible, ments for MS may require not only the mitigation of autoimmunity
and potentially effective in the treatment of re- but also the stimulation of oligodendrocyte regeneration and resto-
lapsing-remitting multiple sclerosis (RRMS) patients ration of a functional myelin sheath. Periodic cycles of prolonged
(NCT01538355). fasting (PF) or of a fasting mimicking diet (FMD) lasting 2 or more

Cell Reports 15, 111, June 7, 2016 2016 The Author(s) 1

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 Please cite this article in press as: Choi et al., A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple Sclerosis
Symptoms, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.05.009

Figure 1. FMD Cycles Decrease Disease Severity of the MOG3555-Induced EAE Model
(A) Diagram displaying the time course of the immunization and the diet interventions.
(B) The EAE severity scores of the control diet (EAE CTRL; n = 23), ketogenic diet (EAE KD; n = 13), semi-therapeutic FMD cycles (EAE FMD (S); n = 7), or
therapeutic FMD cycles (FMD(T); n = 23).
(C) Incidence rate of EAE CTRL and EAE FMD (S) (n = 723).
(D) EAE severity score in mice for which FMD(T) completely reversed EAE severity, with no observable disease (score = 0; 5 out of 23 mice).
(E) EAE severity score of the best-performing control mice (n = 12) and FMD(T) mice (n = 12).
(F) EAE severity score of the mice treated with FMD after chronic EAE development (EAE CTRL-FMD; n = 6).
(GM) Spinal cord sections of EAE CTRL and EAE FMD (T) mice with quantification of H&E staining (G), solochrome cyanine staining (H), and MBP (myelin basic
protein)/SMI32 (I) double staining of spinal cord sections isolated at day 14.
Data are presented as mean SEM; *p < 0.05, **p < 0.01, and ***p < 0.001, Students t test, one-way or two-way ANOVA, and Bonferroni post test. Scale bar
represents 200 mm.

days can increase protection of multiple systems against a variety Here, we report on the effects of low-calorie and low-protein
of chemotherapy drugs in mice and possibly humans (Fontana FMD cycles as a treatment in MS mouse models, and we inves-
et al., 2010; Guevera-Aguirre et al., 2011; Lee et al., 2010; Longo tigate the mechanisms involved. Furthermore, we report prelim-
and Mattson, 2014). Moreover, PF or an FMD reverses the immu- inary results on the safety and feasibility of a FMD and a KD in
nosuppression or immunosenescence of either chemotherapy patients with relapsing-remitting multiple sclerosis (RRMS).
or aging through hematopoietic stem cell-based regeneration
(Brandhorst et al., 2015; Cheng et al., 2014). Chronic caloric re- RESULTS
striction, a ketogenic diet (KD), and intermittent fasting have
been shown to help prevent EAE by reducing inflammation and FMD Cycles Reduce Disease Severity in the MOG3555-
enhancing neuroprotection when administered prior to disease Induced EAE Model
induction or signs (Esquifino et al., 2007; Kafami et al., 2010; Kim We examined the effects of 3 cycles of a very-low-calorie and
do et al., 2012; Piccio et al., 2008), but dietary interventions have low-protein FMD lasting 3 days every 7 days or a KD continued
not been reported to be effective as therapies for EAE or MS or for 30 days in EAE-induced by active immunization with myelin
to promote myelin regeneration. oligodendrocyte glycoprotein 3555 (MOG3555) (Figure 1A).

2 Cell Reports 15, 111, June 7, 2016

 Please cite this article in press as: Choi et al., A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple Sclerosis
Symptoms, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.05.009

Groups of mice were treated semi-therapeutically (EAE FMD (S), CTRL, with the exception of granulocytes, indicating that the
where FMD treatment started after 10% of the immunized pop- FMD cycles cause both WBC death and regeneration (Figure 2A).
ulation showed signs of EAE) or therapeutically (EAE FMD (T), Next, we measured the inflammatory markers associated with
where FMD treatment started after all of the immunized popula- EAE pathophysiology. Day 3 and day 14 spinal cord sections
tion showed signs of EAE). FMD and KD treatment decreased of the EAE CTRL mice were extensively populated with
disease severity compared to that in the control group (Fig- CD11b+ cells (Figure 2B). However, at day 14, the EAE FMD
ure 1B). However, the FMD reduced the mean severity score mice displayed a 75% reduction (p < 0.05) in spinal cord-associ-
to 1, whereas a KD reduced the severity score to 2 at the later ated CD11b+ cells compared to mice on the control diet (11.7%
stages (Figure 1B). In the EAE FMD (S) group, FMD treatment not versus 2.8%; Figure 2B). Since myelin-specific effector T cells
only delayed the onset of disease but also lowered the incidence migrate into the CNS and initiate demyelination, we investigated
rate (100% versus 45.6%; Figure 1C). In the EAE FMD (T) group, the accumulation of CD4+ or CD8+ T cells in the spinal cord.
FMD cycles completely reversed the severity score to 0 in 21.7% A large number of CD4+ T cells were detected in the white matter
of the cohort (no observable signs; Figure 1D) and reduced the of spinal cord sections from the control diet cohort (Figure 2C). In
severity score to <0.5 in >50% of mice (12 out of 23 mice; Fig- contrast, the FMD-treated cohort displayed a >4-fold reduction
ure 1E). To address whether the FMD cycles also have beneficial (p < 0.01) in CD4+ T cells at day 3 (8.6% versus 1.5%; Figure 2C)
effects in chronic EAE models that have established disease, we compared to the control diet cohort, which remained lower even
initiated FMD treatment 2 weeks after the initial signs of EAE at day 14. The FMD group also had reduced CD8+ T cells (day 3:
were observed (EAE CTRL-FMD). Prior to treatment, the control 1.3% versus 0.4%; p < 0.01; Figure 2D) compared to the control
diet EAE group (EAE CTRL) and EAE CTRL-FMD cohorts had diet group. To investigate whether the FMD affects APCs, we
similar severity scores (3.19 0.52 versus 3.30 0.27; day 24). isolated splenocytes from EAE CTRL and EAE FMD mice at
After three FMD cycles, we observed a significant reduction in day 3, stained them for CD11c and F4/80, and characterized
severity score in the EAE CTRL-FMD cohort compared to the them by flow cytometry. We observed a significant decrease
EAE CTRL cohort (3.3 0.57 versus 2.1 0.89; day 42; p < (p < 0.05) in CD11c+ dendritic cells in the EAE FMD cohort
0.05; Figure 1F). As infiltration of immune cells and demyelination compared to the EAE CTRL cohort (3.08% 0.70% versus
are histopathological hallmarks of EAE and MS, spinal cord sec- 1.46% 0.31%), but we did not observe any changes in the
tions from control and FMD(T) mice were stained with H&E to number of F4/80+ macrophage cells in the control or FMD-
visualize infiltrating immune cells (Figure 1G) or solochrome treated groups (Figures 2E and S2B). To determine the effects
cyanine to visualize myelin (Figure 1H). To assess demyelination of the FMD treatment on T cell infiltration in the spinal cord, we
and axonal damage, immunohistochemistry was performed us- measured T cell activation levels. The number of CD4+ T cells
ing antibodies against myelin basic protein (MBP) or dephos- and CD8+ T cells in EAE CTRL and EAE FMD mice was similar
phorylated neurofilaments (SMI-32; Figure 1I). At day 3, levels (Figures S2C and S2D), but the ratio of splenic naive (CD44low)
of infiltrating immune cells and demyelination were similar in to activated (CD44high) CD4+ T cells was increased (p < 0.05) in
the EAE CTRL and EAE FMD groups (Figures 1J and S1H). At the FMD group compared to the control group (1.95 versus
day 14, sections of EAE CTRL mice displayed severe immune 3.67; Figure 2F). No difference in CD8+ T cells was observed (Fig-
cell infiltration corresponding to demyelinated lesions, reduced ure S2E). Moreover, the total number of effector (CD44high and
MBP expression, and increased SMI-32 expression (Figures CD62Llow) T cells was reduced in the FMD compared to the con-
1J1M). By contrast, sections of EAE FMD mice at day 14 dis- trol group, but the ratio of effector (CD44high and CD62Llow) to
played significantly reduced immune cell infiltration and demye- memory T (CD44high and CD62Lhigh) cells did not change (Fig-
lination (Figures 1J1M). Although MBP staining showed no sig- ures S2FS2H). These results indicate that FMD cycles reduce
nificant difference between EAE CTRL and EAE FMD mice at day the number of dendritic cells and increase the relative number
14 (Figure 1L), neurofilament dephosphorylation in EAE FMD of naive T cells, which may explain the reduced autoimmunity
mice was reduced compared to EAE CTRL mice (Figure 1M). caused by the FMD.
Overall, these results suggest that FMD cycles reduce EAE dis-
ease severity in part by reducing inflammation and preventing FMD Cycles Induce Autoreactive Lymphocyte Apoptosis
demyelination and axonal damage. and Increase the Number of Naive Cells
To determine whether FMD cycles also reduce the number of
FMD Cycles Reduce Infiltration of Immune Cells in the MOG-specific antigen-reactive cells, we used a major histocom-
Spinal Cord patibility complex (MHC) tetramer (MOG3555/IAb) to identify
To investigate the capacity of FMD cycles to reduce potential antigen-reactive cells after an FMD cycle in vivo. The number
autoimmune T cells, we measured circulating white blood cells of CD4+ MOG3555 /IAb+ cells was reduced in the EAE FMD
(WBCs), lymphocytes, monocytes, and granulocytes in naive, cohort compared to the EAE CTRL cohort (5.75% 0.51%
EAE CTRL, EAE FMD, and EAE FMD:RF (measured 4 days after versus 3.83% 0.66% of lymphocytes; * p < 0.05; Figure 2G).
returning to a standard ad lib diet) mice after three cycles of the To determine whether the reduced active T cell number is
FMD regimen (Figure 2A). The FMD resulted in a temporary due to an increase in the number of regulatory T (Treg) cells, we
40%50% reduction in total WBCs, lymphocytes, monocytes, isolated lymphocytes from draining lymph nodes and spleens
and granulocytes. Upon returning to the standard ad lib diet of EAE CTRL or EAE FMD mice and analyzed them for CD4+
(EAE FMD:RF), all complete blood counts (CBCs) returned to CD25+ FoxP3+ Treg cells. The FMD cohort showed a 2-fold in-
either naive or lower levels than those observed in the EAE crease (p < 0.01) in the number of CD25+ FoxP3+-expressing

Cell Reports 15, 111, June 7, 2016 3

 Please cite this article in press as: Choi et al., A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple Sclerosis
Symptoms, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.05.009

(legend on next page)

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 Please cite this article in press as: Choi et al., A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple Sclerosis
Symptoms, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.05.009

Treg cells (13.6% 4.2% versus 25.1% 4.2%; Figure 2H). kine production (IL-17, IFN-g, and tumor necrosis factor a
Moreover, the FMD cohort showed a 27.8% reduction (p < [TNF-a]), we analyzed serum from naive, EAE CTRL, and EAE
0.05) in the number of interferon g (IFN-g)- expressing TH1 cells FMD mice (Figures 2K2M). We observed significant reductions
(2,974.4 708.0 versus 2,148.1 1,396.1; Figure 2I) and a in serum TNF-a (113.3 7.9 versus 79.3 10.5 pg/ml; p < 0.001;
46.5% reduction (p < 0.05) in the number of interleukin-17 Figure 2M), IFN-g (558.43 124.5 versus 296.0 83.4 pg/ml;
(IL-17)-expressing TH17 cells (2,535.9 722.0 versus 1,357.1 p < 0.001; Figure 2N), and IL-17 (36.8 9.67 versus 20.75
256.2; Figure 2J), both of which are known to be central media- 4.2 pg/ml; p < 0.01; Figure 2O). To identify a potential mediator
tors of EAE. Interestingly, upon re-feeding of the control diet, the for the effects of FMD cycles on the suppression of autoimmune
EAE FMD treatment group (EAE FMD:RF) showed a 72.9% responses, we measured serum corticosterone levels. Cortico-
reduction (p < 0.05) in the number of IFN-g-expressing TH1 cells sterone is a glucocorticoid hormone with broad anti-inflamma-
(2,974.4 708.0 versus 805.8 251.5; Figure 2I) and a 82.9% tory and immunosuppressive effects affecting leukocyte dis-
reduction (p < 0.05) in the number of IL-17-expressing TH17 cells tribution, trafficking, and death (Ashwell et al., 2000; Herold
(2,535.9 722.0 versus 432.4 117.4; Figure 2J), suggesting et al., 2006; Planey and Litwack, 2000; Vegiopoulos and Herzig,
that the FMD can prevent autoimmunity in part by reducing the 2007). Serum corticosterone levels were elevated in association
levels of pro-inflammatory T cells implicated in EAE. with the first signs of EAE (EAE day 1, before treatment) (data not
In order to assess how FMD cycles may reduce the number of shown). FMD treatment caused a further increase in corticoste-
T cells, we measured apoptosis in MOG-specific T cells (CD3+ rone levels at day 3 compared to those of controls (245.9 38.8
MOG3555/IAb) in vivo. We observed a significant increase (p < versus 375.0 94.1 ng/ml; p < 0.01), which returned to EAE basal
0.05) in apoptotic CD3+ MOG3555/IAb levels in the EAE FMD levels by day 14 in both groups (Figure 2P). These results indi-
cohort compared to the EAE CTRL cohort (28.3% 4.94% cate that FMD cycles reduce the number of TH1 and TH17
versus 39.1% 4.79%; Figure 2K), which was consistent with effector cells and the production of pro-inflammatory cytokines.
the major reduction in the number of WBCs and lymphocytes These effects of the FMD may be regulated in part by the tempo-
observed in the FMD group (Figure 2A). To investigate whether rary elevation of corticosterone levels, dampening of T cell acti-
these apoptotic cells are replaced by newly generated cells, vation, and reduced APC and T cell infiltration in the spinal cord.
we treated the mice with bromodeoxyuridine (BrdU) during the
re-feeding period (four injections within 48 hr, at 1 mg of BrdU FMD Reverses EAE Symptoms by Reducing the Level
per injection). Splenocytes were isolated 4 days after the re- and Reactivity of Established Autoimmune Cells
feeding of the regular diet and stained for BrdU (Figure S2I). To determine how the FMD affects the initiation of EAE, spleno-
We observed no difference in levels of total BrdU+ lymphocytes cytes were isolated from EAE CTRL and EAE FMD mice, re-acti-
(8.11% 1.99% versus 12.02% 2.72%; Figure S2J), but we vated with MOG3555 peptide and IL-23 ex vivo, and transferred
observed a significantly reduced proliferation of TH1 (BrdU+ into naive recipient mice to induce EAE. The mice were then sub-
CD4+IFNg+) (5.74% 1.07% versus 3.65% 0.63%; *p < jected to either a control diet or FMD cycles (Figure 3A). The
0.05; Figure 2L) and no difference in proliferation of TH17 supernatant from ex vivo splenocyte cultures derived from
(BrdU+CD4+IL17+) (4.71% 1.53% versus 5.01% 1.66%; Fig- EAE FMD mice showed no difference in TNF-a levels (110.8
ure S2K). Taken together, these data indicate that FMD cycles 14.9 pg/ml versus 97.1 8.4 pg/ml; Figure 3B) but a major reduc-
may promote apoptosis of autoreactive T cells, leading to an tion (p < 0.01) in the levels of IFN-g (342.0 29.8 pg/ml versus 46.6
increase in the proportion of naive T cells and regulatory 16.6 pg/ml Figure 3C) and IL-17 (850.5 442.0 pg/ml versus
T cells. In addition, FMD cycles may interfere with proliferation 257.4 36.4 pg/ml; Figure 3D). Interestingly, upon in vitro reactiva-
and differentiation of TH1 cells, but not TH17 cells. To investigate tion, both EAE CTRL and EAE FMD had similar levels of TH1 and
whether the FMDs effects on CNS infiltrating immune cells are TH17 differentiated cells (Figures 3E and 3F). To determine
associated with suppression of TH1- and TH17-dependent cyto- whether the immune cells from EAE CTRL and EAE FMD mice

Figure 2. FMD Cycles Decrease the Number of Infiltrating T Cells in the Spinal Cord
(A) Total white blood cell (WBC), lymphocyte, monocyte, and granulocyte counts of naive, EAE-CTRL, EAE-FMD, and EAE-FMD:RF (after 3 days of re-feeding)
mice after three cycles of the FMD and a matched time point for EAE-CTRL mice.
(BD) Spinal cord sections (day 14) and quantification at days 3 and 14 after the first sign of EAE for CD11b+ (B), CD4+ (C), and CD8+ (D) (at least six sections per
mouse).
(E) CD11c+ isolated from EAE CTRL or EAE FMD mice on day 3, and quantification of cells from the total isolated splenocyte.
(F) CD4+ gated for CD44low or CD44high cells isolated from EAE CTRL or EAE FMD mice, and quantification of percent splenocytes in CD4+ CD44low (inactive) or
CD4+ CD44high (active) cells.
(G) CD3+ lymphocytes gated for CD4 and MOG3555/IAb from EAE CTRL or EAE FMD mice, and quantification of MOG-specific CD4+ cells.
(H) CD4+ CD25+ FoxP3+ isolated from EAE CTRL or EAE FMD mice, and quantification of CD25+ FoxP3+ in CD4+ cells.
(I and J) Intracellular staining for either IFNg (I) or IL17(J) after gated for CD4+ of the naive, EAE CTRL, EAE FMD, EAE FMD:RF and quantification of cell counts.
(K) Quantification of Annexin V+ apoptotic CD3+ MOG35-55/IAb cells.
(L) Quantification of CD4+IFNg+ of BrdU+ lymphocytes.
(MO) Serum TNF-a (M), IFN-g (N), and IL-17 levels (O) (pg/ml) in naive, EAE CTRL, and EAE FMD mice on day 3 after the first sign of EAE.
(P) Serum corticosterone levels (ng/ml) before immunization, at the time of symptom occurrence, or 3 or 14 days after the initial symptom appeared in the control
or FMD group.
n = 48 per group; mean SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, Students t test, one-way ANOVA, and Bonferroni post test. Scale bar represents 200 mm.

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 Please cite this article in press as: Choi et al., A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple Sclerosis
Symptoms, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.05.009

Figure 3. Antigen-Activated Splenocytes

from EAE-CTRL and the EAE-FMD Mice
Had Similar Encephalitogenic Effects
(A) Diagram for the adoptive transfer EAE model.
(BD) Quantification of TNF-a (B), IFN-g (C), and
IL-17 (D) (pg/ml) in the supernatant from ex vivo
cultures of splenocytes from naive, EAE CTRL,
and EAE FMD mice either with or without MOG3555
and IL-23 re-activation.
(E and F) Quantification of TH1 or TH17 (repre-
sented by percentage of CD4+) from lymphocyte
culture of EAE CTRL and EAE FMD mice with or
without MOG3555 and IL-23 re-activation.
(G) Incidence rate of adoptive transfer EAE
groups.
(H) EAE severity score of adoptive transfer EAE
groups.
n = 56 per group; mean SEM. *p < 0.05, **p <
0.01, and ***p < 0.001, Students t test, one-way
ANOVA, and Bonferroni post test.

post-transfer; Figure 3G) and a major

reduction in EAE severity scores
compared to control mice (2.38 0.48
versus 0.75 0.87; Figure 3H). Taken
together, these results suggest that T cell
priming in response to myelin antigen
occurred normally in the EAE CTRL and
EAE FMD groups, but the FMD can reduce
the level of the existing autoimmunity.

FMD Cycles Stimulate

Remyelination by Promoting
Oligodendrocyte Regeneration
To investigate whether the reduced demy-
elination in FMD mice may also be related
to enhanced oligodendrocyte regenera-
tion, we first carried out a quantitative im-
age analysis of NG2+ (an oligodendrocyte
progenitor cell [OPC] marker) and GST-
p+ (a mature oligodendrocyte marker) in
spinal cord sections from control or FMD
mice (Figure 4A). We observed no differ-
ence in the number of NG2+ OPCs in sec-
tions taken from EAE CTRL and EAE FMD
mice (Figure S3A). However, at day 14, the
number of GST-p+ oligodendrocytes was
have similar encephalitogenic effects, we transferred splenocytes reduced in the EAE CTRL group, but not in the EAE FMD group
from either donor group (EAE CTRL or EAE FMD) into naive recip- (886.7 41.6 versus 1,273 200.3; cells per spinal cord section
ients (A, EAE CTRL donor to control diet recipient; and C, EAE FMD area; p < 0.01; Figure 4B). To assess whether the normal levels
donor to control diet recipient). This resulted in a similar disease of mature oligodendrocytes in the EAE FMD group were due to
incidence rate (Figure 3G) and an equally severe EAE disease enhanced regeneration and/or differentiation, EAE CTRL or EAE
severity by day 20 (2.38 0.48 versus 2.70 0.75; Figure 3H), indi- FMD mice were injected with BrdU at the time of re-feeding
cating that the FMD did not affect the development and function of (day 10). We observed a major increase (p < 0.01) in the percent-
reactive immune cells in vivo or ex vivo. However, when FMD treat- age of cells that are double positive for BrdU+ and GST-p+ in the
ment was initiated after transfer of control donor splenocytes (B, EAE FMD group compared to the EAE CTRL group (42.9%
EAE CTRL donor to naive mice with FMD treatment), recipient 11.2% versus 83.0% 13.2%; p < 0.01), suggesting that the
mice displayed a delayed disease onset (day 12 versus day 16 FMD promotes oligodendrocyte differentiation from precursor

6 Cell Reports 15, 111, June 7, 2016

 Please cite this article in press as: Choi et al., A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple Sclerosis
Symptoms, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.05.009

Figure 4. FMD, which Protects the Mouse Spinal Cord from Loss of Oligodendrocytes and Enhances Remyelination, Is Safe and Potentially
Effective in the Treatment of MS Patients
(AC) Spinal cord sections isolated at day 14 and quantification for GST-p (mature oligodendrocyte) and BrdU (A), TUNEL and NG2 (oligodendrocyte precursor
cells) (B), and TUNEL and GST-p (C) in naive, EAE-CTRL, or EAE-FMD mice.
(FH) Sections from the corpus callosum region and quantification of cuprizone treated brains, stained with Luxol Fast Blue of the naive control, end of 5 weeks of
cuprizone diet (week 0), cuprizone (5 weeks) plus regular chow (2 weeks), and cuprizone (5 weeks) plus FMD cycle (2 weeks).
(I and J) Section from the corpus callosum region and its quantification of the cuprizone treated brains stained with GST-p+ of cuprizone (5 weeks) plus regular
chow (2 weeks), and cuprizone (5 weeks) plus FMD (2 weeks). Quantification is normalized to percent naive GST-p+ level.
(KN) Change in quality of life at 3 months in terms of overall quality of life (K), change in health (L), physical health composite (M), and mental health composite (N).
The dotted line represents a threshold that is thought to be clinically important (R5 points). Data represent mean standard error of the difference (SED);
*p < 0.05, Mann-Whitney U test. An increase of R5 points is considered clinically important.
At least 12 sections per mouse were used for quantification; n = 4; mean SEM, *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA and Bonferroni post test.

Cell Reports 15, 111, June 7, 2016 7

 Please cite this article in press as: Choi et al., A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple Sclerosis
Symptoms, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.05.009

cells (Figure 4C). To assess the effects of the FMD on either OPCs was lower urinary tract infection. No indication of an increase in
or mature oligodendrocytes, sections were stained with TUNEL, liver enzymes exceeding the normal range was observed in any
an apoptotic marker, and GST-p+ or NG2+ (Figure 4D). We of the three treatment groups. Also, the interventions were well
observed a significant increase in the number of TUNEL+ NG2+ tolerated, as evidenced by high compliance rates (CD, 60%;
(11.2 12.2 versus 1.9 1.4 cells/section) and TUNEL+ GST-p+ KD, 90%; and FMD, 100%). During the 6-month study period,
(18.8 15.2 versus 2.9 5.3 cells/section) cells in the control group we observed a total of eight relapses: four in the CD group, one
compared to the FMD group (p < 0.05; Figures 4E and 4F). Taken in the KD group, and three in the FMD group. In addition to
together, these results indicate that the FMD not only stimulates increased b-hydroxybutyrate levels in plasma, we observed a
regeneration and differentiation of oligodendrocytes but also pro- slight reduction in lymphocytes and WBC counts and detected
tects OPCs and mature oligodendrocytes from apoptosis. a mild reduction in expanded disability status scale (EDSS) scores
To investigate whether the FMD-dependent stimulation of in the FMD and KD groups (measured at baseline, month 3, and
oligodendrocyte differentiation and remyelination can occur in- month 6; Tables S1 and S6). Thus, there was an inverse associa-
dependent of the observed effects on T cell number and activity, tion between EDSS and HRQOL scores (Table S7). In MS patients
we used the cuprizone-induced demyelinating mouse model the FMD treatment lead to an over 20% drop in the total lympho-
(Ransohoff, 2012; Torkildsen et al., 2008). Addition of 0.2% cyte count (baseline versus day 8; Table S4) in 72% of the patients
(w/w) cuprizone to the regular mouse diet for 56 weeks results (13 of 18 FMD-treated patients). WBC counts returned to the
in demyelination in the corpus callosum followed by sponta- baseline levels after these patients were switched to the Mediter-
neous remyelination upon re-feeding with regular chow. After ranean diet (month 3). Based on the mouse studies, these results
5 weeks of cuprizone treatment, mice were switched to either raise the possibility that the FMD alleviated symptoms in MS
the control diet or FMD cycles for 5 weeks, and some were patients by reducing the number of autoimmune lymphocytes.
euthanized weekly to assess the degree of myelination by Luxol Overall, our study indicates that the administration of FMD and
fast staining and GST-p+ (Figures 4G and 4I). As expected, after KD is safe, feasible, and potentially effective, but further studies,
5 weeks of the cuprizone diet, a significant reduction in myelin including analyses such as magnetic resonance imaging (MRI),
staining was observed in the corpus callosum compared to the blinded clinical assessments, and immune assays, are required
naive controls (Figures 4H and 4J). After two cycles, the FMD- to determine efficacy.
treated group displayed increased myelin staining and an
increased number of GST-p+ oligodendrocytes compared to DISCUSSION
the control diet group (Figures 4H and 4J). However, at later
time points, we did not observe differences in spontaneous An FMD administered every week was effective in ameliorating
re-myelination between the control diet and FMD cohorts, as it EAE symptoms in all mice and completely reversed disease pro-
is well established that cuprizone-dependent myelin damage gression in a portion of animals after the onset of EAE signs. By
can be fully reversed after removal of the toxin (Figures S3C contrast, the KD had more modest effects and did not reverse
and S3D). These results indicate that the FMD promotes OPC- EAE progression in mice. FMD cycles appear to be effective in
dependent regeneration and accelerates OPC differentiation the treatment of EAE in mice by (1) promoting oligodendrocyte
into oligodendrocytes while enhancing remyelination indepen- precursor-dependent regeneration and (2) reducing the levels
dently of its modulation of the inflammatory response. of microglia/monocytes and T cells contributing to autoimmunity
and encephalomyelitis. Our results support an FMD-mediated
A Randomized Pilot Trial to Test the Effects of a FMD or anti-inflammatory effect possibly involving the upregulation of
KD in Relapsing-Remitting MS Patients: Evidence for AMPK or the downregulation of mTORC1, which sense nutrient
Safety and Feasibility availability and dictate cell fate (Laplante and Sabatini, 2012). It
A randomized, parallel-group, three-arm pilot trial (NCT01538355) was shown that mTORC1 couples immune signals and metabolic
was conducted to assess the safety and feasibility of FMD or KD programming to establish Treg cell function (Zeng et al., 2013). In
treatment on health-related quality of life (HRQOL) in RRMS pa- fact, treatment with the mTORC1 inhibitor rapamycin or the
tients. 60 patients were randomly assigned to a control diet (CD; AMPK activator metformin attenuates EAE symptoms by modu-
n = 20), KD for 6 months (n = 20), or a single cycle of a modified lating effector T cells and Treg cells and restricting the infiltration of
human FMD for 7 days (n = 20) followed by a Mediterranean diet mononuclear cells into the CNS (Esposito et al., 2010; Nath et al.,
for 6 months (Figure S4). Baseline characteristics were balanced 2009). Therefore, FMD treatment could interfere with T cell prolif-
among the three groups (Tables S1 and S2). The FMD and KD co- eration and differentiation and with recruitment of other immune
horts displayed clinically meaningful improvements in the HRQOL cells, resulting in a decreased recruitment at lesion sites (Fig-
summary scales at 3 months, which included the overall quality of ure 5). Some of these effects of the FMD may be triggered by
life (Figure 4K) change in health (Figure 4L), a physical health com- endogenous glucocorticoid production. Glucocorticoids are
posite (Figure 4M), and a mental health composite (Figure 4N). used to treat MS relapses, but they are generally administered
Also, similar changes were observed in the total HRQOL scales in short bursts, since they can cause AEs such as osteoporosis
at different time points (Figure S5). Adverse events (AEs) and and metabolic syndrome (Brusaferri and Candelise, 2000; Ce
serious adverse events (SAEs) were reported for 92% (8%) of et al., 2006; Roth et al., 2010; Uttner et al., 2005). The FMD may
CD cohort individuals, 78% (16%) of FMD cohort individuals, avoid these adverse effects by promoting additional and coordi-
and 78% (11%) of KD cohort individuals (Table S5). The most nated endogenous responses. Importantly, FMD cycles also acti-
common AE was airway infection, and the most frequent SAE vated OPCs, resulting in myelin regeneration, as demonstrated

8 Cell Reports 15, 111, June 7, 2016

 Please cite this article in press as: Choi et al., A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple Sclerosis
Symptoms, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.05.009

Figure 5. A Simplified Model of FMD-Mediated Effects on Immune Suppression, Oligodendrocyte Regeneration, and Differentiation in MS
FMD treatment promotes endogenous glucocorticoid production, increases Treg cell numbers, blocks T cell activation, and promotes T cell death. In the lesion
area, FMD treatment reduces autoimmune T cell and microglia infiltration and promotes oligodendrocyte-precursor-dependent regeneration and differentiation
of myelinating oligodendrocytes, which engage with demyelinated axons to promote the formation of myelin sheaths.

by accelerated remyelination rate in the cuprizone model (Fig- mixed 1:1 with supplemented complete Freunds adjuvant followed by
ure 5). Notably, because it is the alternation of FMD cycles and 200 ng pertussis toxin (PTX; List Biological Laboratories) intraperitoneally
(i.p.) at days 0 and 2. For adoptive transfer, spleens from active immunized
re-feeding and not the FMD alone that promotes the regeneration
mice were isolated and red blood cells (RBCs) were lysed. Spleen cells were
and replacement of autoimmune cells with naive cells, the use of cultured in the presence of MOG3555 (20 mg/ml) with rmIL-23 (20 ng/ml) for
chronic restriction or even a chronic KD may not be effective, or 48 hr. Cells were collected and re-suspended in PBS, and 15 million cells
as effective, in the treatment of EAE and MS. were injected intravenously. See Supplemental Experimental Procedures
Finally, we report that the administration of the FMD and for a detailed description of disease severity scoring. All experiments
KD in MS patients was safe and well tolerated and resulted were performed in accordance with approved Institutional Animal Care
and Use Committee (IACUC) protocols of the University of Southern
in high compliance. We observed positive effects of FMD cy-
California.
cles or KD treatment in RRMS based on changes in self-re-
ported HRQOL and a mild improvement in EDSS (Table S6). Mouse Fasting Mimicking Diet
However, the lack of a proper Mediterranean diet control Mice were fed ad lib with irradiated TD.7912 rodent chow (Harlan Teklad),
makes it difficult to establish whether FMD cycles alone containing 15.69 kJ/g digestible energy (animal-based protein 3.92 kJ/g,
are sufficient to produce these effects. In addition, MRI ana- carbohydrate 9.1 kJ/g, and fat 2.67 kJ/g). The experimental FMD is based
lyses and adequately blinded clinical assessments (EDSS on a nutritional screen that identified ingredients that allow high nourish-
ment during periods of low calorie consumption. The FMD diet consists
and multiple sclerosis functional composite [MSFC]), as well
of two different components, day 1 diet and day 23 diet, that were fed
as immune function analyses would greatly enhance the in this order, respectively. See Supplemental Experimental Procedures
strength of the clinical findings. Because, unlike for the mouse for a detailed explanation of the FMD. Mice consumed all the supplied
experiments, the FMD was only administered to patients only food on each day of the FMD regimen and showed no signs of food aver-
once, it will be important to test the effects of multiple FMD sion. After the end of FMD, we supplied TD.7912 chow ad lib for 4 days
cycles on MS patients in larger, randomized, and controlled before starting another FMD cycle. Prior to supplying the FMD, animals
were transferred into fresh cages to avoid feeding on residual chow and
trials.
coprophagy.

EXPERIMENTAL PROCEDURES Clinical Trial Design

This study was a three-armed, parallel-group, single-center, controlled,
EAE Model and randomized clinical pilot trial to assess the effects of dietary interven-
C57Bl/6 (10-week-old female) mice were purchased from The Jackson Lab- tions on HRQOL in RRMS patients. The permuted-block randomization
oratory and immunized subcutaneously with 200 mg MOG3555 (GenScript) was generated online at http://randomization.com. An investigator blind

Cell Reports 15, 111, June 7, 2016 9

 Please cite this article in press as: Choi et al., A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple Sclerosis
Symptoms, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.05.009

to the randomization plan determined the patients randomization number Brandhorst, S., Choi, I.Y., Wei, M., Cheng, C.W., Sedrakyan, S., Navarrete, G.,
before they underwent the randomization step. This study is registered at Dubeau, L., Yap, L.P., Park, R., Vinciguerra, M., et al. (2015). A periodic diet
http://www/clicaltrials.gov as NCT01538355. The study was approved by that mimics fasting promotes multi-system regeneration, enhanced cognitive
the local ethics committee. All participants gave informed written consent performance, and healthspan. Cell Metab. 22, 8699.
according to the 1964 Declaration of Helsinki. See Supplemental Experi- Brusaferri, F., and Candelise, L. (2000). Steroids for multiple sclerosis and
mental Procedures for detailed descriptions of the clinical trial and diet optic neuritis: a meta-analysis of randomized controlled clinical trials.
compositions. J. Neurol. 247, 435442.
Ce, P., Gedizlioglu, M., Gelal, F., Coban, P., and Ozbek, G. (2006). Avascular
SUPPLEMENTAL INFORMATION necrosis of the bones: an overlooked complication of pulse steroid treatment
of multiple sclerosis. Eur. J. Neurol. 13, 857861.
Supplemental Information includes Supplemental Experimental Procedures,
Chang, A., Tourtellotte, W.W., Rudick, R., and Trapp, B.D. (2002). Premyelinat-
five figures, and seven tables and can be found with this article online at
ing oligodendrocytes in chronic lesions of multiple sclerosis. N. Engl. J. Med.
http://dx.doi.org/10.1016/j.celrep.2016.05.009.
346, 165173.
AUTHOR CONTRIBUTIONS Cheng, C.W., Adams, G.B., Perin, L., Wei, M., Zhou, X., Lam, B.S., Da Sacco,
S., Mirisola, M., Quinn, D.I., Dorff, T.B., et al. (2014). Prolonged fasting reduces
I.Y.C., L.P., M.W., and V.D.L. designed mouse experiments. I.Y.C., S.B., and IGF-1/PKA to promote hematopoietic-stem-cell-based regeneration and
P.C. performed the mouse experiment. I.Y.C., L.P., P.C., B.B., and A.G. reverse immunosuppression. Cell Stem Cell 14, 810823.
performed and processed immunohistochemistry. I.Y.C., L.P., and B.B. per- Dhib-Jalbut, S. (2007). Pathogenesis of myelin/oligodendrocyte damage in
formed qualitative and quantitative analysis. I.Y.C. and J.S. performed fluores- multiple sclerosis. Neurology 68, S13S21, discussion S43S54.
cence-activated cell sorting (FACS) analysis. I.Y.C. processed the cytokine
Esposito, M., Ruffini, F., Bellone, M., Gagliani, N., Battaglia, M., Martino, G.,
assay. A.M., F.P., and M.B. designed the human study; M.B. acquired human
and Furlan, R. (2010). Rapamycin inhibits relapsing experimental autoim-
clinical data; A.M., F.P., and M.B., analyzed and interpreted data; and M.B.
mune encephalomyelitis by both effector and regulatory T cells modulation.
performed, interpreted, and presented the statistical analysis. A.M., F.P.,
J. Neuroimmunol. 220, 5263.
M.B., A.H.C., T.E.M., M.W., and V.D.L. were involved in discussing the results
Esquifino, A.I., Cano, P., Jimenez-Ortega, V., Fernandez-Mateos, M.P., and
and editorial support. I.Y.C., M.B., and V.D.L. wrote the paper. All authors
Cardinali, D.P. (2007). Immune response after experimental allergic encepha-
discussed the results and commented on the manuscript.
lomyelitis in rats subjected to calorie restriction. J. Neuroinflammation 4, 6.
CONFLICTS OF INTEREST Fletcher, J.M., Lalor, S.J., Sweeney, C.M., Tubridy, N., and Mills, K.H. (2010).
T cells in multiple sclerosis and experimental autoimmune encephalomyelitis.
The University of Southern California has licensed intellectual property to Clin. Exp. Immunol. 162, 111.
L-Nutra that is under study in this research. As part of this license agreement, Fontana, L., Partridge, L., and Longo, V.D. (2010). Extending healthy life span
the University has the potential to receive royalty payments from L-Nutra. from yeast to humans. Science 328, 321326.
V.D.L. has equity interest in L-Nutra, a company that develops medical food.
Friese, M.A., and Fugger, L. (2005). Autoreactive CD8+ T cells in multiple scle-
rosis: a new target for therapy? Brain 128, 17471763.
ACKNOWLEDGMENTS
Goverman, J. (2009). Autoimmune T cell responses in the central nervous
We thank Dr. Stephen Hauser for insightful comments, Dr. Pinchas Cohen for system. Nat. Rev. Immunol. 9, 393407.
assistance with fluorescence microscopy, and Nadine Krueger and Gabi Rahn Guevara-Aguirre, J., Balasubramanian, P., Guevara-Aguirre, M., Wei, M.,
for technical assistance. L.P. is a Harry Weaver Neuroscience Scholar of the Na- Madia, F., Cheng, C.W., Hwang, D., Martin-Montalvo, A., Saavedra, J., Ingles,
tional Multiple Sclerosis Society (NMSS, JF 2144A2/1) and is funded by Fonda- S., et al. (2011). Growth hormone receptor deficiency is associated with a ma-
zione Italiana Sclerosi Multipla (FISM; 2014/R/15) and the Office of the Assistant jor reduction in pro-aging signaling, cancer, and diabetes in humans. Sci.
Secretary of Defense for Health Affairs, through the Multiple Sclerosis Research Transl. Med. 3, 70ra13.
Program, under award number W81XWH-14-1-0156. The opinions, interpreta- Hemmer, B., Archelos, J.J., and Hartung, H.P. (2002). New concepts in the
tions, conclusions, and recommendations express in this article are those of immunopathogenesis of multiple sclerosis. Nat. Rev. Neurosci. 3, 291301.
the author and are not necessarily endorsed by the Department of Defense.
Herold, M.J., McPherson, K.G., and Reichardt, H.M. (2006). Glucocorticoids in
The mouse study was funded by National Institutes of Health (NIH)/National Insti-
T cell apoptosis and function. Cell. Mol. Life Sci. 63, 6072.
tute on Aging (NIA) grant AG034906 (to V.D.L.). The human study was funded by
Meylin Projekt e.V. and Familie Ernst Wendt Stiftung Stadt Koeln, which were not Kafami, L., Raza, M., Razavi, A., Mirshafiey, A., Movahedian, M., and Khorra-
involved in any decision-making processes relating the study or its participants. mizadeh, M.R. (2010). Intermittent feeding attenuates clinical course of exper-
The work of F.P. is supported by Deutsche Forschungsgemeinschaft (DFG Exc imental autoimmune encephalomyelitis in C57BL/6 mice. Avicenna J. Med.
257). The content is solely the responsibility of the authors and does not neces- Biotechnol. 2, 4752.
sarily represent the official views of the NIA or NIH. The University of Southern Cal- Kim do, Y., Hao, J., Liu, R., Turner, G., Shi, F.D., and Rho, J.M. (2012). Inflam-
ifornia has licensed intellectual property to L-Nutra that is under study in this mation-mediated memory dysfunction and effects of a ketogenic diet in a
research. As part of this license agreement, the university has the potential to murine model of multiple sclerosis. PLoS ONE 7, e35476.
receive royalty payments from L-Nutra. V.D.L. has equity interest in L-Nutra, a Laplante, M., and Sabatini, D.M. (2012). mTOR signaling in growth control and
company that develops medical food. disease. Cell 149, 274293.
Lee, C., Safdie, F.M., Raffaghello, L., Wei, M., Madia, F., Parrella, E., Hwang,
Received: May 22, 2015
D., Cohen, P., Bianchi, G., and Longo, V.D. (2010). Reduced levels of IGF-I
Revised: February 20, 2016
mediate differential protection of normal and cancer cells in response to fast-
Accepted: April 26, 2016
ing and improve chemotherapeutic index. Cancer Res. 70, 15641572.
Published: May 26, 2016
Longo, V.D., and Mattson, M.P. (2014). Fasting: molecular mechanisms and
REFERENCES clinical applications. Cell Metab. 19, 181192.
ck, W., Parisi, J., Scheithauer, B., Rodriguez, M., and Lass-
Lucchinetti, C., Bru
Ashwell, J.D., Lu, F.W., and Vacchio, M.S. (2000). Glucocorticoids in T cell mann, H. (1999). A quantitative analysis of oligodendrocytes in multiple scle-
development and function*. Annu. Rev. Immunol. 18, 309345. rosis lesions. A study of 113 cases. Brain 122, 22792295.

10 Cell Reports 15, 111, June 7, 2016

 Please cite this article in press as: Choi et al., A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple Sclerosis
Symptoms, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.05.009

Nath, N., Khan, M., Paintlia, M.K., Singh, I., Hoda, M.N., and Giri, S. (2009). Torkildsen, O., Brunborg, L.A., Myhr, K.M., and B, L. (2008). The cuprizone
Metformin attenuated the autoimmune disease of the central nervous system model for demyelination. Acta Neurol. Scand. Suppl. 188, 7276.
in animal models of multiple sclerosis. J. Immunol. 182, 80058014.
ller, S., Zinser, C., Maier, M., Su
Uttner, I., Mu ssmuth, S., Claus, A., Ostermann,
Pender, M.P., and Greer, J.M. (2007). Immunology of multiple sclerosis. Curr. B., Elitok, E., Ecker, D., Brettschneider, J., et al. (2005). Reversible impaired
Allergy Asthma Rep. 7, 285292. memory induced by pulsed methylprednisolone in patients with MS. Neurology
Piccio, L., Stark, J.L., and Cross, A.H. (2008). Chronic calorie restriction at- 64, 19711973.
tenuates experimental autoimmune encephalomyelitis. J. Leukoc. Biol. 84,
Vegiopoulos, A., and Herzig, S. (2007). Glucocorticoids, metabolism and
940948.
metabolic diseases. Mol. Cell. Endocrinol. 275, 4361.
Planey, S.L., and Litwack, G. (2000). Glucocorticoid-induced apoptosis in
lymphocytes. Biochem. Biophys. Res. Commun. 279, 307312. Wingerchuk, D.M., and Carter, J.L. (2014). Multiple sclerosis: current and
Raine, C.S., and Wu, E. (1993). Multiple sclerosis: remyelination in acute emerging disease-modifying therapies and treatment strategies. Mayo Clin.
lesions. J. Neuropathol. Exp. Neurol. 52, 199204. Proc. 89, 225240.

Ransohoff, R.M. (2012). Animal models of multiple sclerosis: the good, the bad Wolswijk, G. (1998). Chronic stage multiple sclerosis lesions contain a rela-
and the bottom line. Nat. Neurosci. 15, 10741077. tively quiescent population of oligodendrocyte precursor cells. J. Neurosci.
Roth, P., Wick, W., and Weller, M. (2010). Steroids in neurooncology: actions, 18, 601609.
indications, side-effects. Curr. Opin. Neurol. 23, 597602. Zeng, H., Yang, K., Cloer, C., Neale, G., Vogel, P., and Chi, H. (2013). mTORC1
Sospedra, M., and Martin, R. (2005). Immunology of multiple sclerosis. Annu. couples immune signals and metabolic programming to establish T(reg)-cell
Rev. Immunol. 23, 683747. function. Nature 499, 485490.

Cell Reports 15, 111, June 7, 2016 11