Anne-Hilde Muris

Effects of vitamin D on the peripheral adaptive immune system: A review

Epidemiological studies have shown that a poor vitamin D status is associated with an increased risk of several diseases, including autoimmune diseases. The immune regulatory function of vitamin D is thought to have an important role in these associations. Cells of the adaptive immune system have shown to be direct targets of the vitamin D metabolites. Besides being direct targets, cells of the adaptive immune system express the enzymes involved in the metabolism of vitamin D, enabling them to locally convert 25(OH)D into its active metabolite 1,25(OH)2D. In this review, the effects of vitamin D on cells of the adaptive immune system are described. Experimental data in vitro show that vitamin D skews cells of the adaptive immune system toward a more tolerogenic status which might be exploited in the treatment of autoimmune diseases. However, it should be noticed that in vivo effects may differ from in vitro effects due to the cross-talk between different vitamin D sensitive cells, but data support the view that vitamin D is positively involved in maintaining or restoring immune homeostasis. Upcoming vitamin D supplementation trials will further elucidate the in vivo effects of vitamin D on the immune system and its potency to serve as an immune regulating agent in autoimmune diseases.

Vitamin D effects on B cell function in autoimmunity.

Vitamin D seems to be implicated in the pathophysiology of autoimmune disorders as a natural immune modulator. Beneficial effects of vitamin D have been associated with different cells of the immune system; however, thus far, B cells seem to be somewhat neglected. In this paper, we describe the possible direct effects of vitamin D on B cells, with a focus on antibody production and the more recently identified regulatory B (Breg) cells. B cells upregulate the vitamin D receptor (VDR) upon activation. Furthermore, due to regulated expression of the metabolizing enzymes CYP27B1 and CYP24A1, B cells have the potential to control the local availability of active vitamin D. B cells, therefore, may participate in vitamin D–mediated immune homeostasis, including plasma cell generation. Whether or not other B cell subsets, such as Breg cells, are equally responsive to vitamin D remains to be established.

Illuminating vitamin D effects on B cells – the multiple sclerosis perspective

Vitamin D is associated with many immune‐mediated disorders. In multiple sclerosis (MS) a poor vitamin D status is a major environmental factor associated with disease incidence and severity. The inflammation in MS is primarily T‐cell‐mediated, but increasing evidence points to an important role for B cells. This has paved the way for investigating vitamin D effects on B cells. In this review we elaborate on vitamin D interactions with antibody production, T‐cell‐stimulating capacity and regulatory B cells. Although in vitro plasma cell generation and expression of co‐stimulatory molecules are inhibited and the function of regulatory B cells is promoted, this is not supported by in vivo data. We speculate that differences might be explained by the B‐cell–Epstein–Barr virus interaction in MS, the exquisite role of germinal centres in B‐cell biology, and/or in vivo interactions with other hormones and vitamins that interfere with the vitamin D pathways. Further research is warranted to illuminate this tube‐versus‐body paradox.

A low vitamin D status at diagnosis is associated with an early conversion to secondary progressive multiple sclerosis

Low circulating 25-hydroxyvitamin D (25(OH)D) levels have been associated with an increased risk of relapses in relapsing remitting multiple sclerosis (RRMS), but an association with disability progression is uncertain. Lower 25(OH)D levels are found in secondary progressive MS (SPMS) when compared to RRMS. We hypothesized that a poor vitamin D status in RRMS is associated with an increased risk of conversion to SPMS. In a retrospective longitudinal study we measured 25(OH)D levels at the start of a 3-year follow-up, and analyzed whether these levels predict the risk of RRMS to SPMS conversion. In 338 RRMS patients, vitamin D status did not predict the 3-year risk of conversion to SPMS (n=51; OR 0.970; p=0.65). However, in diagnostic blood samples of SPMS patients with a relatively short RRMS duration (n=19) 25(OH)D levels were significantly lower (38nmol/L; Q1-Q3: 24-50) than in diagnostic samples of matched RRMS patients with no progression to SPMS ((n=38; 55nmol/L; Q1-Q3: 40-70) (p<0.01). These data indicate an association between a low vitamin D status at the start of RRMS and the early conversion to SPMS. Therefore, time to SPMS conversion is of interest as clinical measure in (follow-up of) clinical vitamin D supplementation studies.

Fraction of IL-10 + and IL-17 + CD8 T cells is increased in MS patients in remission and during a relapse, but is not influenced by immune modulators

In the present study, circulating proportions of CD8(+) T (Tc) cell subsets, including IL-17 (Tc17) and IL-10 (Tc10) producing cells, were assessed in relapsing-remitting MS (RRMS) patients and a possible effect of beta interferon (IFN-β), glatiramer acetate (GA), and vitamin D (VitD) on these cell subsets was investigated. We show that both Tc17 and Tc10 cell fractions are elevated in the circulation of RRMS patients in remission compared to healthy subjects and that these Tc subsets remain unaffected by current immune modulating regimens.

Vitamin D Status Does Not Affect Disability Progression of Patients with Multiple Sclerosis over Three Year Follow-Up.

Background and Objective The risk of developing multiple sclerosis (MS) as well as MS disease activity is associated with vitamin D (25(OH)D) status. The relationship between the main functional disability hallmark of MS, disability progression, and 25(OH)D status is less well established though, especially not in MS patients with progressive disease. Methods This retrospective follow-up study included 554 MS patients with a serum baseline 25(OH)D level and Expanded Disability Status Scale (EDSS) with a minimum follow-up of three years. Logistic regressions were performed to assess the effect of baseline 25(OH)D status on relapse rate. Repeated measures linear regression analyses were performed to assess the effect on disability and disability progression. Results Baseline deseasonalized 25(OH)D status was associated with subsequent relapse risk (yes/no), but only in the younger MS patients (≤ 37.5 years; OR = 0.872, per 10 nmol/L 25(OH)D, p = 0.041). Baseline 25(OH)D status was not significantly associated with either disability or disability progression, irrespective of MS phenotype. Conclusion Within the physiological range, 25(OH)D status is just significantly associated with the occurrence of relapses in younger MS patients, but is not associated with disability or disability progression over three years follow-up. Whether high dose supplementation to supra physiological 25(OH)D levels prevents disability progression in MS should become clear from long term follow-up of supplementation studies.

Immune regulatory effects of high dose vitamin D3 supplementation in a randomized controlled trial in relapsing remitting multiple sclerosis patients receiving IFNβ; the SOLARIUM study

Multiple sclerosis (MS) is characterized by a disturbed immune homeostasis and low serum vitamin D levels are associated with an increased disease activity. While vitamin D has been hypothesized to promote the maintenance of immune homeostasis, vitamin D supplementation could be of benefit to patients with MS. The SOLAR study investigated the effects of high dose vitamin D3 supplementation on clinical outcomes in a randomized controlled trial. Here we present the immune regulatory effects, investigated in the SOLARIUM sub-study. Thirty Dutch relapsing remitting (RR) MS patients treated with IFNβ-1a received high dose vitamin D3 supplementation and 23 patients received placebo during a period of 48weeks. Lymphocytes were phenotypically characterized by flow cytometry and in vitro cytokine secretion was assessed in the presence or absence of 1,25(OH)2D3 using Luminex technology. Changes in immune regulatory parameters were determined within subjects as well as between treatment groups. The proportion of cells in the immune regulatory cell compartment (nTreg, iTreg and Breg) was not altered upon high dose vitamin D3 supplementation. Proportions of T helper subsets were not affected by vitamin D3, except for the proportion of IL4+ Th cells, which decreased in the placebo but not in the vitamin D3 group. T cell cytokine secretion increased, most pronounced for IL5 and latency activated protein of TGFβ, in the placebo group but not in the vitamin D3 group. Lymphocytes remained equally reactive to in vitro 1,25(OH)2D3. In conclusion, high dose vitamin D3 supplementation did not result in a relative increase in lymphocytes with a regulatory phenotype. However, this study supports the hypothesis that vitamin D contributes to the maintenance of immune homeostasis by preventing further disturbance of the T cell compartment early in the disease course of MS.

Intracellular IL-10 detection in T cells by flowcytometry: the use of protein transport inhibitors revisited.

In the past two decades, interleukin-10 (IL-10) has gained much attention as an important regulatory cytokine involved in self-tolerance. Functional assessment of IL-10 producing immune cells is traditionally done by stimulation and measurement of cytokine production by flowcytometry. Thereby a protein transport inhibitor like monensin is used to accumulate the cytokine of interest intracellularly. In this study we elaborated on the monensin effect on cytokine detection and focused on IL-10 detection in human T cells. Peripheral blood mononuclear cells (PBMC) of 32 study subjects were isolated and stimulated with PMA/ionomycin, in the absence and presence of monensin, and stained intracellularly for IFN-γ, IL-4, IL-10 and IL-17A. Our results re-established that detection of IFN-γ+ and IL-4+ T cells benefited from the presence of monensin during stimulation. However, stimulation in the presence of monensin yielded lower proportions of IL-10+ T cells (0.45% (0.28-0.80) versus 0.80% (0.50-1.50) of CD4+ T cells, p<0.01), although monensin addition did result in an increased MFI (2431 (1273-4959) versus 1928 (1147-3760), p<0.01). Detectable fractions of IL-17A+ CD4+ T cells were not affected by monensin. A shorter incubation time, but not lower monensin concentrations, was effective in improving the detection of IL-10+ T cells. We found a strong correlation between the fraction of IL-10+ CD4+ T cells in the presence and absence of monensin (R=0.80 p<0.01). Next to this, also the detection of IL-10+ NK-T cells and IL-10+ monocytes, but not IL-10+ B cells, is impaired in the presence of monensin. This study shows that the effect of monensin on cytokine accumulation is time and cytokine dependent. Due to the use of monensin, previous research may have underestimated the number of IL-10+ leukocytes or may even have not been able to detect them at all. It is important to consider this for future research or when interpreting historical IL-10 data.

GM-CSF production by CD4+ T cells in MS patients: Regulation by regulatory T cells and vitamin D

BACKGROUND/OBJECTIVE Data from animal models of MS suggest that GM-CSF(+)CD4(+)T cells are pathogenic cells. Therefore, GM-CSF production by CD4(+)T cells of MS patients and their susceptibility to regulatory mechanisms were investigated. METHODS Intracellular flowcytometry was performed to determine the GM-CSF(+)CD4(+)T cell fraction in PBMC and CSF of MS patients and controls. The effect of regulatory T cells (Tregs) on GM-CSF production by CD4(+)T cells was studied in MS patients using a proliferation-suppression assay. Finally, GM-CSF(+)CD4(+)T cell fraction and GM-CSF protein levels in supernatant were assessed in anti-CD3-stimulated CD4(+)T cell cultures derived from healthy controls and MS patients, in the presence or absence of the active vitamin D metabolite calcitriol. RESULTS The GM-CSF(+)CD4(+)T cell fraction in the peripheral blood did not differ between controls and MS patients. This T cell population could also be detected in the CSF of both subjects with MS as well as subjects with another diagnosis. In the CSF, it comprised a significant fraction of the T cell population. Upon in vitro stimulation of PBMC with anti-CD3 antibody, no differences were observed in GM-CSF(+)CD4(+)T cell frequencies. GM-CSF secretion was susceptible to regulation by Treg and vitamin D. Suppression of GM-CSF secretion by vitamin D was reduced in MS patients. CONCLUSIONS Our study showed no elevation in GM-CSF(+)CD4(+)T cell fractions in MS patients compared to controls. Furthermore, GM-CSF secretion was prone to regulation by Treg and vitamin D, the latter being less effective in MS patients.

Reduced thymic expression of ErbB receptors without auto-antibodies against synaptic ErbB in myasthenia gravis

In myasthenia gravis (MG), the neuromuscular transmission is impaired mainly by auto-antibodies against the acetylcholine receptor (AChR) or MuSK. In about 5% of the MG patients, however, the auto-antigen is still unknown. We investigated whether these idiopathic MG patients (iMG) have auto-antibodies against ErbB proteins, which influence the AChR density at the NMJ. Our results show reduced mRNA expression levels of ErbB4 in thymus tissue of iMG patients compared to AChR-MG and non-MG patients, but we could not detect anti-ErbB antibodies in sera of iMG patients. Therefore, our results do not support a role for ErbB receptors as auto-antigens in iMG patients.