Fingolimod Increases CD39-Expressing Regulatory T Cells in Multiple Sclerosis Patients

Background

Multiple sclerosis (MS) likely results from an imbalance between regulatory and inflammatory immune processes. CD39 is an ectoenzyme that cleaves ATP to AMP and has been suggested as a novel regulatory T cells (Treg) marker. As ATP has numerous proinflammatory effects, its degradation by CD39 has anti-inflammatory influence. The purpose of this study was to explore regulatory and inflammatory mechanisms activated in fingolimod treated MS patients.

Methods and Findings

Peripheral blood mononuclear cells (PBMCs) were isolated from relapsing-remitting MS patients before starting fingolimod and three months after therapy start. mRNA expression was assessed in ex vivo PBMCs. The proportions of CD8, B cells, CD4 and CD39-expressing cells were analysed by flow cytometry. Treg proportion was quantified by flow cytometry and methylation-specific qPCR. Fingolimod treatment increased mRNA levels of CD39, AHR and CYP1B1 but decreased mRNA expression of IL-17, IL-22 and FOXP3 mRNA in PBMCs. B cells, CD4⁺ cells and Treg proportions were significantly reduced by this treatment, but remaining CD4⁺ T cells were enriched in FOXP3⁺ cells and in CD39-expressing Tregs.

Conclusions

In addition to the decrease in circulating CD4⁺ T cells and CD19⁺ B cells, our findings highlight additional immunoregulatory mechanisms induced by fingolimod.     

Oligoclonal Band Status in Scandinavian Multiple Sclerosis Patients Is Associated with Specific Genetic Risk Alleles

The presence of oligoclonal bands (OCB) in cerebrospinal fluid (CSF) is a typical finding in multiple sclerosis (MS). We applied data from Norwegian, Swedish and Danish (i.e. Scandinavian) MS patients from a genome-wide association study (GWAS) to search for genetic differences in MS relating to OCB status. GWAS data was compared in 1367 OCB positive and 161 OCB negative Scandinavian MS patients, and nine of the most associated SNPs were genotyped for replication in 3403 Scandinavian MS patients. HLA-DRB1 genotypes were analyzed in a subset of the OCB positive (n = 2781) and OCB negative (n = 292) MS patients and compared to 890 healthy controls. Results from the genome-wide analyses showed that single nucleotide polymorphisms (SNPs) from the HLA complex and six other loci were associated to OCB status. In SNPs selected for replication, combined analyses showed genome-wide significant association for two SNPs in the HLA complex; rs3129871 (p = 5.7×10⁻¹⁵) and rs3817963 (p = 5.7×10⁻¹⁰) correlating with the HLA-DRB1*15 and the HLA-DRB1*04 alleles, respectively. We also found suggestive association to one SNP in the Calsyntenin-2 gene (p = 8.83×10⁻⁷). In HLA-DRB1 analyses HLA-DRB1*15∶01 was a stronger risk factor for OCB positive than OCB negative MS, whereas HLA-DRB1*04∶04 was associated with increased risk of OCB negative MS and reduced risk of OCB positive MS. Protective effects of HLA-DRB1*01∶01 and HLA-DRB1*07∶01 were detected in both groups. The groups were different with regard to age at onset (AAO), MS outcome measures and gender. This study confirms both shared and distinct genetic risk for MS subtypes in the Scandinavian population defined by OCB status and indicates different clinical characteristics between the groups. This suggests differences in disease mechanisms between OCB negative and OCB positive MS with implications for patient management, which need to be further studied.     

Kinetics of IL-6 Production Defines T Effector Cell Responsiveness to Regulatory T Cells in Multiple Sclerosis

In multiple sclerosis (MS) autoaggressive T effector cells (Teff) are not efficiently controlled by regulatory T cells (Treg) but the underlying mechanisms are incompletely understood. Proinflammatory cytokines are key factors facilitating Teff activity in chronic inflammation. Here we investigated the influence of IL-6 on Treg sensitivity of Teff from therapy-naïve MS patients with or without active disease. Compared to healthy volunteers and independent of disease course CD4⁺ and especially CD8⁺ MS-Teff were insensitive against functional active Treg from healthy controls. This unresponsiveness was caused by accelerated production of IL-6, elevated IL-6 receptor expression and phosphorylation of protein kinase B (PKB)/c-Akt in MS-Teff. In a positive feedback loop, IL-6 itself induced its accelerated synthesis and enhanced phosphorylation of PKB/c-Akt that finally mediated Treg resistance. Furthermore, accelerated IL-6 release especially by CD8⁺ Teff prevented control of surrounding Teff, described here as “bystander resistance”. Blockade of IL-6 receptor signaling or direct inhibition of PKB/c-Akt phosphorylation restored Treg responsiveness of Teff and prevented bystander resistance. In Teff of healthy controls (HC) exogenous IL-6 also changed the kinetics of IL-6 production and induced Treg unresponsiveness. This modulation was only transient in Teff from healthy volunteers, whereas accelerated IL-6 production in MS-Teff maintained also in absence of IL-6. Hence, we showed that the kinetics of IL-6 production instead of elevated IL-6 levels defines the Teff responsiveness in early Treg-T cell communication in MS independent of their disease course and propose IL-6 and associated PKB/c-Akt activation as effective therapeutic targets for modulation of Teff activity in MS.     

Peripheral Blood T Cell Dynamics Predict Relapse in Multiple Sclerosis Patients on Fingolimod

Background

Fingolimod efficiently reduces multiple sclerosis (MS) relapse by inhibiting lymphocyte egress from lymph nodes through down-modulation of sphingosine 1-phosphate (S1P) receptors. We aimed to clarify the alterations in peripheral blood T cell subsets associated with MS relapse on fingolimod.

Methods/Principal Findings

Blood samples successively collected from 23 relapsing-remitting MS patients before and during fingolimod therapy (0.5 mg/day) for 12 months and 18 healthy controls (HCs) were analysed for T cell subsets by flow cytometry. In MS patients, the percentages of central memory T (CCR7⁺CD45RO⁺) cells (TCM) and naïve T (CCR7⁺CD45RO^-) cells decreased significantly, while those of effector memory T (CCR7^-CD45RA^-) and suppressor precursor T (CD28^-) cells increased in both CD4⁺T and CD8⁺T cells from 2 weeks to 12 months during fingolimod therapy. The percentages of regulatory T (CD4⁺CD25^highCD127^low) cells in CD4⁺T cells and CCR7^-CD45RA⁺T cells in CD8⁺T cells also increased significantly. Eight relapsed patients demonstrated greater percentages of CD4⁺TCM than 15 non-relapsed patients at 3 and 6 months (p=0.0051 and p=0.0088, respectively). The IL17-, IL9-, and IL4-producing CD4⁺T cell percentages were significantly higher at pre-treatment in MS patients compared with HCs (p<0.01 for all), while the IL17-producing CD4⁺T cell percentages tended to show a transient increase at 2 weeks of fingolimod therapy (p^corr=0.0834).

Conclusions

The CD4⁺TCM percentages at 2 weeks to 12 months during fingolimod therapy are related to relapse.     

Human Regulatory T Cell Suppressive Function Is Independent of Apoptosis Induction in Activated Effector T Cells

Background

CD4⁺CD25⁺FOXP3⁺ Regulatory T cells (Treg) play a central role in the immune balance to prevent autoimmune disease. One outstanding question is how Tregs suppress effector immune responses in human. Experiments in mice demonstrated that Treg restrict effector T cell (Teff) responses by deprivation of the growth factor IL-2 through Treg consumption, resulting in apoptosis of Teff.

Principal Findings

In this study we investigated the relevance of Teff apoptosis induction to human Treg function. To this end, we studied naturally occurring Treg (nTreg) from peripheral blood of healthy donors, and, to investigate Treg function in inflammation in vivo, Treg from synovial fluid of Juvenile Idiopathic Arthritis (JIA) patients (SF-Treg). Both nTreg and SF-Treg suppress Teff proliferation and cytokine production efficiently as predicted. However, in contrast with murine Treg, neither nTreg nor SF-Treg induce apoptosis in Teff. Furthermore, exogenously supplied IL-2 and IL-7 reverse suppression, but do not influence apoptosis of Teff.

Significance

Our functional data here support that Treg are excellent clinical targets to counteract autoimmune diseases. For optimal functional outcome in human clinical trials, future work should focus on the ability of Treg to suppress proliferation and cytokine production of Teff, rather than induction of Teff apoptosis.     

The Impact of Vitamin D on Dendritic Cell Function in Patients with Systemic Lupus Erythematosus

Background

Excessive activity of dendritic cells (DCs) is postulated as a central disease mechanism in Systemic Lupus Erythematosus (SLE). Vitamin D is known to reduce responsiveness of healthy donor DCs to the stimulatory effects of Type I IFN. As vitamin D deficiency is reportedly common in SLE, we hypothesized that vitamin D might play a regulatory role in the IFNα amplification loop in SLE. Our goals were to investigate the relationship between vitamin D levels and disease activity in SLE patients and to investigate the effects of vitamin D on DC activation and expression of IFNα-regulated genes in vitro.

Methodology/Principal Findings

In this study, 25-OH vitamin D (25-D) levels were measured in 198 consecutively recruited SLE patients. Respectively, 29.3% and 11.8% of African American and Hispanic SLE patient had 25-D levels <10 ng/ml. The degree of vitamin D deficiency correlated inversely with disease activity; R = −.234, p = .002. In 19 SLE patients stratified by 25-D levels, there were no differences between circulating DC number and phenotype. Monocyte-derived DCs (MDDCs) of SLE patients were normally responsive to the regulatory effects of vitamin D in vitro as evidenced by decreased activation in response to LPS stimulation in the presence of 1,25-D. Additionally, vitamin D conditioning reduced expression of IFNα-regulated genes by healthy donor and SLE MDDCs in response to factors in activating SLE plasma.

Conclusions/Significance

We report on severe 25-D deficiency in a substantial percentage of SLE patients tested and demonstrate an inverse correlation with disease activity. Our results suggest that vitamin D supplementation will contribute to restoring immune homeostasis in SLE patients through its inhibitory effects on DC maturation and activation. We are encouraged to support the importance of adequate vitamin D supplementation and the need for a clinical trial to assess whether vitamin D supplementation affects IFNα activity in vivo and, most importantly, improves clinical outcome.     

Increased B Cell and Cytotoxic NK Cell Proportions and Increased T Cell Responsiveness in Blood of Natalizumab-Treated Multiple Sclerosis Patients

Background

Changes in the blood lymphocyte composition probably both mediate and reflect the effects of natalizumab treatment in multiple sclerosis, with implications for treatment benefits and risks.

Methods

A broad panel of markers for lymphocyte populations, including states of activation and co-stimulation, as well as functional T cell responses to recall antigens and mitogens, were assessed by flow cytometry in 40 patients with relapsing multiple sclerosis before and after one-year natalizumab treatment.

Results

Absolute numbers of all major lymphocyte populations increased after treatment, most markedly for NK and B cells. The fraction of both memory and presumed regulatory B cell subsets increased, as did CD3^-CD56^dim cytotoxic NK cells, whereas CD3^-CD56^bright regulatory NK cells decreased. The increase in cell numbers was further associated with a restored T cell responsiveness to recall antigens and mitogens in functional assays.

Conclusions

Our data confirms that natalizumab treatment increases the number of lymphocytes in blood, likely mirroring the expression of VLA-4 being highest on NK and B cells. This finding supports reduction of lymphocyte extravasation as a main mode of action, although the differential effects on subpopulation composition suggests that cell-signalling may also be affected. The systemic increase in T cell responsiveness reflects the increase in numbers, and while augmenting anti-infectious responses systemically, localized responses may become correspondingly decreased.     

Poor Sleep in Patients with Multiple Sclerosis

Background

Poor sleep is a frequent symptom in patients with multiple sclerosis (MS). Sleep may be influenced by MS-related symptoms and adverse effects from immunotherapy and symptomatic medications. We aimed to study the prevalence of poor sleep and the influence of socio-demographic and clinical factors on sleep quality in MS- patients.

Methods

A total of 90 MS patients and 108 sex-and age- matched controls were included in a questionnaire survey. Sleep complaints were evaluated by Pittsburgh Sleep Quality Index (PSQI) and a global PSQI score was used to separate good sleepers (≤5) from poor sleepers (>5). Excessive daytime sleepiness, the use of immunotherapy and antidepressant drugs, symptoms of pain, depression, fatigue and MS-specific health related quality of life were registered. Results were compared between patients and controls and between good and poor sleepers among MS patients.

Results

MS patients reported a higher mean global PSQI score than controls (8.6 vs. 6.3, p = 0.001), and 67.1% of the MS patients compared to 43.9% of the controls (p = 0.002) were poor sleepers. Pain (p = 0.02), fatigue (p = 0.001), depression (p = 0.01) and female gender (p = 0.04) were associated with sleep disturbance. Multivariate analyses showed that female gender (p = 0.02), use of immunotherapy (p = 005) and a high psychological burden of MS (p = 0.001) were associated with poor sleep among MS patients.

Conclusions

Poor sleep is common in patients with MS. Early identification and treatment of modifiable risk factors may improve sleep and quality of life in MS.     

Vitamin D and Risk of Multiple Sclerosis: A Mendelian Randomization Study

BackgroundObservational studies have demonstrated an association between decreased vitamin D level and risk of multiple sclerosis (MS); however, it remains unclear whether this relationship is causal. We undertook a Mendelian randomization (MR) study to evaluate whether genetically lowered vitamin D level influences the risk of MS.ConclusionsA genetically lowered 25OHD level is strongly associated with increased susceptibility to MS. Whether vitamin D sufficiency can delay, or prevent, MS onset merits further investigation in long-term randomized controlled trials.     

T Cell Vaccination Benefits Relapsing Progressive Multiple Sclerosis Patients: A Randomized,Double-Blind Clinical Trial

Background

T-cell vaccination (TCV) for multiple sclerosis (MS) refers to treatment with autologous anti-myelin T-cells, attenuated by irradiation. Previously published clinical trials have been all open-labeled.

Aim

To evaluate the safety and efficacy of TCV in progressive MS, in a double-blind, controlled clinical trial.

Methodology

Twenty-six patients with relapsing-progressive MS were enrolled in the study (mean age: 39±9.8 years; mean EDSS: 4.4±1.7). T-cell lines reactive to 9 different peptides of the myelin antigens, MBP, MOG and PLP were raised from the patients'' peripheral blood. The patients were randomized into two groups: 19 were treated with TCV (four subcutaneous injections of 10–30×10⁶ T-cells, attenuated by irradiation, on days 1, 30, 90 and 180) and 7 patients were treated with sham injections. Twenty-four patients (17 in the TCV group and 7 in the placebo) were eligible for per-protocol analysis.

Results

At one year following the inclusion, an increase in the EDSS (+0.50) and an increase in 10-meter walking time (+0.18 sec), were observed in the placebo group; in the TCV group there was a decrease in the EDSS (−0.44; p<0.01) and in the 10-meter walking time (0.84 sec; p<0.005). Sixteen of the 17 patients (94.1%) in the TCV group remained relapse-free during the year of the study, as compared to 42.9% in the placebo group (p = 0.01 and p = 0.03 with adjustment). The proportion of patients with any relapse during the year of the study in the TCV-group, was reduced by 89.6%., as compared to the placebo-treated group. MRI parameters did not change significantly.

Conclusions

This is the first controlled, double-blind trial with TCV in progressive MS. The results demonstrate the feasibility and safety of the procedure, and provide significant indications of clinical efficacy. Further studies with larger groups of subjects are warranted.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01448252","term_id":"NCT01448252"}}NCT01448252     

The Association of Vitamin D Receptor Polymorphisms with Multiple Sclerosis in a Case-Control Study from Kuwait

Vitamin D deficiency is associated with several diseases including multiple sclerosis (MS). Several factors influence vitamin D levels and its optimal multi-function maintenance. Our objective was to assess quantifiable variables influencing vitamin D level and metabolism in MS patients from Kuwait. In a case-control study involving 50 MS patients, and 50 healthy control individuals for which plasma vitamin D levels, supplement use, vitamin D receptor (VDR) variants, and skin pigmentation indices were ascertained; we found overall vitamin D levels to be deficient in both groups, and supplement use to be common practice. VDR variants TaqI and BsmI associated with MS risk, and ApaI associated with low disease progression. VDR variant FokI associated with higher vitamin D levels in both groups. We conclude that several quantifiable variables related to vitamin D associate with MS suggesting a possible clinical immuno-modulatory application of vitamin D for MS patients in Kuwait.     

Vitamin D Status Is an Independent Risk Factor for Global Cognitive Impairment in Peritoneal Dialysis Patients

Objective

Vitamin D (VD) deficiency is an independent risk factor for cognitive impairment (CI) in the general population, but VD status in peritoneal dialysis (PD) patients has not been investigated. In this study, we aimed to investigate the relationship between serum VD levels and global and specific cognitive functions in PD patients.

Design and Setting

Cross-sectional study, simultaneously conducted at two PD centers.

Patients

Clinically stable patients (n = 273) undergoing PD for at least 3 months were enrolled over a period of one year.

Main outcome Measures

Demographic and comorbidity data were recorded, and routine biochemical parameters and serum 25-hydroxyvitamin D (25(OH) D) levels of overnight fasted patients were determined. Global cognitive function was assessed by the Modified Mini-Mental State Examination (3MS) score; executive function, by the trail making tests (Trails A and B); and immediate memory, delayed memory, and language ability by the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) sub-tests.

Results

In the univariate analysis, serum 25(OH) D levels significantly correlated with 3MS scores (r = -0.139; P = 0.02), and Trail A (r = -0.188; P = 0.002) and B (r = -0.154; P = 0.01) completion times. In the multivariate analysis, 25(OH) D was found to be independently associated with global CI, but not with executive dysfunction. Serum 25(OH) D could not predict scores of immediate/delayed memory and language ability.

Conclusions

VD deficiency is highly prevalent in PD patients and is an independent risk factor for global CI in this patient cohort.     

个体化补充维生素D与多发性硬化疾病治疗的探讨

维生素D不仅在骨骼代谢及钙平衡中发挥着重要作用,而且具有免疫调节、抗炎及神经细胞保护功能等。这提示我们,VD在自身免疫性疾病如多发性硬化中可能发挥着重要的作用。流行病学调查及临床数据显示,低VD水平或VD代谢失调是诱发多发性硬化的危险因素之一。另有研究报道,VD血清浓度与多发性硬化的疾病活动和进展呈反相关。但是,这些数据并不是没有争议的,VD在多发性硬化治疗和预防中的作用还有着很多需要回答的问题。现有的以VD补充作为治疗MS措施的临床干预研究中得到的临床数据并不具备得出结论的能力,并且有些数据之间甚至是相互矛盾。     

EBNA2 Binds to Genomic Intervals Associated with Multiple Sclerosis and Overlaps with Vitamin D Receptor Occupancy

Epstein-Barr virus (EBV) is a non-heritable factor that associates with multiple sclerosis (MS). However its causal relationship with the disease is still unclear. The virus establishes a complex co-existence with the host that includes regulatory influences on gene expression. Hence, if EBV contributes to the pathogenesis of MS it may do so by interacting with disease predisposing genes. To verify this hypothesis we evaluated EBV nuclear antigen 2 (EBNA2, a protein that recent works by our and other groups have implicated in disease development) binding inside MS associated genomic intervals. We found that EBNA2 binding occurs within MS susceptibility sites more than expected by chance (factor of observed vs expected overlap [O/E] = 5.392-fold, p < 2.0e-05). This remains significant after controlling for multiple genomic confounders. We then asked whether this observation is significant per se or should also be viewed in the context of other disease relevant gene-environment interactions, such as those attributable to vitamin D. We therefore verified the overlap between EBNA2 genomic occupancy and vitamin D receptor (VDR) binding sites. EBNA2 shows a striking overlap with VDR binding sites (O/E = 96.16-fold, p < 2.0e-05), even after controlling for the chromatin accessibility state of shared regions (p <0.001). Furthermore, MS susceptibility regions are preferentially targeted by both EBNA2 and VDR than by EBNA2 alone (enrichment difference = 1.722-fold, p = 0.0267). Taken together, these findings demonstrate that EBV participates in the gene-environment interactions that predispose to MS.     

Glutathione Restricts Serine Metabolism to Preserve Regulatory T Cell Function

1. Download : Download high-res image (120KB)
2. Download : Download full-size image

     

Ischemic Preconditioning in the Liver Is Independent of Regulatory T Cell Activity

Ischemic preconditioning (IPC) protects organs from ischemia reperfusion injury (IRI) through unknown mechanisms. Effector T cell populations have been implicated in the pathogenesis of IRI, and T regulatory cells (Treg) have become a putative therapeutic target, with suggested involvement in IPC. We explored the role of Treg in hepatic IRI and IPC in detail. IPC significantly reduced injury following ischemia reperfusion insults. Treg were mobilized rapidly to the circulation and liver after IRI, but IPC did not further increase Treg numbers, nor was it associated with modulation of circulating pro-inflammatory chemokine or cytokine profiles. We used two techniques to deplete Treg from mice prior to IRI. Neither Treg depleted FoxP3.LuciDTR mice, nor wildtyoe mice depleted of Tregs with PC61, were more susceptible to IRI compared with controls. Despite successful enrichment of Treg in the liver, by adoptive transfer of both iTreg and nTreg or by in vivo expansion of Treg with IL-2/anti-IL-2 complexes, no protection against IRI was observed.We have explored the role of Treg in IRI and IPC using a variety of techniques to deplete and enrich them within both the liver and systemically. This work represents an important negative finding that Treg are not implicated in IPC and are unlikely to have translational potential in hepatic IRI.     

Polymorphonuclear Cell Functional Impairment in Relapsing Remitting Multiple Sclerosis Patients: Preliminary Data

Multiple Sclerosis patients run an increased risk of microbial infections, which leads to high rates of hospitalization and infection-related mortality. Although immunotherapy may increase infection risk in some cases, data as to the relationship among microbial factors, immunotherapy and alterations in the innate immunity of these patients are still scanty. On these grounds, this interdisciplinary study aims at investigating the role the functional activity of polymorphonuclear cells (PMNs) play in relapsing remitting multiple sclerosis at different stages. The in vitro ability of PMNs from patients, either untreated or treated with immunosuppressant or immunomodulatory drugs to kill Klebsiella pneumonia or Candida albicans, were investigated and compared to PMNs from healthy subjects. The release of various cytokines was also assessed, as was the production of reactive oxygen species and their ability to regulate apoptosis after microbial stimulation. Our results indicate that although patients have a normal number of PMNs, they have a statistically significant (p<0.05) reduction in intracellular killing activity. Although variations are strongly related to the therapeutic management of patients, they are independent from their disease stage. As no statistically significant differences were observed between patients and controls in cytokine release values, reactive oxygen species production or apoptosis, we came to the conclusion that other factors may be involved. Supportive validation of these results from further studies might well help in identifying a subset of patients at high risk of infection who could benefit from a closer follow-up and/or antibiotic prophylaxis.     

Role of Vitamin D in the Onset,Progression, and Severity of Multiple Sclerosis

ObjectiveTo review and assess the role of vitamin D in the onset, progression, and relapse of multiple sclerosis (MS), based on evidence acquired from the analysis of preclinical, observational, and interventional studies.MethodsAll English language literature in MEDLINE (January 1969 through April 2012) was searched for observational and interventional studies on the dosage effect of vitamin D on the onset, progression, and relapse rate of MS. The medical subject heading (MeSH) terms used in the search included Vitamin D and Multiple Sclerosis. Additional publications and abstracts were identified from review articles and from the references cited in the previously found articles. In addition to the experimental studies, only those human studies that specified the population size, doses of vitamin D used, and the resulting effect on MS were considered.ResultsVitamin D deficiency is very common among MS patients. Multiple preclinical studies have shown that vitamin D is a potent regulator of inflammation in MS. Most observational studies support an association between high vitamin D levels and a reduced risk of developing MS. However, conflicting results have been reported by observational studies on the correlation between vitamin D and MS severity and by interventional studies using vitamin D as a therapeutic agent for MS.ConclusionVitamin D deficiency in MS patients should be avoided. In addition, the risk of developing MS might be reduced by maintaining optimal vitamin D levels in the healthy population. Larger randomized interventional trials are needed to clarify the therapeutic effect of vitamin D in MS. (Endocr Pract. 2013;19:129-136)