Danny Bruce

Failure of T cell homing, reduced CD4/CD8αα intraepithelial lymphocytes, and inflammation in the gut of vitamin D receptor KO mice

Specific pathogen-free IL-10 KO mice failed to develop inflammatory bowel disease (IBD), whereas IL-10/vitamin D receptor (VDR) double KO mice developed fulminating IBD. WT CD4 T cells inhibited experimental IBD, while VDR KO CD4 T cells failed to suppress IBD. VDR KO mice had normal numbers and functions of regulatory T cells. The percentages of IL-17- and IFN-γ-secreting T cells in the gut of mice reconstituted with WT and VDR KO CD4 T cells were also not different. Instead, there were twice as many CD8αα intraepithelial lymphocytes (IEL) in mice that were reconstituted with WT CD4 T cells than in mice reconstituted with VDR KO CD4 T cells. Furthermore, VDR KO mice had reduced numbers of CD8αα IEL, absent CD4/CD8αα populations, and as a result low IL-10 production in the IEL. The lack of CD8αα IEL was due in part to decreased CCR9 expression on T cells that resulted in the failure of the VDR KO T cells to home to the small intestine. We conclude that the VDR mediates T cell homing to the gut and as a result the VDR KO mouse has reduced numbers of CD8αα IEL with low levels of IL-10 leading to increased inflammatory response to the normally harmless commensal flora.

Converging pathways lead to overproduction of IL-17 in the absence of vitamin D signaling

Multiple pathways converge to result in the overexpression of T(h)17 cells in the absence of either vitamin D or the vitamin D receptor (VDR). CD4(+) T cells from VDR knockout (KO) mice have a more activated phenotype than their wild-type (WT) counterparts and readily develop into T(h)17 cells under a variety of in vitro conditions. Vitamin D-deficient CD4(+) T cells also overproduced IL-17 in vitro and 1,25 dihydroxyvitamin D(3) inhibited the development of T(h)17 cells in CD4(+) T-cell cultures. Conversely, the induction of inducible (i) Tregs was lower in VDR KO CD4(+) T cells than WT and the VDR KO iTregs were refractory to IL-6 inhibition. Host-specific effects of the VDR were evident on in vivo development of naive T cells. Development of naive WT CD4(+) T cells in the VDR KO host resulted in the overexpression of IL-17 and more severe experimental inflammatory bowel disease (IBD). The increased expression of T(h)17 cells in the VDR KO mice was associated with a reduction in tolerogenic CD103(+) dendritic cells. The data collectively demonstrate that T(h)17 and iTreg cells are direct and indirect targets of vitamin D. The increased propensity for development of T(h)17 cells in the VDR KO host results in more severe IBD.

The paradoxical effects of vitamin D on Type 1 mediated immunity

Low vitamin D status is associated with an increased risk of Th1 mediated autoimmune diseases like inflammatory bowel disease. 1,25(OH)(2)D(3) treatments have been shown to suppress Th1 mediated immunity and protect animals from experimental autoimmunity. Th1 mediated immunity is important for clearance of a number of different infectious diseases. For tuberculosis 1,25(OH)(2)D(3) treatment is associated with decreased Th1 mediated immunity but increased bactericidal activity. Systemic candidiasis is unaffected by 1,25(OH)(2)D(3) treatment. The seemingly paradoxical effects of 1,25(OH)(2)D(3) and vitamin D on Th1 mediated autoimmunity versus infectious immunity point to a broad array of vitamin D targets in the immune system. The interplay of these vitamin D targets and their impact on the host-immune response then dictate the outcome.

Vitamin D and host resistance to infection? Putting the cart in front of the horse.

Vitamin D is being touted as an anti-infective agent and it has even been suggested that vitamin D supplementation could be effective against the H1N1 influenza virus. The claims are largely based on the ability of vitamin D to induce antibacterial peptides and evidence that the immune system produces active vitamin D (1,25(OH)2D3) in situ. While there are many examples of immune production of 1,25(OH)2D3 in vitro, there is little in vivo evidence. In addition, it is not clear what role immune production of 1,25(OH)2D3 has on the course of disease. Vitamin D and 1,25(OH)2D3 inhibit T helper type 1 (Th1)/Th17-mediated immune responses and autoimmune diseases by acting on the innate and acquired immune system to inhibit the function of Th1 and Th17 cells. Th1 and Th17 cells are important in host resistance to many infections including tuberculosis (TB) caused by Mycobacterium tuberculosis. Paradoxically the innate immune system is induced to produce antibacterial peptides that are effective against TB in vitro. Data from several models of infection have so far not supported a role for vitamin D in affecting the course of disease. There is also very little evidence that vitamin D affects the course of human TB infection. Experiments have not been done in cells, mice or humans to evaluate the effect of vitamin D on influenza virus. At this time it would be premature to claim that vitamin D has an effect on TB, influenza or any other infection.

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 TCRαβ+ T cells in the gut of VDR knockout (KO) mice, and that reduction was largely in the CD8αα+ TCRαβ+ cells. Conversely TCRγδ+ T cells were normal in the VDR KO mice. The thymic precursors of CD8αα+ TCRαβ+ cells (triple-positive for CD4, CD8αα, and CD8αβ) were reduced and less mature in VDR KO mice. In addition, VDR KO mice had a higher frequency of the CD8αα+ TCRαβ+ precursors (double-negative [DN] TCRαβ+ T cells) in the gut. The proliferation rates of the DN TCRαβ+ gut T cells were less in the VDR KO compared with those in wild type. Low proliferation of DN TCRαβ+ 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+ TCRαβ+ cells was cell intrinsic. Decreased maturation and proliferation of CD8αα+ TCRαβ+ cells in VDR KO mice results in fewer functional CD8αα+ TCRαβ+ T cells, which likely explains the increased inflammation in the gastrointestinal tract of VDR KO and vitamin D-deficient mice.

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

BackgroundVitamin 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.ResultsVDR 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.ConclusionsOur 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.

Chemico-Genetic Identification of Drebrin as a Regulator of Calcium Responses

Store-operated calcium channels are plasma membrane Ca(2+) channels that are activated by depletion of intracellular Ca(2+) stores, resulting in an increase in intracellular Ca(2+) concentration, which is maintained for prolonged periods in some cell types. Increases in intracellular Ca(2+) concentration serve as signals that activate a number of cellular processes, however, little is known about the regulation of these channels. We have characterized the immuno-suppressant compound BTP, which blocks store-operated channel mediated calcium influx into cells. Using an affinity purification scheme to identify potential targets of BTP, we identified the actin reorganizing protein, drebrin, and demonstrated that loss of drebrin protein expression prevents store-operated channel mediated Ca(2+) entry, similar to BTP treatment. BTP also blocks actin rearrangements induced by drebrin. While actin cytoskeletal reorganization has been implicated in store-operated calcium channel regulation, little is known about actin-binding proteins that are involved in this process, or how actin regulates channel function. The identification of drebrin as a mediator of this process should provide new insight into the interaction between actin rearrangement and store-operated channel mediated calcium influx.

Elevated non-specific immunity and normal Listeria clearance in young and old vitamin D receptor knockout mice

1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and the vitamin D receptor (VDR) are important regulators of autoimmunity. The effect of the VDR on the ability of mice to fight a primary or secondary infection has not been determined. Young and old VDR knockout (KO) mice were able to clear both primary and secondary infections with Listeria monocytogenes. However, the kinetics of clearance was somewhat delayed in the absence of the VDR. Memory T cell development was not different in young VDR KO and wild-type (WT) mice; however, old VDR KO mice had significantly less memory T cells than their WT counterparts but still mounted an adequate immune response as determined by the complete clearance of L. monocytogenes. Although the primary and secondary immune responses were largely intact in the VDR KO mice, the old VDR KO mice had increased cytokines and antibody responses compared with the old WT mice. In particular, old VDR KO mice had elevated antigen non-specific antibodies; however, these magnified immune responses did not correspond to more effective Listeria clearance. The increased antibody and cytokine responses in the old VDR KO mice are consistent with the increased susceptibility of these mice to autoimmunity.

Vitamin D and Inflammatory Bowel Disease

Publisher Summary This chapter reviews the immunoregulatory role of vitamin D and its effect on the pathology of inflammatory bowel disease (IBD). Following this, it discusses the epidemiological evidence connecting vitamin D deficiency to IBD severity and the data from animal models of experimental IBD. It also proposes the vitamin D hypothesis, which suggests that vitamin D status may be an environmental factor involved in the development of IBD. Finally the chapter reviews the current treatment options for IBD patients and explains how vitamin D might be used as an alternative or a supplemental treatment for patients with IBD. Vitamin D, in vitamin-D-insufficient patients, and its active metabolites and analogs could be a safe and effective adjunct to the therapies available to treat or prevent IBD. Unfortunately, clinical interventions have not been done to look at the effects of vitamin D on IBD disease in humans; based on the extensive data in animal models several studies have been proposed. Issues that need to be addressed include—defining the appropriate dose of the vitamin D compound to be used, effect of other therapies a patient is on, and whether or not patients with either Ulcerative colitis or celiac disease or both can benefit.

Vitamin D and host resistance to infection? Putting the cart in front of the horse: authors reply

We appreciate the interest in our review and the comments made by Drs Grant, Goldstein and Mascitelli on our manuscript ‘Vitamin D and host resistance to infection? Putting the cart in front of the horse’. However, we disagree with the premise of their comments and their characterization of our review. While it is correct that we based our conclusions on our knowledge of animal experiments and the mechanisms underlying the effects of vitamin D on immune function, we did not overlook relevant human studies. Careful examination of the Aloia study that was referred to as a successful randomized controlled trial of vitamin D supplementation reveals that it was a letter to the editor, where the authors did a secondary analysis of a randomized control trial that was negative for the original outcomes (i.e. bone mineral changes). The letter to the editor did not contain enough information to evaluate the analysis presented and since letters to the editor are not peer-reviewed, this reference should not be used as such. The Urashima et al. study was a randomized control study using vitamin D supplementation and very carefully looked at the incidence of influenza A and B. The cases of influenza A in the vitamin D group were 18 of 167, while the placebo group had 31 of 167. Indeed, there were significantly fewer cases in the vitamin D group; however, influenza B cases were 39 of 167 in the vitamin D group and 28 of 167 in the placebo group. The increase in cases in the vitamin D-supplemented group was not significantly different. Dr Grant and colleagues state that ‘No effect of vitamin D was found for type B influenza, which is generally less common than type A influenza’. However, in this case this may be misleading since in these children there were more cases of type B influenza (67 cases) than type A influenza (49 cases). In addition, if you add the cases of type A and B influenza together the vitamin D-treated children had 57 cases and the placebo group had 59 cases. Lastly, there is no evidence that the immune response to type A or type B influenza differs in a way that would explain vitamin D influencing one strain versus another. The review article by Dr Cannell and colleagues does not provide any new data on the topic and is largely a hypothesis that needs to be tested. We conclude that these two human studies fail to provide ‘ample’ evidence that vitamin D reduces the risk of human infection with influenza. As for the other references cited by Dr Grant and colleagues, they fail to provide new and/or direct evidence to support a role for vitamin D in humans infected with influenza and or other infections. Associations between season and vitamin D and/or UV light and the 1918–1919 pandemic are not especially helpful. UV light does increase vitamin D production in the skin, but has a number of other effects that should be independently tested. In addition, UV light is immunosuppressive and this immunosuppression still exists in mice that are deficient in the ability to use vitamin D. Similarly, review articles and expert opinions do not provide new data and, therefore, our original conclusion that there is no evidence to support the anti-infective effects of vitamin D does not overlook human studies. We, therefore, maintain our conclusion that there is to date no published, peer-reviewed evidence in any system to directly support a role for vitamin D and host resistance to infection. Unfortunately, the various letters to the editors, reviews, etc. that suggest a connection are no substitute for direct proof. The hypotheses have been proposed and now the critical experiments need to be performed in all areas (cells, animals and humans) to either prove or disprove the hypothesis. While healthy debate is good, over-interpretation of the current peer reviewed data only serves to confuse the public and other scientists who work in the area of vitamin D nutrition.