Effects of repeated infections with non-typeable Haemophilus influenzae on lung in vitamin D deficient and smoking mice.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), exacerbations cause acute inflammatory flare-ups and increase the risk for hospitalization and mortality. Exacerbations are common in all disease stages and are often caused by bacterial infections e.g., non-typeable Heamophilus influenzae (NTHi). Accumulating evidence also associates vitamin D deficiency with the severity of COPD and exacerbation frequency. However, it is still unclear whether vitamin D deficiency when combined with cigarette smoking would worsen and prolong exacerbations caused by repeated infections with the same bacterial strain. METHODS: Vitamin D sufficient (VDS) and deficient (VDD) mice were exposed to nose-only cigarette smoke (CS) for 14 weeks and oropharyngeally instilled with NTHi at week 6, 10 and 14. Three days after the last instillation, mice were assessed for lung function, tissue remodeling, inflammation and immunity. The impact of VDD and CS on inflammatory cells and immunoglobulin (Ig) production was also assessed in non-infected animals while serum Ig production against NTHi and dsDNA was measured in COPD patients before and 1 year after supplementation with Vitamin D3. RESULTS: VDD enhanced NTHi eradication, independently of CS and complete eradication was reflected by decreased anti-NTHi Ig's within the lung. In addition, VDD led to an increase in total lung capacity (TLC), lung compliance (Cchord), MMP12/TIMP1 ratio with a rise in serum Ig titers and anti-dsDNA Ig's. Interestingly, in non-infected animals, VDD exacerbated the CS-induced anti-NTHi Ig's, anti-dsDNA Ig's and inflammatory cells within the lung. In COPD patients, serum Ig production was not affected by vitamin D status but anti-NTHi IgG increased after vitamin D3 supplementation in patients who were Vitamin D insufficient before treatment. CONCLUSION: During repeated infections, VDD facilitated NTHi eradication and resolution of local lung inflammation through production of anti-NTHi Ig, independently of CS whilst it also promoted autoantibodies. In COPD patients, vitamin D supplementation could be protective against NTHi infections in vitamin D insufficient patients. Future research is needed to decipher the determinants of dual effects of VDD on adaptive immunity. TRAIL REGISTRATION: ClinicalTrials, NCT00666367. Registered 23 April 2008, https://www.clinicaltrials.gov/ct2/show/study/NCT00666367 .

High Serum Vitamin D Concentrations, Induced via Diet, Trigger Immune and Intestinal Microbiota Alterations Leading to Type 1 Diabetes Protection in NOD Mice.

The hormonally-active form of vitamin D, 1,25-dihydroxyvitamin D3, can modulate both innate and adaptive immunity, through binding to the nuclear vitamin D receptor expressed in most immune cells. A high dose of regular vitamin D protected non-obese diabetic (NOD) mice against type 1 diabetes (T1D), when initiated at birth and given lifelong. However, considerable controversy exists on the level of circulating vitamin D (25-hydroxyvitamin D3, 25(OH)D3) needed to modulate the immune system in autoimmune-prone subjects and protect against T1D onset. Here, we evaluated the impact of two doses of dietary vitamin D supplementation (400 and 800 IU/day), given to female NOD mice from 3 until 25 weeks of age, on disease development, peripheral and gut immune system, gut epithelial barrier function, and gut bacterial taxonomy. Whereas serum 25(OH)D3 concentrations were 2.6- (400 IU/day) and 3.9-fold (800 IU/day) higher with dietary vitamin D supplementation compared to normal chow (NC), only the 800 IU/day vitamin D-supplemented diet delayed and reduced T1D incidence compared to NC. Flow cytometry analyses revealed an increased frequency of FoxP3+ Treg cells in the spleen of mice receiving the 800 IU/day vitamin D-supplemented diet. This vitamin D-induced increase in FoxP3+ Treg cells, also expressing the ecto-5'-nucleotidase CD73, only persisted in the spleen of mice at 25 weeks of age. At this time point, the frequency of IL-10-secreting CD4+ T cells was also increased in all studied immune organs. High-dose vitamin D supplementation was unable to correct gut leakiness nor did it significantly modify the increased gut microbial diversity and richness over time observed in NOD mice receiving NC. Intriguingly, the rise in alpha-diversity during maturation occurred especially in mice not progressing to hyperglycaemia. Principal coordinates analysis identified that both diet and disease status significantly influenced the inter-individual microbiota variation at the genus level. The abundance of the genera Ruminoclostridium_9 and Marvinbryantia gradually increased or decreased, respectively in faecal samples of mice on the 800 IU/day vitamin D-supplemented diet compared to mice on the 400 IU/day vitamin D-supplemented diet or NC, irrespective of disease outcome. In summary, dietary vitamin D reduced T1D incidence in female NOD mice at a dose of 800, but not of 400, IU/day, and was accompanied by an expansion of Treg cells in various lymphoid organs and an altered intestinal microbiota signature.

Screening for hypovitaminosis D: cost-effective or not?

Few topics have elicited more emotion than the issue of screening for vitamin D status and the discussion on the need for global supplementation with vitamin D metabolites. The importance of the problem is highlighted by the USPSTF posted draft research plan with the aim of making an update recommendations statement, possibly next year. Here, we discuss two different viewpoints on screening for vitamin D status: for and against. In the literature there are scientifically sound opinions supporting pro and cons positions. However, we believe that the best way to definitively elucidate this issue is the implementation of a randomized controlled trial evaluating clinical outcomes or harms in persons screened versus those not screened for vitamin D deficiency. The feasibility of such a trial is probably questionable owing to uncertainties still present concerning threshold for vitamin D sufficiency and end points (that is, for example, improved bone mineral density, reduced risk of falls and so on) to be reached.

Vitamin D-modulated dendritic cells delay lethal graft-versus-host disease through induction of regulatory T cells.

Graft-versus-host disease (GVHD) is the most lethal complication after allogeneic bone marrow transplantation (allo-BMT). Current approaches to prevent GVHD rely on donor lymphocyte/T cell depletion or general immunosuppression, leading to opportunistic infections and cancer relapse. Tolerogenic dendritic cells can induce regulatory T cells (Tregs) with the ability to suppress inflammation and prevent transplant rejection, making them an attractive cellular therapy to control GVHD. Active vitamin D (1α,25-dihydroxyvitamin D3; 1α,25(OH)2D3) promotes the generation of tolerogenic dendritic cells (1,25D3-DCs). This study aimed to determine the ability of ex vivo generated 1,25D3-DCs to trigger the expansion of Tregs that are able to control lethal xenogeneic GVHD in humanized NOD/LtSz-PrkdcscidIL2rγtm1Wjl (NSG) mice. We demonstrate that 1,25D3-DCs express lower levels of HLA-DR and costimulatory molecules, such as CD80 and CD86, and produce higher levels of IL-10 and TNF-α and lower amounts of IL-12, compared to vehicle-treated DCs. Moreover, these cells express increased levels of various co-inhibitory molecules such as PD-L1 and ILT-3 and the glycoprotein CD52 that is known to suppress T cell activation. Consequently, 1,25D3-DCs are poor stimulators of alloantigen-primed T cells, but foster the generation of antigen-specific suppressive Tregs. When adoptively transferred in humanized NSG mice, these 1,25D3-DC-induced Tregs delayed GVHD caused by the co-transferred autologous human peripheral blood mononuclear cells (PBMCs). These results indicate that 1,25D3-DC-induced Tregs can inhibit xenogeneic GVHD and maintain their immunomodulatory function under conditions of inflammation.

Vitamin D controls the capacity of human dendritic cells to induce functional regulatory T cells by regulation of glucose metabolism.

Tolerogenic dendritic cells (tolDCs) instruct regulatory T cells (Tregs) to dampen autoimmunity. Active vitamin D3 (1α,25-dihydroxyvitamin D3; 1α,25(OH)2D3) imprints human monocyte-derived DCs with tolerogenic properties by reprogramming their glucose metabolism. Here we identify the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) as a critical checkpoint and direct transcriptional target of 1α,25(OH)2D3 in determining the tolDC profile. Using tracer metabolomics, we show that PFKFB4 activity is essential for glucose metabolism, especially for glucose oxidation, which is elevated upon 1α,25(OH)2D3 exposure. Pharmacological inhibition of PFKFB4 reversed the 1α,25(OH)2D3-mediated shift in metabolism, DC profile and function, as determined by expression of inhibitory surface markers and secretion of regulatory cytokines and factors. Moreover, PFKFB4 inhibition in 1α,25(OH)2D3-treated DCs blocked their hallmark capacity to induce suppressive Tregs. This work demonstrates that alterations in the bioenergetic metabolism of immune cells are central to the immunomodulatory effects induced by 1α,25(OH)2D3.

Reversal of Diabetes in NOD Mice by Clinical-Grade Proinsulin and IL-10-Secreting Lactococcus lactis in Combination With Low-Dose Anti-CD3 Depends on the Induction of Foxp3-Positive T Cells.

The introduction of ß-cell autoantigens via the gut through Lactococcus lactis (L. lactis) has been demonstrated to be a promising approach for diabetes reversal in NOD mice. Here we show that a combination therapy of low-dose anti-CD3 with a clinical-grade self-containing L. lactis, appropriate for human application, secreting human proinsulin and interleukin-10, cured 66% of mice with new-onset diabetes, which is comparable to therapy results with plasmid-driven L. lactis Initial blood glucose concentrations (<350 mg/dL) and insulin autoantibody positivity were predictors of the stable reversal of hyperglycemia, and decline in insulin autoantibody positivity was an immune biomarker of therapeutic outcome. The assessment of the immune changes induced by the L. lactis-based therapy revealed elevated frequencies of CD4+Foxp3+ T cells in the pancreas-draining lymph nodes, pancreas, and peripheral blood of all treated mice, independent of metabolic outcome. Neutralization of cytotoxic T-lymphocyte antigen 4 and transforming growth factor-ß partially abrogated the suppressive function of therapy-induced regulatory T cells (Tregs). Ablation or functional impairment of Foxp3+ Tregs in vivo at the start or stop of therapy impaired immune tolerance, highlighting the dependence of the therapy-induced tolerance in mice with new-onset diabetes on the presence and functionality of CD4+Foxp3+ T cells. Biomarkers identified in this study can potentially be used in the future to tailor the L. lactis-based combination therapy for individual patients.