Vitamin A and vitamin D regulate the microbial complexity, barrier function, and the mucosal immune responses to ensure intestinal homeostasis.

Diet is an important regulator of the gastrointestinal microbiota. Vitamin A and vitamin D deficiencies result in less diverse, dysbiotic microbial communities and increased susceptibility to infection or injury of the gastrointestinal tract. The vitamin A and vitamin D receptors are nuclear receptors expressed by the host, but not the microbiota. Vitamin A- and vitamin D-mediated regulation of the intestinal epithelium and mucosal immune cells underlies the effects of these nutrients on the microbiota. Vitamin A and vitamin D regulate the expression of tight junction proteins on intestinal epithelial cells that are critical for barrier function in the gut. Other shared functions of vitamin A and vitamin D include the support of innate lymphoid cells that produce IL-22, suppression of IFN-γ and IL-17 by T cells, and induction of regulatory T cells in the mucosal tissues. There are some unique functions of vitamin A and D; for example, vitamin A induces gut homing receptors on T cells, while vitamin D suppresses gut homing receptors on T cells. Together, vitamin A- and vitamin D-mediated regulation of the intestinal epithelium and mucosal immune system shape the microbial communities in the gut to maintain homeostasis.

Control of Circulating IgE by the Vitamin D Receptor In Vivo Involves B Cell Intrinsic and Extrinsic Mechanisms.

Vitamin D deficiency is associated with the development of asthma and allergy. The active form of vitamin D [1,25(OH)2D] regulates B cells in vitro and mice without the vitamin D receptor (VDR knockout [KO]) have high serum IgE. Whole-body VDR KO, T cell-specific VDR (T-VDR) KO, B cell-specific VDR (B-VDR) KO, and vitamin D deficient mice were used to determine the targets of vitamin D in the regulation of IgE in vivo. Vitamin D deficient, VDR KO, and B-VDR KO mice developed hyper-IgE, whereas T-VDR KO mice did not. The data show that IL-10 secretion by B cells and CD1d expression on IL-10 secreting B cells was lower in VDR KO mice. Mesenteric lymph node cultures from VDR KO and B-VDR KO mice secreted higher IgE ex vivo than wild-type (WT) cultures, and the addition of IL-10 eliminated the difference in IgE production between VDR KO and WT cultures. The increase in IgE in VDR KO mice was 2-fold greater than in the B-VDR KO mice, suggesting that VDR deficiency in non-B cells contributes to hyper-IgE in vivo. Antibiotic depletion of the microbiota raised serum IgE 4-fold in both WT and VDR KO mice. The VDR directly and indirectly regulates IgE production in B cells. Through the VDR, vitamin D is an environmental factor that helps to maintain low serum IgE responses.

The role of UVR and vitamin D on T cells and inflammatory bowel disease.

Vitamin D deficiency is associated with the development of inflammatory bowel disease (IBD). In experimental IBD the targets of vitamin D that result in protection from IBD include gut epithelial cells, innate immune cells, T cells, and the microbiota. Ultraviolet radiation (UVR) induces production of vitamin D in the skin and suppresses T cell responses in the host. There is limited data demonstrating an effect of UVR on experimental IBD but the mechanisms of UVR suppression in IBD have not been defined. There are several shared effects of vitamin D and UVR on T cells including inhibition of proliferation and suppression of IFN-γ and IL-17 producing T cells. Conversely UVR decreases and vitamin D increases IL-4 production from T cells. Together the data suggest that UVR suppression of T cells and potentially IBD are both vitamin D dependent and independent.

Vitamin D and Lung Infection.

Available data suggest that vitamin D plays a role in controlling inflammation in the lungs. However, to date vitamin D-induced production of cathelicidin has not been shown to have an effect on the burden of either viruses or bacteria. Future work should continue to determine the effects of vitamin D-regulated mechanisms in the lung and the possible role of cathelicidin against different pulmonary pathogens in vivo.

NKT cells can help mediate the protective effects of 1,25-dihydroxyvitamin D3 in experimental autoimmune encephalomyelitis in mice.

Active vitamin D [1,25-dihydroxyvitamin D3 (1,25D3)] blocks the development of experimental autoimmune diseases. However, the molecular and immunobiological mechanisms underlying 1,25D3's anti-inflammatory properties are not fully understood. We employed a murine model of experimental autoimmune encephalomyelitis (EAE) in order to determine the role of NKT cells in 1,25D3-mediated protection from EAE. Wild-type (WT) mice or mice lacking all NKT cells (CD1d(-/-)) or invariant NKT cells (Jα18(-/-)) were fed control or 1,25D3-supplemented diets. All mice fed with the control diet developed severe EAE. 1,25D3 treatment of WT mice protected them from developing EAE. CD1d(-/-) and Jα18(-/-) mice treated with 1,25D3 were not protected to the same extent as WT mice. Myelin oligodendrocyte glycoprotein-specific IL-17 and IFN-γ production was significantly reduced in 1,25D3 WT mice compared with WT but was not decreased in 1,25D3 CD1d(-/-) mice compared with CD1d(-/-) mice. IL-4(-/-) mice were utilized to determine how IL-4 deficiency affects susceptibility to EAE. IL-4(-/-) mice were not protected from developing EAE by α-galactosylceramide (α-GalCer) or 1,25D3 treatment. Furthermore, 1,25D3 treatment of splenocytes in vitro decreased α-GalCer-induced IL-17 and increased IL-4, IL-5 and IL-10 production. 1,25D3 alters the cytokine profile of invariant NKT cells in vitro. These studies demonstrate that NKT cells are important mediators of 1,25D3-induced protection from EAE in mice and NKT cell-derived IL-4 may be an important factor in providing this protection.

Crucial role of macrophage selenoproteins in experimental colitis.

Inflammation is a hallmark of inflammatory bowel disease (IBD) that involves macrophages. Given the inverse link between selenium (Se) status and IBD-induced inflammation, our objective was to demonstrate that selenoproteins in macrophages were essential to suppress proinflammatory mediators, in part, by the modulation of arachidonic acid metabolism. Acute colitis was induced using 4% dextran sodium sulfate in wild-type mice maintained on Se-deficient (<0.01 ppm Se), Se-adequate (0.08 ppm; sodium selenite), and two supraphysiological levels in the form of Se-supplemented (0.4 ppm; sodium selenite) and high Se (1.0 ppm; sodium selenite) diets. Selenocysteinyl transfer RNA knockout mice (Trsp(fl/fl)LysM(Cre)) were used to examine the role of selenoproteins in macrophages on disease progression and severity using histopathological evaluation, expression of proinflammatory and anti-inflammatory genes, and modulation of PG metabolites in urine and plasma. Whereas Se-deficient and Se-adequate mice showed increased colitis and exhibited poor survival, Se supplementation at 0.4 and 1.0 ppm increased survival of mice and decreased colitis-associated inflammation with an upregulation of expression of proinflammatory and anti-inflammatory genes. Metabolomic profiling of urine suggested increased oxidation of PGE2 at supraphysiological levels of Se that also correlated well with Se-dependent upregulation of 15-hydroxy-PG dehydrogenase (15-PGDH) in macrophages. Pharmacological inhibition of 15-PGDH, lack of selenoprotein expression in macrophages, and depletion of infiltrating macrophages indicated that macrophage-specific selenoproteins and upregulation of 15-PGDH expression were key for Se-dependent anti-inflammatory and proresolving effects. Selenoproteins in macrophages protect mice from dextran sodium sulfate-colitis by enhancing 15-PGDH-dependent oxidation of PGE2 to alleviate inflammation, suggesting a therapeutic role for Se in IBD.

Vitamin D inhibits the occurrence of experimental cerebral malaria in mice by suppressing the host inflammatory response.

In animal models of experimental cerebral malaria (ECM), neuropathology is associated with an overwhelming inflammatory response and sequestration of leukocytes and parasite-infected RBCs in the brain. In this study, we explored the effect of vitamin D (VD; cholecalciferol) treatment on host immunity and outcome of ECM in C57BL/6 mice during Plasmodium berghei ANKA (PbA) infection. We observed that oral administration of VD both before and after PbA infection completely protected mice from ECM. VD administration significantly dampened the inducible systemic inflammatory responses with reduced circulating cytokines IFN-γ and TNF and decreased expression of these cytokines by the spleen cells. Meanwhile, VD also resulted in decreased expression of the chemokines CXCL9 and CXCL10 and cytoadhesion molecules (ICAM-1, VCAM-1, and CD36) in the brain, leading to reduced accumulation of pathogenic T cells in the brain and ultimately substantial improvement of the blood-brain barriers of PbA-infected mice. In addition, VD inhibited the differentiation, activation, and maturation of splenic dendritic cells. Meanwhile, regulatory T cells and IL-10 expression levels were upregulated upon VD treatment. These data collectively demonstrated the suppressive function of VD on host inflammatory responses, which provides significant survival benefits in the murine ECM model.

Vitamin A-Deficient Hosts Become Nonsymptomatic Reservoirs of Escherichia coli-Like Enteric Infections.

Vitamin A deficiency (A(-)) remains a public health concern in developing countries and is associated with increased susceptibility to infection. Citrobacter rodentium was used to model human Escherichia coli infections. A(-) mice developed a severe and lethal (40%) infection. Vitamin A-sufficient (A(+)) mice survived and cleared the infection by day 25. Retinoic acid treatment of A(-) mice at the peak of the infection eliminated C. rodentium within 16 days. Inflammation levels were not different between A(+) and A(-) mouse colons, although the A(-) mice were still infected at day 37. Increased mortality of A(-) mice was not due to systemic cytokine production, an inability to clear systemic C. rodentium, or increased pathogenicity. Instead, A(-) mice developed a severe gut infection with most of the A(-) mice surviving and resolving inflammation but not eliminating the infection. Improvements in vitamin A status might decrease susceptibility to enteric pathogens and prevent potential carriers from spreading infection to susceptible populations.

Vitamin D receptor expression controls proliferation of naïve CD8+ T cells and development of CD8 mediated gastrointestinal inflammation.

BACKGROUND: Vitamin D receptor (VDR) deficiency contributes to the development of experimental inflammatory bowel disease (IBD) in several different models. T cells have been shown to express the VDR, and T cells are targets of vitamin D. In this article we determined the effects of VDR expression on CD8+ T cells. RESULTS: VDR KO CD8+ T cells, but not WT CD8+ T cells, induced colitis in Rag KO recipients. In addition, co-transfer of VDR KO CD8+ T cells with naïve CD4+ T cells accelerated colitis development. The more severe colitis was associated with rapidly proliferating naïve VDR KO CD8+ T cells and increased IFN-γ and IL-17 in the gut. VDR KO CD8+ T cells proliferated in vitro without antigen stimulation and did not downregulate CD62L and upregulate CD44 markers following proliferation that normally occurred in WT CD8+ T cells. The increased proliferation of VDR KO CD8+ cells was due in part to the higher production and response of the VDR KO cells to IL-2. CONCLUSIONS: Our data indicate that expression of the VDR is required to prevent replication of quiescent CD8+ T cells. The inability to signal through the VDR resulted in the generation of pathogenic CD8+ T cells from rapidly proliferating cells that contributed to the development of IBD.

Streptococcus pneumoniae-induced pneumonia and Citrobacter rodentium-induced gut infection differentially alter vitamin A concentrations in the lung and liver of mice.

In the developing world, vitamin A (VA) deficiency is endemic in populations that are also at great risk of morbidity and mortality because of pneumococcal pneumonia and enteric infections. To better understand how lung and gastrointestinal pathogens affect VA status, we assessed VA concentrations in serum, lung, and liver during an invasive pneumonia infection induced by Streptococcus pneumoniae serotype 3, and a noninvasive gut infection induced by Citrobacter rodentium, in vitamin A-adequate (VAA) and vitamin A-deficient (VAD) mice. For pneumonia infection, mice were immunized with pneumococcal polysaccharide serotype 3 (PPS3), or not (infected-control), 5 d prior to intranasal inoculation with S. pneumoniae. Two days post-inoculation, immunization was protective against systemic infection regardless of VA status as PPS3 immunization decreased bacteremia compared with infected-control mice (P < 0.05). Retinol concentrations in the lung were higher in infected-control VAA mice (15.7 nmol/g: P < 0.05) compared with PPS3-immunized mice (8.23 nmol/g), but this was not associated with increased lung bacterial burden. VAA mice had reduced severity of C. rodentium-induced gut infection as measured by fecal bacterial shedding compared with VAD mice (P < 0.05). Liver retinol and retinyl ester concentrations in VAA mice decreased at the peak of infection (retinol, 8.1 nmol/g; retinyl esters, 985 nmol/g; P < 0.05, compared with uninfected mice; retinol, 29.5 nmol/g; retinyl esters, 1730 nmol/g), whereas tissue VA concentrations were low in VAD mice during both infections. Colonic mucin gene expression was also depressed at peak infection compared with uninfected mice (P < 0.05). Overall, pneumonia had less effect on VA status than gastrointestinal infection, predominantly owing to reduced hepatic VA storage at the peak of gut infection.

Mushrooms and Health Summit proceedings.

The Mushroom Council convened the Mushrooms and Health Summit in Washington, DC, on 9-10 September 2013. The proceedings are synthesized in this article. Although mushrooms have long been regarded as health-promoting foods, research specific to their role in a healthful diet and in health promotion has advanced in the past decade. The earliest mushroom cultivation was documented in China, which remains among the top global mushroom producers, along with the United States, Italy, The Netherlands, and Poland. Although considered a vegetable in dietary advice, mushrooms are fungi, set apart by vitamin B-12 in very low quantity but in the same form found in meat, ergosterol converted with UV light to vitamin D2, and conjugated linoleic acid. Mushrooms are a rare source of ergothioneine as well as selenium, fiber, and several other vitamins and minerals. Some preclinical and clinical studies suggest impacts of mushrooms on cognition, weight management, oral health, and cancer risk. Preliminary evidence suggests that mushrooms may support healthy immune and inflammatory responses through interaction with the gut microbiota, enhancing development of adaptive immunity, and improved immune cell functionality. In addition to imparting direct nutritional and health benefits, analysis of U.S. food intake survey data reveals that mushrooms are associated with higher dietary quality. Also, early sensory research suggests that mushrooms blended with meats and lower sodium dishes are well liked and may help to reduce intakes of red meat and salt without compromising taste. As research progresses on the specific health effects of mushrooms, there is a need for effective communication efforts to leverage mushrooms to improve overall dietary quality.

Vitamin D regulates the gut microbiome and protects mice from dextran sodium sulfate-induced colitis.

The active form of vitamin D [1,25-dihydroxycholecalciferol, 1,25(OH)2D3] and the vitamin D receptor (VDR) regulate susceptibility to experimental colitis. The effect of the bacterial microflora on the susceptibility of C57BL/6 mice to dextran sodium sulfate-induced colitis was determined. Mice that cannot produce 1,25(OH)2D3 [Cyp27b1 (Cyp) knockout (KO)], VDR KO as well as their wild-type littermates were used. Cyp KO and VDR KO mice had more bacteria from the Bacteroidetes and Proteobacteria phyla and fewer bacteria from the Firmicutes and Deferribacteres phyla in the feces compared with wild-type. In particular, there were more beneficial bacteria, including the Lactobacillaceae and Lachnospiraceae families, in feces from Cyp KO and VDR KO mice than in feces from wild-type. Helicobacteraceae family member numbers were elevated in Cyp KO compared with wild-type mice. Depletion of the gut bacterial flora using antibiotics protected mice from colitis. 1,25(OH)2D3 treatment (1.25 µg/100 g diet) of Cyp KO mice decreased colitis severity and reduced the numbers of Helicobacteraceae in the feces compared with the numbers in the feces of untreated Cyp KO mice. The mechanisms by which the dysbiosis occurs in VDR KO and Cyp KO mice included lower expression of E-cadherin on gut epithelial and immune cells and fewer tolerogenic dendritic cells that resulted in more gut inflammation in VDR and Cyp KO mice compared with wild-type mice. Increased host inflammation has been shown to provide pathogens with substrates to out-compete more beneficial bacterial species. Our data demonstrate that vitamin D regulates the gut microbiome and that 1,25(OH)2D3 or VDR deficiency results in dysbiosis, leading to greater susceptibility to injury in the gut.

White button mushrooms increase microbial diversity and accelerate the resolution of Citrobacter rodentium infection in mice.

The effect of feeding C57BL/6 mice white button (WB) mushrooms or control (CTRL) diets for 6 wk was determined on the bacterial microflora, urinary metabolome, and resistance to a gastrointestinal (GI) pathogen. Feeding mice a diet containing 1 g WB mushrooms/100 g diet resulted in changes in the microflora that were evident at 2 wk and stabilized after 4 wk of WB feeding. Compared with CTRL-fed mice, WB feeding (1 g/100 g diet) increased the diversity of the microflora and reduced potentially pathogenic (e.g., Clostridia) bacteria in the GI tract. Bacteria from the Bacteroidetes phylum increased and the Firmicutes phylum decreased in mushroom-fed mice compared with CTRL. The changes in the microflora were also reflected in the urinary metabolome that showed a metabolic shift in the WB-fed compared with the CTRL-fed mice. The WB feeding and changes in the microbiome were associated with fewer inflammatory cells and decreased colitis severity in the GI mucosa following Citrobacter rodentium infection compared with CTRL. Paradoxically, the clearance of C. rodentium infection did not differ even though Ifn-γ and Il-17 were higher in the colons of the WB-fed mice compared with CTRL. Adding modest amounts of WB mushrooms (1 g/100 g diet) to the diet changed the composition of the normal flora and the urinary metabolome of mice and these changes resulted in better control of inflammation and resolution of infection with C. rodentium.

Intrinsic requirement for the vitamin D receptor in the development of CD8αα-expressing T cells.

Vitamin D and vitamin D receptor (VDR) deficiency results in severe symptoms of experimental inflammatory bowel disease in several different models. The intraepithelial lymphocytes of the small intestine contain large numbers of CD8αα(+) T cells that have been shown to suppress the immune response to Ags found there. In this study, we determined the role of the VDR in the development of CD8αα(+) T cells. There are fewer total numbers of TCRαß(+) T cells in the gut of VDR knockout (KO) mice, and that reduction was largely in the CD8αα(+) TCRαß(+) cells. Conversely TCRγδ(+) T cells were normal in the VDR KO mice. The thymic precursors of CD8αα(+) TCRαß(+) cells (triple-positive for CD4, CD8αα, and CD8αß) were reduced and less mature in VDR KO mice. In addition, VDR KO mice had a higher frequency of the CD8αα(+) TCRαß(+) precursors (double-negative [DN] TCRαß(+) T cells) in the gut. The proliferation rates of the DN TCRαß(+) gut T cells were less in the VDR KO compared with those in wild type. Low proliferation of DN TCRαß(+) T cells was a result of the very low expression of the IL-15R in this population of cells in the absence of the VDR. Bone marrow transplantation showed that the defect in VDR KO CD8αα(+) TCRαß(+) cells was cell intrinsic. Decreased maturation and proliferation of CD8αα(+) TCRαß(+) cells in VDR KO mice results in fewer functional CD8αα(+) TCRαß(+) T cells, which likely explains the increased inflammation in the gastrointestinal tract of VDR KO and vitamin D-deficient mice.

Epigenetic reduction in invariant NKT cells following in utero vitamin D deficiency in mice.

Vitamin D status changes with season, but the effect of these changes on immune function is not clear. In this study, we show that in utero vitamin D deficiency in mice results in a significant reduction in invariant NKT (iNKT) cell numbers that could not be corrected by later intervention with vitamin D or 1,25-dihydroxy vitamin D(3) (active form of the vitamin). Furthermore, this was intrinsic to hematopoietic cells, as vitamin D-deficient bone marrow is specifically defective in generating iNKT cells in wild-type recipients. This vitamin D deficiency-induced reduction in iNKT cells is due to increased apoptosis of early iNKT cell precursors in the thymus. Whereas both the vitamin D receptor and vitamin D regulate iNKT cells, the vitamin D receptor is required for both iNKT cell function and number, and vitamin D (the ligand) only controls the number of iNKT cells. Given the importance of proper iNKT cell function in health and disease, this prenatal requirement for vitamin D suggests that in humans, the amount of vitamin D available in the environment during prenatal development may dictate the number of iNKT cells and potential risk of autoimmunity.

Invariant NKT cell defects in vitamin D receptor knockout mice prevents experimental lung inflammation.

Vitamin D receptor (VDR) deficiency (knockout [KO]) results in a failure of mice to generate an airway hyperreactivity (AHR) response on both the BALB/c and C57BL/6 background. The cause of the failed AHR response is the defective population of invariant NKT (iNKT) cells in the VDR KO mice because wild-type (WT) iNKT cells rescued the AHR response. VDR KO mice had significantly fewer iNKT cells and normal numbers of T cells in the spleen compared with WT mice. In BALB/c VDR KO mice, the reduced frequencies of iNKT cells were not apparent in the liver or thymus. VDR KO and WT Th2 cells produced similar levels of IFN-γ and IL-5. On the BALB/c background, Th2 cells from VDR KO mice produced less IL-13, whereas on the C57BL/6 background, Th2 cells from VDR KO mice produced less IL-4. Conversely, VDR KO iNKT cells were defective for the production of multiple cytokines (BALB/c: IL-4, IL-5, and IL-13; C57BL/6: IL-4 and IL-17). Despite relatively normal Th2 responses, BALB/c and C57BL/6 VDR KO mice failed to develop AHR responses. The defect in iNKT cells as a result of the VDR KO was more important than the highly susceptible Th2 background of the BALB/c mice. Defective iNKT cell responses in the absence of the VDR result in the failure to generate AHR responses in the lung. The implication of these mechanistic findings for human asthma requires further investigation.

Two lineages of immune cells that differentially express the vitamin D receptor.

Since 1983 it has been known that monocytes and activated T and B cells expressed the vitamin D receptor (VDR) and are therefore vitamin D targets. New data identified two lineages of immune cells that can be differentiated by the expression of the VDR. Monocytes, macrophages, neutrophils, and hematopoietic stem cells were mostly from VDR positive lineages. T cells, ILC1 and ILC3 were also largely VDR positive, which is consistent with the known effects of vitamin D as regulators of type-1 and type-3 immunity. Activation of the VDR negative T cells did not induce the expression of the VDR reporter, suggesting that perhaps only a subset of the T cells in the periphery express the VDR. When activated, the VDR negative T cells responded as if they were VDR knockout T cells in that they made more IFN-γ and proliferated faster than the VDR positive T cells. The ability of vitamin D to regulate immune function will depend on which cells express the VDR and a better understanding of the signals that regulate VDR expression in immune cells.

Highlights from the 24th workshop on vitamin D in Austin, September 2022.

The 24th Workshop on Vitamin D was held September 7-9, 2022 in Austin, Texas and covered a wide diversity of research in the vitamin D field from across the globe. Here, we summarize the meeting, individual sessions, awards and presentations given.

Vitamin D, multiple sclerosis and inflammatory bowel disease.

It has now been more than 20years since the vitamin D receptor was identified in cells of the immune system. The immune system has now been established as an important target of vitamin D. Vitamin D receptor knockout and vitamin D deficient mice have a surplus of effector T cells that have been implicated in the pathology of multiple sclerosis (MS) and inflammatory bowel disease (IBD). The active form of vitamin D directly and indirectly suppresses the function of these pathogenic T cells while inducing several regulatory T cells that suppress MS and IBD development. There is reason to believe that vitamin D could be an environmental factor that may play a role in the development of these immune mediated diseases in the clinic but at present there has not been a causal relationship established. Nonetheless, current evidence suggests that improving vitamin D status and/or using vitamin D receptor agonists may be useful in MS and IBD.