Multiple sclerosis (MS) is more common in western countries with diet being a potential contributing factor. Here we show that intermittent fasting (IF) ameliorated clinical course and pathology of the MS model, experimental autoimmune encephalomyelitis (EAE). IF led to increased gut bacteria richness, enrichment of the Lactobacillaceae, Bacteroidaceae, and Prevotellaceae families and enhanced antioxidative microbial metabolic pathways. IF altered T cells in the gut with a reduction of IL-17 producing T cells and an increase in regulatory T cells. Fecal microbiome transplantation from mice on IF ameliorated EAE in immunized recipient mice on a normal diet, suggesting that IF effects are at least partially mediated by the gut flora. In a pilot clinical trial in MS patients, intermittent energy restriction altered blood adipokines and the gut flora resembling protective changes observed in mice. In conclusion, IF has potent immunomodulatory effects that are at least partially mediated by the gut microbiome.

We therefore undertook studies of IF in the EAE model and in MS patients experiencing a relapse and showed that IF ameliorated EAE through effects at least in part mediated by changes in the gut flora. IF induced protective changes in gut microbiome metabolic pathways and lamina propria lymphocytes as demonstrated by the fact that gut microbiome transplantation from mice on IF ameliorated EAE in recipient mice after immunization. To translate our findings in patients, we performed a small pilot randomized controlled trial. IF in MS patients having a relapse was a safe and feasible intervention associated with short-term metabolic and gut microbiome changes that recapitulated what was observed in the animal model.

MS is more common in western countries. Dietary habits have been considered as a potential factor contributing to MS epidemiology (). Different diets and dietary supplements have been implicated in MS risk, but the field is lacking robust scientific data to support this risk. Indeed, many studies highlight the importance of the complex interplay between nutrition, metabolic state, and immune-inflammatory responses in MS (). Obesity during childhood/young adulthood is a risk factor for MS development as shown in several recent studies (). This might be related to a low-grade chronic inflammatory state in obesity that could promote autoimmunity through altered adipokine production (). An additional link between nutrition and immune-inflammatory responses is the gut microbiome. Diet is a critical determinant of the gut microbial composition. Gut commensal bacteria and their metabolites have the potential to exert both pro- and anti-inflammatory responses by regulating T cell differentiation and immune responses in the gut (). Ultimately, this can have systemic effects and either drive or protect from autoimmune diseases, including in the EAE model (). Recently it has been reported that the gut microbiome in RRMS patients is altered compared with healthy controls (). Further, calorie restriction (CR) has potent anti-inflammatory effects (). Studies, including our own, demonstrated that chronic CR significantly inhibited EAE (). However, chronic CR is not likely to be feasible for most people. Intermittent fasting (IF) induces many of the same changes observed by chronic CR and would possibly be more acceptable.

High body mass index before age 20 is associated with increased risk for multiple sclerosis in both men and women.

Multiple sclerosis (MS) is a central nervous system (CNS) inflammatory demyelinating disease characterized by varying degrees of axon and neuron injury triggered presumably by autoimmune mechanisms. MS affects 2.5 million people worldwide, with significant personal and socioeconomic burden. Clinically, MS can be relapsing-remitting (RRMS), or can have a progressive course characterized by accumulating neurological disability with or without superimposed relapse activity (). Genetic risk factors do not account fully for disease development. Environmental factors, including some infections, low vitamin D levels, smoking, and obesity, have each been associated with increased MS risk (). Experimental autoimmune encephalomyelitis (EAE) is an MS animal model that has been instrumental in the development of several MS therapies (). T lymphocytes, in particular CD4T cells, are key players in EAE and are thought to be major contributors to MS pathogenesis. CD4T helper (Th) cells are characterized by distinct cytokine profiles and expression of master transcription factors, which are used to define T cell subsets (). Several lines of evidence indicate the pathogenic role in MS and EAE of CD4T cells producing IL-17 (Th17), interferon (IFN)-γ (Th1), and granulocyte-macrophage colony-stimulating factor (GM-CSF) while regulatory T cells (Tregs) have immunomodulatory and protective functions.

Stool samples were collected from the MS patients in the control and IER groups at baseline and 15 days after IER. Mice and humans harbor different gut microbiomes at lower taxonomic levels, but the vast majority of the microbes in both species belong to two phyla, Bacteroidetes and Firmicutes (). Less abundant, Actinobacteria and Verrucomicrobia are also shared by mouse and human. Change of the relative abundances of four phyla in mice undergoing IF and in RRMS patients after 15 days of IER were compared. Similar directions of relative abundance changes of the four phyla were observed in mice on IF and humans on IER ( Figure 7 B). It should be noted that corticosteroid treatment in the human study may have influenced the changes in gut microbiome observed. However, the magnitude of change was much lower in the corticosteroid group than IER plus corticosteroid. No bacteria were significantly different at day 15 between the two groups, but the abundance of Faecalibacterium, Lachnospiracea incertae sedis, and Blautia showed an increasing trend after 15 days of IER. Moreover, Faecalibacterium was strongly correlated with the level of adiponectin in blood (r = 0.86, p = 0.0009) ( Figure 7 C).

At baseline and day 15, blood was also collected to better characterize any possible effects of IER on circulating immune cell subsets, including naive and memory T lymphocytes, B lymphocytes, monocytes, natural killer cells, myeloid and plasmacytoid dendritic cells, and Tregs ( Table S6 ). Absolute numbers of T and B lymphocytes increased on day 15 in the ad libitum control diet group, whereas in the IER group, absolute numbers of T and B cells were stable or decreased compared with baseline. B cell numbers on day 15 were significantly lower in the IER group compared with the ad libitum group (p = 0.02) and some suggestion for a similar difference in CD4and CD8T cells (p = 0.07 and 0.08, respectively) were observed (controlling for baseline levels and change in BMI). This effect seemed to be mainly driven by changes in the numbers of naive CD4and CD8T cells, which on day 15 were lower in the IER groups compared with the ad libitum group, with a difference that was statistically significant for the CD4(p = 0.04) and close to for CD8(p = 0.07). Absolute counts of circulating Tregs were unchanged between the two groups at day 15, although the percentage of Tregs decreased slightly in the control group, but remained stable in the IER group, leading to a significant difference on day 15 between the two groups (p = 0.03). Interestingly, in vitro functional studies of Tregs on day 15 showed significantly more suppression of effector T cell proliferation at different Teff:Treg ratios in the IER compared with the ad libitum group (p = 0.012 by two-way ANOVA), while no differences were noted at baseline (p = 0.18; Figure S6 ). No differences in memory and effector T cells and the other immune cell subsets between the two groups on day 15 were noted ( Table S6 ).

Differences in serum metabolites at day 15 between the IER and control groups were assessed after controlling for baseline (day 1) levels and change in BMI, focusing on metabolites altered by IF in the EAE studies. These included leptin, adiponectin, and β-hydroxybutyrate. Cortisol was not measured, since all patients were being treated with corticosteroids. Both groups showed reduced leptin levels at day 15 compared with baseline, but a significantly greater reduction of leptin levels at day 15 was observed in the IER group (p = 0.006) after adjusting for baseline levels and change in BMI ( Figure 7 A; Table S5 ). Adiponectin levels increased in both groups; no significant difference between the IER and the ad libitum group was detected (p = 0.46). No effect of intermittent low calorie intake on β-hydroxybutyrate was seen after adjusting for baseline and change in BMI ( Table S5 ). This was not unexpected given that it takes multiple consecutive days of low carbohydrate intake to increase serum levels of ketone bodies in humans ().

(C) Levels of serum adiponectin are strongly positively correlated with the relative abundance of Faecalibacterium in the MS study participants (r = 0.86, p = 0.0009 Pearson correlation).

(B) At phylum level, the alteration of the gut microbiome after IER for 15 days in RRMS patients (n = 5 patients/group, top panel) shows similar trends as that in mice (n = 8 mice/group, bottom panel). The y axis represents the percentage of change of the relative abundance of the gut microbiome. In human studies, the percentage change is calculated based on the microbial abundance at baseline and day 15. In mouse studies, the percent change is calculated based on the microbial abundance on T2 and T3. The box in the graphs extends from the 25th to 75th percentiles, the bars are medians, the whiskers indicate the smallest and largest values.

(A) Comparison of serum levels of leptin and adiponectin on day 15 in RRMS patients in the ad libitum and IER groups in the human trial. Each dot represents a mouse and the bars are medians with interquartile range.

Based on our beneficial results using CR and IF in murine EAE, we initiated a small randomized controlled pilot trial to examine the effects on laboratory and clinical measures of intermittent energy restriction (IER) in RRMS patients. Goals of this pilot study were to assess safety, feasibility, and compliance with IER in MS patients and to obtain preliminary data on IER effects on blood adipokine levels and other metabolic or immune-inflammatory markers in RRMS patients. Seventeen MS subjects undergoing relapse were enrolled and randomized 50:50 to IER versus ad libitum control diet between 2014 and 2016, of which 16 completed the trial (one person randomized to control diet withdrew). The groups were similar with no significant differences in age, race, BMI and expanded disability status scale (EDSS) ( Table S4 ). IER was well tolerated; no person in the IER group dropped out. The average number of days that the subjects in the IER group fasted was 6 ± 1 (SD) days of the 7 intended during the 2 study weeks (86 ± 17 [SD] % adherence to the diet) based on the dietary recall diary. No safety concerns were uncovered by routine complete blood count and comprehensive metabolic panel testing. We observed an increase of white blood cells, mainly driven by an increase in neutrophils on day 15 compared with baseline in both groups, which was expected given the steroid treatment. Furthermore, there was a significant difference in BMIs on day 15 in the two groups after controlling for baseline BMI (p = 0.03 by analysis of covariance [ANCOVA]; Table S5 ). EDSS improvement was seen in both groups (likely due to both natural recovery and corticosteroid treatment of the MS relapse) without any significant difference in the degree of amelioration. No significant differences between the groups were observed for changes in the MS functional composite (which components are the 25-foot timed walk, paced auditory serial addition test, and the 9-hole peg test) and the symbol digit modality test, which was expected for a small patient-unblinded trial of short duration.

Gut microbiota are believed to directly influence immune cells residing in the gut lamina propria (), which may in turn modulate local and systemic immune responses, including in EAE (). IL-17-producing T cells and Tregs normally reside in the intestinal lamina propria (). Thus, phenotype and cytokine profiles of small intestine lamina propria (SI LP) T cells were characterized after 4 weeks on IF versus ad libitum diet. Numbers of CD4and CD8T cells and level of CD44 expression on effector T cells in the SI LP were not different between the two groups (data not shown). However, the proportion of Th17 CD4cells in the SI LP from the IF group was reduced versus the ad libitum group ( Figure 6 A and Table S3 ). No significant differences were noted in the percentages of GM-CSF and IFN-γ producing CD4T cells. Assay of CD4CD25Foxp3Tregs in the SI LP at T2 ( Figures 6 B and 6C) revealed significantly increased proportions of Tregs in SI LP and peripheral lymph node (PLN) of the IF group ( Figures 6 B and 6C). A trend toward increased percentages of Tregs in mesenteric lymph nodes (MLNs) of IF mice was noted, whereas no significant differences were seen in the spleen ( Figure 6 C).

(C) Proportion of Tregs in MLNs, PLNs, and spleen. Each dot represents a mouse, bars are mean ± SD. These results are from one experiment out of two (in A) or three (in B and C) performed with similar results ( Table S3 reports the results of all experiments performed).

(B) Proportion of Tregs in the SI LP in the two groups. Representative flow cytometry plots for one mouse/group are shown on the right panel.

(A) Intracellular cytokine production by CD4 + T cells in the SI LP after 4 weeks on IF or normal ad libitum analyzed by flow cytometry.

Reduced Proportion of IL-17- Producing T Cells and Increased Proportion of Tregs in the Gut Lamina Propria after 4 Weeks of IF

Figure 6 Reduced Proportion of IL-17- Producing T Cells and Increased Proportion of Tregs in the Gut Lamina Propria after 4 Weeks of IF

The mechanisms by which IF decreases the severity of EAE is not fully understood. Fecal microbiota transplantation (FMT) experiments were performed to test the hypothesis that EAE amelioration by IF may, at least in part, be mediated by changes in the gut microbiota. Fecal matter from mice on IF or ad libitum were transferred by oral gavage into antibiotic-treated recipients, which were subsequently immunized to induce EAE (the study design is presented in Figure 5 A). Overall disease severity and spinal cord pathology were significantly reduced in recipients of FMT from the IF mice compared with recipients of FMT from ad libitum fed mice ( Figures 5 B–5D and Table S3 ). Thus, modulation of the gut microbiome likely plays a mechanistic role in the beneficial effects of IF in EAE.

(D) Quantification of inflammation, demyelination (evaluated by histology and MBP staining), and axonal damage (evaluated by SMI-32 + staining) in the spinal cord in the two groups (n = 10/group). Each dot represents a mouse and the bars are means ± SD. ∗ p < 0.05; ∗∗ p < 0.0005. All p values were calculated by Mann-Whitney test.

(C) Spinal cord pathology: the upper panel shows histological staining (solochrome cyanine) for myelin (in blue) and the lower panel shows immuno-staining for SMI-32 + damaged axons (in red) and MBP (in green). Scale bars, 200 μm.

(B) EAE clinical course in mice transferred with fecal matter from mice on IF or fed ad libitum. Shown is one representative experiment out of two performed with similar results (each dot represent the mean clinical scores for all five mice in each group; error bars are SEM; p < 0.0001 by two-way ANOVA; Table S3 reports clinical characteristics for the two experiments performed).

Mice were pre-treated with an antibiotic cocktail for a week, and then subjected to FMT from donor mice that were on IF (for 4 weeks) or fed ad libitum. FMT was administered for a week before and a week after EAE immunization.

The gut microbiome is involved in nutrient absorption, metabolism, and storage. mWGS of the gut microbiome allows for characterization of bacterial metabolic pathways to infer the functional potential of the whole stool bacterial community. Here, mWGS reads from two groups at T3 were mapped to the KEGG database to determine the metabolic potential. Twenty-seven metabolic pathways implicated in carbohydrate metabolism, lipid metabolism, and amino acid metabolism were differentially represented in the microbiome between IF and ad libitum diet groups (LEfSe, p < 0.01; Figure 4 ). Eleven of these pathways were enriched in stool samples of intermittently fasted mice versus the ad libitum group. Interestingly, IF was associated with increased relative abundance of the synthesis and degradation of ketone bodies (ko00072) and glutathione metabolism (ko00480) pathways, suggesting that IF might modulate the antioxidant signaling pathway of the gut microbiome, which in turn could also affect the host. Sixteen pathways were less represented in stool samples of IF mice and these were mainly implicated in metabolism of cofactors and vitamins. Notably, the lipopolysaccharide biosynthesis pathway (ko00540) was significantly decreased in the IF group which could potentially benefit EAE, through less stimulation of innate immune system.

Metagenomic whole genome shotgun was performed for stool samples collected at T3 in each group (n = 5/group). A linear discriminate analysis (LDA) was conducted to identify differentially represented pathways in the two groups. Pathways with LDA score >2.5 (x axis) and p < 0.01 are shown. The right side of the figure represents pathways whose abundance was significantly higher in the IF group. The left side of the figure represents pathways whose abundance was significantly higher in the ad libitum group. The absolute LDA value is the effect size between two groups for a particular pathway. The color of the bar represents main metabolic functions to which the different pathways belong, as indicated in the legend.

Next, the specific taxa that were differentially represented in IF and ad libitum groups were identified. Several families displayed significantly different abundance in the two groups at T2 and/or T3 ( Figure S5 ). Among these, the abundance of the Bacteroidaceae, Lactobacillaceae, and Prevotellaceae microbial families increased in the IF compared with the ad libitum group at T2, 4 weeks on the specific diet (q = 0.05, q = 0.04, and q = 0.05 for Bacteroidaceae, Lactobacillaceae, and Prevotellaceae, respectively) and even more striking during clinical EAE (T3: q = 0.02, q = 0.006, and q = 0.006 for Bacteroidaceae, Lactobacillaceae, and Prevotellaceae, respectively) ( Figure 3 D). The abundance of these microbes did not differ between groups at baseline. Serum corticosterone levels correlated positively with the abundance of Lactobacillaceae. Unclassified Lactobacillaceae and Bacteroidetes were negatively associated with leptin level, while serum leptin levels were positively correlated with the abundance of Rikenellaceae and Lachnospiraceae ( Figure 3 E). Probiotic strains, such as Lactobacillus species, have been shown to lessen EAE severity (). Although not all the bacteria can be classified to the species level using 16S rRNA gene sequencing, the hypervariable regions of 16S gene allow distinction of different species in Lactobacillaceae (). Lactobacillaceae species that were over-represented in the IF samples were Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus murinus, and Lactobacillus sp. ASF360, identified by aligning the Lactobacillaceae sequence to the NCBI database (with >99% identity and 100% coverage). Strikingly, the relative abundance of the four Lactobacillus species showed different trajectories over the T1, T2, and T3 time points between the two diet groups ( Figure 3 F). Specifically, the relative abundances of the Lactobacillus species (except L. murinus) decreased or remained the same in the ad libitum group, but increased in the IF group, especially Lactobacillus sp. ASF360. Bacteroides fragilis was reported to be protective in EAE (). No B. fragilis was found in the samples. However, Bacteroides caecimuris, a major species in the Bacteroidaceae family, was undetectable at T1 in either group while it increased to 0.4% and 0.9% (median relative abundance) at T2 and T3 in the IF group, respectively, while remaining undetectable in the ad libitum group. These species classification was further supported by our whole genome shotgun sequencing (mWGS) (n = 5 mice/group, at T3). In addition, mWGS data revealed that Bifidobacterium pseudolongum in IF was more than twice as abundant in the ad libitum group at T3 (2.1% versus 0.8%). 16S sequencing did not identify Bifidobacterium, likely because of the 16S primers bias toward its underrepresentation (). Overall, IF had a profound impact on the gut microbiome composition and abundance, with particular enrichment of beneficial bacteria, which are also used as probiotics.

Stool samples were collected at the time points T1, T2, and T3 ( Figure 2 A). No significant differences in the gut microbiota from the 16S rRNA gene sequencing were seen at baseline between the two groups ( Figure 3 A-T1). The gut microbiota were significantly different in the two groups at T2 and T3 (18 days pi; Figure 3 A-T2, T3) (p < 0.01 by PERMANOVA analysis). Richness is a measure of alpha diversity for microbial community (). It reflects complexity of a microbial community with higher diversity being associated with healthier gut microbiome (). Bacterial richness was not significantly different at baseline (T1) between groups. In the ad libitum group, diversity did not change significantly over the three time points. In contrast, IF increased diversity at the T2 and T3 time points ( Figure 3 B, p < 0.05 by mixed linear regression, and Figure S4 ). Gut microbiota might modulate systemic metabolic responses (). Indeed, blood leptin levels in the IF and ad libitum groups were negatively correlated with gut microbiome diversity (r = −0.51, p < 0.005, Pearson correlation; Figure 3 C), consistent with a previous study (). Adiponectin levels showed a trend toward a positive association with gut microbiome diversity, but did not reach statistical significance (data not shown).

In (B)–(D), IF is in gray and ad libitum is in black. In (B), (D), and (F), the boxes in the graphs extends from the 25th to 75th % percentiles, the bars are medians, the whiskers indicate the smallest and largest values.

(F) Different trajectories of Lactobacillus species in the IF and ad libitum groups were seen over the three time points. In the IF group, Lactobacillus sp. are significantly higher at the T2 and T3 time points (p < 0.05, linear mixed regression).

(E) Pearson or Spearman correlations of corticosterone or leptin and microbiome (data were log transformed). All samples from IF and ad libitum groups are included.

(D) Bacterial families with significantly different relative abundance between the two groups at T2 and T3. Reported here only those bacterial families with the same direction of difference at T2 and T3 between the two groups; y axis is the relative abundance of the bacterial family (q < 0.05, ANCOVA). ∗ q < 0.05; ∗∗ q < 0.01.

(B) Richness is a measure of alpha diversity for microbial community. Bacterial richness increased significantly over the three time points in the IF group, but not in the ad libitum group (linear mixed regression # p < 0.05; y axis is number of different bacteria families).

(A) Non-metric multidimensional scaling (NMDS) plots illustrate microbiome similarity in IF and ad libitum groups (each dot is one sample; x axis and y axis are first and second dimension of microbiome data). At T1, samples from two groups intermingled, indicating similar microbiome (p > 0.05, permutational ANOVA [PERMANOVA] test). At T2 and T3, samples from the two groups clustered separately, indicating two distinct microbiome communities (p < 0.05, PERMANOVA test).

Stool samples were collected from the IF and ad libitum groups at T1 (baseline, n = 10 in the IF group, n = 9 in the ad libitum group), T2 (after 4 weeks on the diet, prior to immunization, n = 9 in IF, n = 10 in ad libitum), and T3 (clinical EAE, n = 10 in IF, n = 8 in ad libitum).

Serum was collected from mice at baseline (before starting IF, named T1), after 4 weeks of IF or ad libitum diet (before immunization-T2) and during clinical EAE (day 18–20 post-immunization [pi]-T3) after a fasting day. The experimental and sample collection timeline is presented in Figure 2 A. Baseline levels of corticosterone, leptin, and adiponectin, each measured by ELISA, were not different in the two groups. Serum leptin levels were significantly decreased in the IF group after 4 weeks (T2) of IF (IF versus ad libitum, respectively: 1.3 ± 0.6 [SD] and 3.6 ± 1.7 ng/mL; p = 0.008), but were not significantly different between the two groups during clinical EAE ( Figure 2 B and Table S3 ). Adiponectin levels were increased in the IF group versus ad libitum (28,425 ± 3,107 and 14,865 ± 4,900 ng/mL, respectively; p = 0.007) at T2 before immunization ( Figure 2 C). Analyses for leptin and adiponectin were also performed controlling for body weights with similar results. Corticosterone levels were higher in the IF group before immunization (T2: 172 ± 89 versus 62 ± 51 ng/mL in the IF and ad libitum groups, respectively) and during EAE (T3: 270 ± 111 versus 147 ± 46 ng/mL) ( Figure 2 D). Serum levels of ketones were measured because they were expected to change with IF and possibly to impact EAE (). Beta-hydroxybutyrate was dramatically increased in mice on IF compared with those on an ad libitum diet at both T2 and T3 (1.8 ± 0.2 versus 0.2 ± 0.1 mM and 1 ± 0.4 versus 0.1 ± 0.08 mM, respectively) ( Figure 2 E). We have also performed measurements in serum samples collected at 4 weeks on the intervention after a feeding day with changes that were comparable to after fasting for all analytes, but for leptin and corticosterone that were decreased and increased respectively, only after a fasting day and not after a feeding day ( Figure S3 ).

(B–E) Serum levels of (B) leptin (n = 6/group), (C) adiponectin (n = 5/group), (D) corticosterone (n = 6/group), and (E) β-hydroxybutyrate (n = 10/group) were measured by ELISA at different time points during the experiment. The box in the graphs extends from the 25th to 75th percentiles, the bars are median, the whiskers indicate the smallest and largest values. Measurements for leptin, adiponectin, and corticosterone were performed in three different experiments with similar results ( Table S3 reports the results of all experiments performed); β-hydroxybutyrate was measured in two different experiments with similar results. In the ELISA assays, each sample was run in duplicate.

(A) Timeline for the experimental procedures including serum and stool sample collection at baseline before starting the diet (IF or ad libitum feeding-T1), after 4 weeks on the diet but before immunization (T2), during clinical EAE (day 18–20 post-immunization-T3).

In accord with the reduced clinical severity, mice in the IF group had less inflammatory cell infiltration and demyelination (evaluated by histology and myelin basic protein staining) in the spinal cord (site of most pathology in the model). SMI-32damaged axons were reduced in the spinal cord in the IF group ( Figures 1 B and 1C). CNS inflammation in EAE is driven by T cells activated in the peripheral lymph nodes draining immunization sites. CD4T cells from draining lymph nodes in IF-treated mice produced less interleukin (IL)-17A and IFN-γ (p < 0.05), cytokines relevant in EAE pathogenesis (). A trend toward reduced production of GM-CSF, another cytokine that is pathogenic in EAE () was noted in the IF group ( Figures 1 D and 1E; Table S3 ). Overall, IF reduced EAE clinical severity and CNS pathology, along with immunomodulation characterized by reduced T cell production of pathogenic cytokines.

RORgammat drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation.

We previously showed that chronic CR ameliorates EAE when initiated before immunization (); however, a high level of CR might be difficult to translate to people with MS on a continuous basis. Thus, the effects on EAE of IF were examined. Mice were fasted every other day (IF group) or fed ad libitum (control group) for 4 weeks. IF group body weights fluctuated depending on whether mice were weighed on fasting or feeding days and were significantly different between groups at time of immunization as they were assessed after a fasting day ( Figure S1 and Table S1 , Experiment 1). Body composition studies by EchoMRI showed a significant reduction of both body fat and lean mass in the IF compared with the control group after 4 weeks on the intervention and after a fasting day ( Figure S2 ). IF resulted in amelioration of EAE clinical course and severity compared with ad libitum diet (all EAE experiments are summarized in Tables S1 and S2 ). In a representative EAE experiment ( Figure 1 A), disease incidence was 100% in the control group, whereas only seven out of ten mice in the IF group developed clinical EAE. Mean EAE day of onset was significantly delayed in the IF group versus the control group (p < 0.05; Table S1 , Exp. 1) and disease course was significantly less severe in the IF group compared with controls (p < 0.005). Results of the sensitivity analysis which summarize all EAE experiments had consistent results with significant differences in delayed onset (p = 0.04), decreased incidence (p < 0.007), and lower cumulative clinical EAE score (p = 0.004) observed between the IF versus ad libitum cages ( Table S2 ).

(E) Representative flow cytometry plots for T cell production of IL-17A and IFN-γ in CD4 + T cells isolated from the draining lymph nodes at day 6 post-immunization in the two groups.

(D) Percentages of CD4T cells producing IL-17A, IFN-γ, and GM-CSF measured by flow cytometry in lymph nodes draining the immunization site on day 6 post-immunization. Each dot represents a mouse and the bars are means ± SD. This is one of three different experiments performed with similar results (n = 4–5/group in each experiment; Table S3 reports the results of all experiments performed).

(C) Quantification of inflammation, demyelination (evaluated by histology and MBP staining), and axonal damage (evaluated by SMI-32 + staining) in the spinal cord in the two groups (n = 10/group) on day 26 post-immunization. Each dot represents a mouse and the bars are means ± SD.

(B) Spinal cord pathology: the upper panel shows a histological staining (solochrome cyanine) for myelin (in blue) and the lower panel is an immuno-staining for SMI-32 + damaged axons (in red) and myelin basic protein (MBP, in green). Scale bars, 200 μm.

(A) EAE clinical course of a representative experiment (dots represent the mean clinical scores for all ten mice in each group, error bars are SEM; p < 0.0001 by two-way ANOVA). Four EAE experiments were performed with similar results ( Table S1 ).

Discussion

Arumugam et al., 2010 Arumugam T.V.

Phillips T.M.

Cheng A.

Morrell C.H.

Mattson M.P.

Wan R. Age and energy intake interact to modify cell stress pathways and stroke outcome. Vasconcelos et al., 2014 Vasconcelos A.R.

Yshii L.M.

Viel T.A.

Buck H.S.

Mattson M.P.

Scavone C.

Kawamoto E.M. Intermittent fasting attenuates lipopolysaccharide-induced neuroinflammation and memory impairment. Johnson et al., 2007 Johnson J.B.

Summer W.

Cutler R.G.

Martin B.

Hyun D.H.

Dixit V.D.

Pearson M.

Nassar M.

Telljohann R.

Maudsley S.

et al. Alternate day calorie restriction improves clinical findings and reduces markers of oxidative stress and inflammation in overweight adults with moderate asthma. Kafami et al., 2010 Kafami L.

Raza M.

Razavi A.

Mirshafiey A.

Movahedian M.

Khorramizadeh M.R. Intermittent feeding attenuates clinical course of experimental autoimmune encephalomyelitis in C57BL/6 mice. Razeghi Jahromi et al., 2016 Razeghi Jahromi S.

Ghaemi A.

Alizadeh A.

Sabetghadam F.

Moradi Tabriz H.

Togha M. Effects of intermittent fasting on experimental autoimmune encephalomyelitis in C57BL/6 mice. Only recently have humans had constant access to food. Early in human evolution, people ate only intermittently. Hundreds of studies have shown that CR without malnutrition exerts a strong anti-inflammatory and immunomodulatory effect. Neuroprotective and anti-inflammatory effects of IF were shown in animal models of stroke () and systemic infection (), as well as in humans with inflammatory systemic conditions (). However, few reports address the effects of IF on immune function and autoimmune diseases, such as MS. In studies herein, we show that IF reduced inflammation, demyelination, and axonal damage in murine EAE, which is in agreement with previous reports (). Immune cells from PLNs had reduced antigen-specific immune responses and reduced pro-inflammatory cytokine production. In addition, IF affected the composition of T cells in the gut lamina propria with a reduction of IL-17-producing T cells and increased numbers of Tregs. IF increased gut bacteria richness and activated microbial metabolic pathways that modulate systemic immune responses. Importantly, we found that transplantation of microbiota from mice on IF into immunized recipient mice reduced myelin antigen-specific lymphocyte proliferation and EAE severity in the normal chow-fed recipients. These data strongly suggest that IF-induced changes in the gut microbiome can mediate a systemic immunomodulatory response to myelin antigens in vivo.

van Nood et al., 2013 van Nood E.

Vrieze A.

Nieuwdorp M.

Fuentes S.

Zoetendal E.G.

de Vos W.M.

Visser C.E.

Kuijper E.J.

Bartelsman J.F.

Tijssen J.G.

et al. Duodenal infusion of donor feces for recurrent Clostridium difficile. Borody et al., 2014 Borody T.J.

Brandt L.J.

Paramsothy S. Therapeutic faecal microbiota transplantation: current status and future developments. Microbiota transplantation from mice on IF into normally fed mice was able to transfer protection from EAE. This striking finding strongly supported that the beneficial effect of IF could be mediated by the gut microbiome, at least in part. FMT is an effective treatment for recurrent C. difficile infection (). Manipulation of the gut microbiota might also be a potential treatment for other diseases. Interestingly, a case report of FMT used to treat gastrointestinal symptoms in three MS patients was reported to improve their neurological symptoms ().

Sonnenburg and Backhed, 2016 Sonnenburg J.L.

Backhed F. Diet-microbiota interactions as moderators of human metabolism. Negrotto et al., 2016 Negrotto L.

Farez M.F.

Correale J. Immunologic effects of metformin and pioglitazone treatment on metabolic syndrome and multiple sclerosis. Piccio et al., 2008 Piccio L.

Stark J.L.

Cross A.H. Chronic calorie restriction attenuates experimental autoimmune encephalomyelitis. Accumulating evidence highlights the close interplay between nutrition, metabolism, and immune responses (). In a small study, treatment of the metabolic syndrome in MS patients with metformin (a CR mimetic) reduced inflammation evidenced by reduced brain MRI activity and peripheral immunological markers (). Our prior work showed that chronic CR inhibited EAE, with an associated modulation of endogenous corticosteroids and systemic adipokines/cytokines (). In humans, chronic CR has been reported to induce similar metabolic changes, including lower concentrations of white blood cells, reduced inflammatory cytokines and leptin, and increased circulating levels of cortisol and adiponectin. However, chronic CR in humans is difficult to sustain, and data from preliminary human studies suggest that IF would be a better and more physiologic option.

Atarashi et al., 2011 Atarashi K.

Tanoue T.

Shima T.

Imaoka A.

Kuwahara T.

Momose Y.

Cheng G.

Yamasaki S.

Saito T.

Ohba Y.

et al. Induction of colonic regulatory T cells by indigenous Clostridium species. Fujimoto et al., 2013 Fujimoto T.

Imaeda H.

Takahashi K.

Kasumi E.

Bamba S.

Fujiyama Y.

Andoh A. Decreased abundance of Faecalibacterium prausnitzii in the gut microbiota of Crohn's disease. Verdam et al., 2013 Verdam F.J.

Fuentes S.

de Jonge C.

Zoetendal E.G.

Erbil R.

Greve J.W.

Buurman W.A.

de Vos W.M.

Rensen S.S. Human intestinal microbiota composition is associated with local and systemic inflammation in obesity. Walters et al., 2014 Walters W.A.

Xu Z.

Knight R. Meta-analyses of human gut microbes associated with obesity and IBD. Miyake et al., 2015 Miyake S.

Kim S.

Suda W.

Oshima K.

Nakamura M.

Matsuoka T.

Chihara N.

Tomita A.

Sato W.

Kim S.W.

et al. Dysbiosis in the gut microbiota of patients with multiple sclerosis, with a striking depletion of species belonging to clostridia XIVa and IV clusters. Taira et al., 2015 Taira R.

Yamaguchi S.

Shimizu K.

Nakamura K.

Ayabe T.

Taira T. Bacterial cell wall components regulate adipokine secretion from visceral adipocytes. Manipulating the gut microbiome may offer a novel way to control autoimmune responses. Remarkably, in our small randomized, controlled study of IER in MS patients undergoing relapse, IER for only 15 days induced changes in leptin levels and gut microbiota that were similar to what was observed in mice with EAE. Our results from the human pilot study showed a trend toward increased abundance of Faecalibacterium and Blautia, which belong to the Clostridium XIV and XIVa (in the Firmicutes phylum), shown to promote Treg accumulation in the colon (), and implicated in disorders such as IBD and obesity (). These bacteria play an important role in producing butyrate in the gut, and have been reported to be reduced in MS patients (). The observed increase of Clostridia clusters XIV and XIVa with IF might serve to counterbalance this reported dysbiosis in MS. The present studies observed a strong correlation between the Faecalibacterium and serum adiponectin in MS patients. Studies suggest that gram-positive cell wall components stimulate adiponectin production (). Whether the presence of Faecalibacterium in the gut microbiome has a direct or indirect role in modulation of serum adiponectin warrants further investigation. Collectively, findings from the present studies demonstrate a consistent trend in metabolic and gut microbiome changes in mice on IF and MS patients undergoing IER, and support the translational potential of IF.