Predominance of Th2 polarization by Vitamin D through a STAT6-dependent mechanism

<p>Abstract</p> <p>Background</p> <p>Vitamin D has several reported immunomodulatory properties including the reduced generation of pro-inflammatory CD4+ T helper 1 (Th1) cells and the increase in levels of the anti-inflammatory Th2 subset. Less clear has been the impact of vitamin D on the pro-inflammatory Th17 subset, and whether and how vitamin D may preferentially drive the polarization of one of the T helper subsets.</p> <p>Methods</p> <p>Using human peripheral blood-derived mononuclear cells and mouse splenocytes and lymph node cells in culture, we examined whether and how vitamin D preferentially skews T cells towards the Th1, Th2 or Th17 subsets. Mice afflicted with the multiple sclerosis-like condition, experimental autoimmune encephalomyelitis (EAE), were examined in vivo for the relevance of the tissue culture-derived results.</p> <p>Results</p> <p>We report that the biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 {1,25(OH)2D3}, consistently generates human and murine Th2 cells in culture, frequently leaving unchanged the levels of Th1/Th17 cytokines. As a result, the ratio of Th2 to Th1 and Th17 is increased by 1,25(OH)2D3. The upregulation of Th2 to Th1 or Th17 subsets by 1,25(OH)2D3 is enabled by an increase of the GATA-3 transcription factor, which itself is promoted upstream by an elevation of the STAT6 transcription factor. In mice, the alleviation of EAE severity by 1,25(OH)2D3 is accompanied by elevation of levels of GATA-3 and STAT6. Significantly, the efficacy of 1,25(OH)2D3 in ameliorating EAE is completely lost in mice genetically deficient for STAT6, which was accompanied by the inability of 1,25(OH)2D3 to raise GATA-3 in STAT6 null lymphocytes.</p> <p>Conclusions</p> <p>These results of vitamin D promoting a Th2 shift through upstream GATA-3 and STAT6 transcription factors shed mechanistic understanding on the utility of vitamin D in MS.</p

Reduction of microglial activity in a model of multiple sclerosis by dipyridamole

BACKGROUND: Despite extensive and persistent activation of microglia in multiple sclerosis (MS), microglia inhibitors have not yet been identified for treatment of the disorder. We sought to identify medications already in clinical use that could inhibit the activation of microglia. On the basis of the reported inhibitory effects of dipyridamole on phosphodiesterase activity that result in the production of various anti-inflammatory outcomes, we selected it for study. Dipyridamole is used clinically for secondary prevention in stroke. In this study, dipyridamole was examined using microglia in culture and in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). RESULTS: We found that dipyridamole attenuated the elevation of several cytokines and chemokines in human microglia caused by Toll-like receptor stimulation. Morphological characteristics of activated microglia in culture were also normalized by dipyridamole. In mice, dipyridamole decreased the clinical severity of EAE and reduced microglial activity and other histological indices of EAE in the spinal cord. CONCLUSIONS: Dipyridamole is an inhibitor of microglia activation and may have a role in MS and other neurological conditions to attenuate microglial activity

1,25-Dihydroxyvitamin D3 Protects against Immune-Mediated Killing of Neurons in Culture and in Experimental Autoimmune Encephalomyelitis.

Several studies have reported that low vitamin D levels are associated with an increased risk of developing multiple sclerosis (MS). As MS is an inflammatory disorder with degeneration of axons and neurons, we examined whether the biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D3), could protect against the T cell-mediated killing of human neurons in culture, and the axonal loss seen in mice with experimental autoimmune encephalomyelitis (EAE). Human neurons were exposed to activated human T lymphocytes and the loss of neurons was documented 24 hours later by counting the number of microtubule-associated protein-2 positive cells. Mice with EAE were harvested for counts of axonal profiles in the spinal cord. 1,25D3 was exposed to T cells in culture or administered to mice from peak EAE clinical severity when axonal loss was already evolving. Activated T lymphocytes killed human neurons prominently within 24 hours but toxicity was significantly attenuated when T cells were exposed to 1,25D3 prior to the co-culture. In EAE, 1,25D3 treatment initiated from peak clinical severity reduced the extent of clinical disability and mitigated the progressive loss of axons. The reduction of axonal and neuronal loss by 1,25D3 in the context of an inflammatory assault to the central nervous system is a potential contributor to the putative benefits of vitamin D in MS

Reduction of microglial activity in a model of multiple sclerosis by dipyridamole

Article deposited according to agreement with BMC, December 2, 2010 and according to publisher policies: http://www.biomedcentral.com/about/copyright [September 5, 2013].YesFunding provided by the Open Access Authors Fund

Treatment of EAE mice at peak disease demonstrates sustained improvement in clinical score and sparing of axons.

<p>Thirty-six mice were immunized with MOG, and 4 mice were sacrificed at day 18 for baseline histology. The remaining 32 mice were equally divided into 1,25D₃ or vehicle treatment group until the end of the experiment where 8 mice each were randomly chosen for histological analyses. <b>(A)</b> 1,25D₃ treatment initiated from peak clinical severity (day 18) caused a significant score separation from day 26 (p<0.05, Mann-Whitney Wilcoxon) that persisted for the remainder of the 56 days. <b>(B,C)</b> A representative image of toluidine blue-stained section (top) that was then processed to highlight axons (false-colored red) within myelin rings (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144084#sec002" target="_blank">Methods</a> for how the imaging analyses were performed). Panel B is from a 1,25D₃-treated mouse, while panel C is from a vehicle-EAE animal. <b>(D)</b> 1,25D₃ treatment of EAE mice leads to significant sparing of axons. Indeed, while control (vehicle) mice at day 56 displayed fewer axons than mice at day 18, indicating progressive axonal loss in the ensuing interval, this was prevented by 1,25D₃. Values are mean ± SD. Asterisks denote significance of 1,25D₃-treated EAE mice and axonal counts at day 18 (peak disease), relative to control day 56 EAE mice. * = p<0.05 compared to control day 56.</p

1,25D₃ protects against T cell killing of neurons in culture.

<p><b>(A,B)</b> Neurons were identified by immunofluorescence for MAP-2 (red). While neurons were not harmed after 24h by unactivated (UA) T cells, exposure to anti-CD3 activated T cells resulted in the loss of over 80% of neurons by 24h (N+A panel). Activated T cells exposed to increasing concentrations of 1,25D₃ (abbreviated “V”) for 3 days prior to their co-culture with neurons were reduced in their capacity to kill neurons. <b>(C)</b> The protection by 1,25D₃ against the T cell killing of neurons is also seen in the “pre- and post-treated” T cell group, but the post-treatment of T cells with 1,25D₃ (i.e. at the time of T cell-neuron co-culture) lacks protective effect. Note that equal number (100,000) of control or 1,25D₃-exposed T cells were added to neurons for their 24h of co-culture. Asterisks denote significance of 1,25D₃ treatment, relative to activated T cells alone. Values are mean ± SD of quadruplicate analyses, and the results of the entire experiment were reproduced in another set; that 1,25D₃-pretreatment of activated T cells alleviates their toxicity to neurons was observed in 3 experiments. * = p<0.05; ** = p<0.01; *** = p<0.001.</p

1,25D₃ alters levels of growth factors in neurons.

<p>VEGF transcript levels were elevated by 1,25D₃, relative to vehicle; there was a trend towards an increase of BDNF transcripts by 1,25D₃ in neurons, but this was not statistically significant. Values are mean ± SD of triplicate analyses. **p<0.01, ***p<0.001 relative to vehicle.</p