Interleukin-3 coordinates glial-peripheral immune crosstalk to incite multiple sclerosis.

Glial cells and central nervous system (CNS)-infiltrating leukocytes contribute to multiple sclerosis (MS). However, the networks that govern crosstalk among these ontologically distinct populations remain unclear. Here, we show that, in mice and humans, CNS-resident astrocytes and infiltrating CD44hiCD4+ T cells generated interleukin-3 (IL-3), while microglia and recruited myeloid cells expressed interleukin-3 receptor-ɑ (IL-3Rɑ). Astrocytic and T cell IL-3 elicited an immune migratory and chemotactic program by IL-3Rɑ+ myeloid cells that enhanced CNS immune cell infiltration, exacerbating MS and its preclinical model. Multiregional snRNA-seq of human CNS tissue revealed the appearance of IL3RA-expressing myeloid cells with chemotactic programming in MS plaques. IL3RA expression by plaque myeloid cells and IL-3 amount in the cerebrospinal fluid predicted myeloid and T cell abundance in the CNS and correlated with MS severity. Our findings establish IL-3:IL-3RA as a glial-peripheral immune network that prompts immune cell recruitment to the CNS and worsens MS.

Bacterial neurotoxic metabolites in multiple sclerosis cerebrospinal fluid and plasma.

The identification of intestinal dysbiosis in patients with neurological and psychiatric disorders has highlighted the importance of gut-brain communication, and yet the question regarding the identity of the components responsible for this cross-talk remains open. We previously reported that relapsing remitting multiple sclerosis patients treated with dimethyl fumarate have a prominent depletion of the gut microbiota, thereby suggesting that studying the composition of plasma and CSF samples from these patients may help to identify microbially derived metabolites. We used a functional xenogeneic assay consisting of cultured rat neurons exposed to CSF samples collected from multiple sclerosis patients before and after dimethyl fumarate treatment to assess neurotoxicity and then conducted a metabolomic analysis of plasma and CSF samples to identify metabolites with differential abundance. A weighted correlation network analysis allowed us to identify groups of metabolites, present in plasma and CSF samples, whose abundance correlated with the neurotoxic potential of the CSF. This analysis identified the presence of phenol and indole group metabolites of bacterial origin (e.g. p-cresol sulphate, indoxyl sulphate and N-phenylacetylglutamine) as potentially neurotoxic and decreased by treatment. Chronic exposure of cultured neurons to these metabolites impaired their firing rate and induced axonal damage, independent from mitochondrial dysfunction and oxidative stress, thereby identifying a novel pathway of neurotoxicity. Clinical, radiological and cognitive test metrics were also collected in treated patients at follow-up visits. Improved MRI metrics, disability and cognition were only detected in dimethyl fumarate-treated relapsing remitting multiple sclerosis patients. The levels of the identified metabolites of bacterial origin (p-cresol sulphate, indoxyl sulphate and N-phenylacetylglutamine) were inversely correlated to MRI measurements of cortical volume and directly correlated to the levels of neurofilament light chain, an established biomarker of neurodegeneration. Our data suggest that phenol and indole derivatives from the catabolism of tryptophan and phenylalanine are microbially derived metabolites, which may mediate gut-brain communication and induce neurotoxicity in multiple sclerosis.

A metabolic perspective on CSF-mediated neurodegeneration in multiple sclerosis.

Multiple sclerosis is an autoimmune demyelinating disorder of the CNS, characterized by inflammatory lesions and an underlying neurodegenerative process, which is more prominent in patients with progressive disease course. It has been proposed that mitochondrial dysfunction underlies neuronal damage, the precise mechanism by which this occurs remains uncertain. To investigate potential mechanisms of neurodegeneration, we conducted a functional screening of mitochondria in neurons exposed to the CSF of multiple sclerosis patients with a relapsing remitting (n = 15) or a progressive (secondary, n = 15 or primary, n = 14) disease course. Live-imaging of CSF-treated neurons, using a fluorescent mitochondrial tracer, identified mitochondrial elongation as a unique effect induced by the CSF from progressive patients. These morphological changes were associated with decreased activity of mitochondrial complexes I, III and IV and correlated with axonal damage. The effect of CSF treatment on the morphology of mitochondria was characterized by phosphorylation of serine 637 on the dynamin-related protein DRP1, a post-translational modification responsible for unopposed mitochondrial fusion in response to low glucose conditions. The effect of neuronal treatment with CSF from progressive patients was heat stable, thereby prompting us to conduct an unbiased exploratory lipidomic study that identified specific ceramide species as differentially abundant in the CSF of progressive patients compared to relapsing remitting multiple sclerosis. Treatment of neurons with medium supplemented with ceramides, induced a time-dependent increase of the transcripts levels of specific glucose and lactate transporters, which functionally resulted in progressively increased glucose uptake from the medium. Thus ceramide levels in the CSF of patients with progressive multiple sclerosis not only impaired mitochondrial respiration but also decreased the bioavailability of glucose by increasing its uptake. Importantly the neurotoxic effect of CSF treatment could be rescued by exogenous supplementation with glucose or lactate, presumably to compensate the inefficient fuel utilization. Together these data suggest a condition of 'virtual hypoglycosis' induced by the CSF of progressive patients in cultured neurons and suggest a critical temporal window of intervention for the rescue of the metabolic impairment of neuronal bioenergetics underlying neurodegeneration in multiple sclerosis patients.

Body Mass Index in Multiple Sclerosis modulates ceramide-induced DNA methylation and disease course.

BACKGROUND: Multiple Sclerosis (MS) results from genetic predisposition and environmental variables, including elevated Body Mass Index (BMI) in early life. This study addresses the effect of BMI on the epigenome of monocytes and disease course in MS. METHODS: Fifty-four therapy-naive Relapsing Remitting (RR) MS patients with high and normal BMI received clinical and MRI evaluation. Blood samples were immunophenotyped, and processed for unbiased plasma lipidomic profiling and genome-wide DNA methylation analysis of circulating monocytes. The main findings at baseline were validated in an independent cohort of 91 therapy-naïve RRMS patients. Disease course was evaluated by a two-year longitudinal follow up and mechanistic hypotheses tested in human cell cultures and in animal models of MS. FINDINGS: Higher monocytic counts and plasma ceramides, and hypermethylation of genes involved in negative regulation of cell proliferation were detected in the high BMI group of MS patients compared to normal BMI. Ceramide treatment of monocytic cell cultures increased proliferation in a dose-dependent manner and was prevented by DNA methylation inhibitors. The high BMI group of MS patients showed a negative correlation between monocytic counts and brain volume. Those subjects at a two-year follow-up showed increased T1 lesion load, increased disease activity, and worsened clinical disability. Lastly, the relationship between body weight, monocytic infiltration, DNA methylation and disease course was validated in mouse models of MS. INTERPRETATION: High BMI negatively impacts disease course in Multiple Sclerosis by modulating monocyte cell number through ceramide-induced DNA methylation of anti-proliferative genes. FUND: This work was supported by funds from the Friedman Brain Institute, NIH, and Multiple Sclerosis Society.

Fumarates target the metabolic-epigenetic interplay of brain-homing T cells in multiple sclerosis.

Cell-permeable formulations of metabolites, such as fumaric acid esters, have been used as highly effective immunomodulators in patients with multiple sclerosis and yet their mechanism of action remains elusive. Since fumaric acid esters are metabolites, and cell metabolism is highly intertwined with the epigenetic regulation of gene expression, we investigated whether this metabolic-epigenetic interplay could be leveraged for therapeutic purposes. To this end we recruited 47 treatment-naïve and 35 fumaric acid ester-treated patients with multiple sclerosis, as well as 16 glatiramer acetate-treated patients as a non-metabolite treatment control. Here we identify a significant immunomodulatory effect of fumaric acid esters on the expression of the brain-homing chemokine receptor CCR6 in CD4 and CD8 T cells of patients with multiple sclerosis, which include T helper-17 and T cytotoxic-17 cells. We report differences in DNA methylation of CD4 T cells isolated from untreated and treated patients with multiple sclerosis, using the Illumina EPIC 850K BeadChip. We first demonstrate that Krebs cycle intermediates, such as fumaric acid esters, have a significantly higher impact on epigenome-wide DNA methylation changes in CD4 T cells compared to amino-acid polymers such as glatiramer acetate. We then define a fumaric acid ester treatment-specific hypermethylation effect on microRNA MIR-21, which is critical for the differentiation of T helper-17 cells. This hypermethylation effect was attributed to the subpopulation of T helper-17 cells using a decomposition analysis and was further validated in an independent prospective cohort of seven patients before and after treatment with fumaric acid esters. In vitro treatment of CD4 and CD8 T cells with fumaric acid esters supported a direct and dose-dependent effect on DNA methylation at the MIR-21 promoter. Finally, the upregulation of miR-21 transcripts and CCR6 expression was inhibited if CD4 or CD8 T cells stimulated under T helper-17 or T cytotoxic-17 polarizing conditions were treated with fumaric acid esters in vitro. These data collectively define a direct link between fumaric acid ester treatment and hypermethylation of the MIR-21 locus in both CD4 and CD8 T cells and suggest that the immunomodulatory effect of fumaric acid esters in multiple sclerosis is at least in part due to the epigenetic regulation of the brain-homing CCR6+ CD4 and CD8 T cells.

Disease-modifying therapies alter gut microbial composition in MS.

Objective: To determine the effects of the disease-modifying therapies, glatiramer acetate (GA) and dimethyl fumarate (DMF), on the gut microbiota in patients with MS. Methods: Participants with relapsing MS who were either treatment-naive or treated with GA or DMF were recruited. Peripheral blood mononuclear cells were immunophenotyped. Bacterial DNA was extracted from stool, and amplicons targeting the V4 region of the bacterial/archaeal 16S rRNA gene were sequenced (Illumina MiSeq). Raw reads were clustered into Operational Taxonomic Units using the GreenGenes database. Differential abundance analysis was performed using linear discriminant analysis effect size. Phylogenetic investigation of communities by reconstruction of unobserved states was used to investigate changes to functional pathways resulting from differential taxon abundance. Results: One hundred sixty-eight participants were included (treatment-naive n = 75, DMF n = 33, and GA n = 60). Disease-modifying therapies were associated with changes in the fecal microbiota composition. Both therapies were associated with decreased relative abundance of the Lachnospiraceae and Veillonellaceae families. In addition, DMF was associated with decreased relative abundance of the phyla Firmicutes and Fusobacteria and the order Clostridiales and an increase in the phylum Bacteroidetes. Despite the different changes in bacterial taxa, there was an overlap between functional pathways affected by both therapies. Interpretation: Administration of GA or DMF is associated with differences in gut microbial composition in patients with MS. Because those changes affect critical metabolic pathways, we hypothesize that our findings may highlight mechanisms of pathophysiology and potential therapeutic intervention requiring further investigation.

Education Research: Resident education through adult learning in neurology: Implementation and impact.

OBJECTIVE: To enhance residency education by implementing the 6 principles of adult learning theory (ALT) in a large academic neurology residency program. METHODS: We implemented a set of curricular interventions aimed at Resident Education through Adult Learning in Neurology (REAL Neurology), in a large, academic neurology residency program. Interventions included didactic reform, increasing resident-as-teacher activities, and enhancing residents' interaction. The primary outcome was the change in mean Residency In-service Training Examination (RITE) percentile between the preintervention and postintervention cohorts, adjusting for US Medical Licensing Examination step 1 and 2 score. Other analysis included evaluating the effect of the duration of intervention exposure on outcome and evaluating the intervention effect on the proportion of advanced performers. RESULTS: A total of 134 RITE score reports were evaluated (87 preintervention and 47 postintervention). The mean RITE score percentile postintervention was 11.7 points higher than preintervention (adjusted, longitudinal analysis: fit linear mixed model, p < 0.0001). Postgraduate year 3 learners who had 1 and 2 years of exposure scored 13.4 and 18.9 points higher than those with no exposure at all, respectively (analysis of covariance, p = 0.04). The adjusted odds of better performance with REAL Neurology was 5.77 (ordinal logistic regression, 95% confidence interval 2.37-14.07, p < 0.05). CONCLUSION: This study evaluated the efficacy and feasibility of an ALT-based curricular program in neurology education. The results show robust and sustainable benefit for residents in training without imposing a financial or logistical burden on programs. REAL Neurology could serve as a model for curricular reform in other programs across subspecialties.

The Microbiome-Gut-Behavior Axis: Crosstalk Between the Gut Microbiome and Oligodendrocytes Modulates Behavioral Responses.

Environmental and dietary stimuli have always been implicated in brain development and behavioral responses. The gut, being the major portal of communication with the external environment, has recently been brought to the forefront of this interaction with the establishment of a gut-brain axis in health and disease. Moreover, recent breakthroughs in germ-free and antibiotic-treated mice have demonstrated the significant impact of the microbiome in modulating behavioral responses in mice and have established a more specific microbiome-gut-behavior axis. One of the mechanisms by which this axis affects social behavior is by regulating myelination at the prefrontal cortex, an important site for complex cognitive behavior planning and decision-making. The prefrontal cortex exhibits late myelination of its axonal projections that could extend into the third decade of life in humans, which make it susceptible to external influences, such as microbial metabolites. Changes in the gut microbiome were shown to alter the composition of the microbial metabolome affecting highly permeable bioactive compounds, such as p-cresol, which could impair oligodendrocyte differentiation. Dysregulated myelination in the prefrontal cortex is then able to affect behavioral responses in mice, shifting them towards social isolation. The reduced social interactions could then limit microbial exchange, which could otherwise pose a threat to the survival of the existing microbial community in the host and, thus, provide an evolutionary advantage to the specific microbial community. In this review, we will analyze the microbiome-gut-behavior axis, describe the interactions between the gut microbiome and oligodendrocytes and highlight their role in the modulation of social behavior.

Diagnostic Criteria, Classification and Treatment Goals in Multiple Sclerosis: The Chronicles of Time and Space.

Multiple sclerosis (MS) is one of the most diverse human diseases. Since its first description by Charcot in the nineteenth century, the diagnostic criteria, clinical course classification, and treatment goals for MS have been constantly revised and updated to improve diagnostic accuracy, physician communication, and clinical trial design. These changes have improved the clinical outcomes and quality of life for patients with the disease. Recent technological and research breakthroughs will almost certainly further change how we diagnose, classify, and treat MS in the future. In this review, we summarize the key events in the history of MS, explain the reasoning behind the current criteria for MS diagnosis, classification, and treatment, and provide suggestions for further improvements that will keep enhancing the clinical practice of MS.

Safety and immunologic effects of high- vs low-dose cholecalciferol in multiple sclerosis.

OBJECTIVE: To study the safety profile and characterize the immunologic effects of high- vs low-dose cholecalciferol supplementation in patients with multiple sclerosis (MS). METHODS: In this double-blind, single-center randomized pilot study, 40 patients with relapsing-remitting MS were randomized to receive 10,400 IU or 800 IU cholecalciferol daily for 6 months. Assessments were performed at baseline and 3 and 6 months. RESULTS: Mean increase of 25-hydroxyvitamin D levels from baseline to final visit was larger in the high-dose group (34.9 ng/mL; 95% confidence interval [CI] 25.0-44.7 ng/mL) than in the low-dose group (6.9 ng/mL; 95% CI 1.0-13.7 ng/mL). Adverse events were minor and did not differ between the 2 groups. Two relapses occurred, one in each treatment arm. In the high-dose group, we found a reduction in the proportion of interleukin-17(+)CD4(+) T cells (p = 0.016), CD161(+)CD4(+) T cells (p = 0.03), and effector memory CD4(+) T cells (p = 0.021) with a concomitant increase in the proportion of central memory CD4(+) T cells (p = 0.018) and naive CD4(+) T cells (p = 0.04). These effects were not observed in the low-dose group. CONCLUSIONS: Cholecalciferol supplementation with 10,400 IU daily is safe and tolerable in patients with MS and exhibits in vivo pleiotropic immunomodulatory effects in MS, which include reduction of interleukin-17 production by CD4(+) T cells and decreased proportion of effector memory CD4(+) T cells with concomitant increase in central memory CD4(+) T cells and naive CD4(+) T cells. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that cholecalciferol supplementation with 10,400 IU daily is safe and well-tolerated in patients with MS and exhibits in vivo pleiotropic immunomodulatory effects.

Bromodomains: Translating the words of lysine acetylation into myelin injury and repair.

Bromodomains are evolutionarily highly conserved α-helical structural motifs that recognize and bind acetylated lysine residues. Lysine acetylation is being increasingly recognized as a major posttranslational modification involved in diverse cellular processes and protein interactions and its deregulation has been implicated in the pathophysiology of various human diseases, such as multiple sclerosis and cancer. Bromodomain-containing proteins can have a wide variety of functions, ranging from histone acetyltransferase activity and chromatin remodeling to transcriptional mediation and co-activation. The role of bromodomains in translating a deregulated cell acetylome into disease phenotypes was recently unveiled by the development of small molecule bromodomain inhibitors. This breakthrough discovery highlighted bromodomain-containing proteins as key players of inflammatory pathways responsible for myelin injury and also demonstrated their role in several aspects of myelin repair including oligodendrocyte differentiation and axonal regeneration.

FTY720 impairs CD8 T-cell function independently of the sphingosine-1-phosphate pathway.

Fingolimod (FTY720) is a multiple sclerosis (MS) therapeutic that upon phosphorylation causes the internalization of sphingosine-1-phosphate receptors (S1PR) and traps CCR7+ T-cells in lymph nodes but relatively spares CCR7-effector T-cells. Nonetheless, FTY720-treated patients are more susceptible to viral infections, indicating a CD8 T-cell defect. Thus, the effects of FTY720 on CD8 T-cells were investigated. To this end, we utilized experimental autoimmune encephalomyelitis (EAE) and a murine influenza model. CD8 T-cell trafficking, IFNγ and Granzyme B (GrB) production were assessed by flow cytometry. CD8 T-cell cytotoxic function was assessed in vitro by an LDH release assay. FTY720 not only ameliorated EAE by sequestering T-cells, but also reduced IFNγ and Granzyme B (GrB) in splenic CD8 T-cells. Murine influenza infection was exacerbated and mortality was increased, as FTY720 inhibited CD8 T-cell GrB production and lung infiltration. Remarkably, only the unphosphorylated compound was able to reduce IFNγ and GrB levels in CD8 T-cells and inhibits their cytotoxic function in vitro. The phosphorylated moiety had no effect in vitro, indicating that CD8 T-cell suppression by FTY720 is independent of S1PR modulation. The addition of arachidonic acid rescued CD8 T-cell function, suggesting that this effect may be mediated via inhibition of cytosolic phospholipase A2. Herein, we demonstrate that FTY720 suppresses CD8 T-cells independently of its trafficking effects and S1PR modulation. This provides a novel explanation not only for the increased rate of viral infections in FTY720-treated patients, but also for its efficacy in MS, as CD8 T-cells have emerged as crucial mediators of MS pathogenesis.

Helper T cells down-regulate CD4 expression upon chronic stimulation giving rise to double-negative T cells.

Double-negative T (DNT) cells are αßTCR(+)CD3(+)CD4(-)CD8(-)NK1.1(-) cells that constitute a small but significant proportion of the αßTCR(+) T cells. Their developmental pathway and pathological significance remain unclear. In the present study, we utilized chronic in vitro stimulation of CD4(+) T cells to mimic immune hyper-activation of autoimmune lymphoproliferative syndrome and systemic lupus erythematosus, conditions characterized by DNT cells accumulation. After approximately 4-5 rounds of stimulation, the CD3(+)CD4(-) population became apparent. These cells did not express CD8, NK1.1, γδTCR, or B220, exhibited a highly proliferative effector phenotype, and were dependent on T cell receptor (TCR) stimulation for survival. Moreover, CD3(+)CD4(-) cells expressed MHC class II-restricted αßTCR, indicative of their origin from a CD4(+) T cell population. The results presented herein illustrate a novel method of DNT cell generation in vitro and suggest that immune hyper-activation could also be implicated in the genesis of the disease-associated DNT cells in vivo.

Expression of CCR7 and CD45RA in CD4+ and CD8+ subsets in cerebrospinal fluid of 134 patients with inflammatory and non-inflammatory neurological diseases.

We investigated CD45RA and CCR7 expression in CD4+ and CD8+ subsets of cerebrospinal fluid (CSF) lymphocytes, both immediately ex vivo and after stimulation, from 134 patients with a variety of inflammatory and non-inflammatory neurological diseases. Most inflammatory diseases had a higher CD4+:CD8+ ratio and higher percentage of effector memory T cells (T(EM)) than non-inflammatory controls, excluding active infection. Moreover, we found that patients with highly elevated cell counts in the CSF tended to have a lower percentage of central memory T cells (T(CM)) than patients with low or absent pleocytosis, with a concomitant increase in T(EM). We also found that samples with elevated IgG index or presence of oligoclonal bands had a significantly higher CD4+:CD8+ ratio than normal samples, consistent with increased CD4+ help for intrathecal IgG synthesis by B cells.