Lutgart Overbergh

The vitamin D receptor gene FokI polymorphism: Functional impact on the immune system

1α,25‐Dihydroxyvitaminu2004D3 (1,25(OH)2D3) has important effects on the growth and function of multiple cell types. These pleiotropic effects of 1,25(OH)2D3 are mediated through binding to the vitaminu2004D receptor (VDR). Several polymorphisms of the human VDR gene have been identified, with the FokI polymorphism resulting in VDR proteins with different structures, a long f‐VDR or a shorter F‐VDR. The aim of this study was to investigate the functional consequences of the FokI polymorphism in immune cells. In transfection experiments, the presence of the shorter F‐VDR resulted in higher NF‐κB‐ and NFAT‐driven transcription as well as higher IL‐12p40 promoter‐driven transcription. Marginal differences were observed for AP‐1‐driven transcription, and no differential effects were observed for transactivation of a classical vitaminu2004D‐responsive element. Concordantly, in human monocytes and dendritic cells with a homozygous short FFu2004VDR genotype, expression of IL‐12 (mRNA and protein) was higher than in cells with a long ffu2004VDR genotype. Additionally, lymphocytes with a short FFu2004VDR genotype proliferated more strongly in response to phytohemagglutinin. Together, these data provide the first evidence that the VDR FokI polymorphism affects immune cell behavior, with a more active immune system for the short F‐VDR, thus possibly playing a role in immune‐mediated diseases.

Regulation of vitamin D homeostasis: implications for the immune system

Vitamin D homeostasis in the immune system is the focus of this review. The production of both the activating (25- and 1alpha-hydroxylase) and the metabolizing (24-hydroxylase) enzymes by cells of the immune system itself, indicates that 1,25(OH)(2)D(3) can be produced locally in immune reaction sites. Moreover, the strict regulation of these enzymes by immune signals is highly suggestive for an autocrine/paracrine role in the immune system, and opens new treatment possibilities.

Vitamin D signaling in immune-mediated disorders: Evolving insights and therapeutic opportunities

1,25(OH)(2)D(3), the active form of vitamin D, is a central player in calcium and bone metabolism. More recently, important immunomodulatory effects have been attributed to this hormone. The widespread presence of the vitamin D receptor (VDR) in the immune system and the expression of the enzymes responsible for the synthesis of the active 1,25(OH)(2)D(3) regulated by specific immune signals, even suggest a paracrine immunomodulatory role for 1,25(OH)(2)D(3). Additionally, the different molecular mechanisms used by 1,25(OH)(2)D(3) to exert its immunomodulatory effects prove of a broad action radius for this compound. Both, the effects of vitamin D deficiency and/or absence of the VDR as well as intervention with pharmacological doses of 1,25(OH)(2)D(3) or one of its less-calcemic analogs, affects immune system behavior in different animal models of immune-mediated disorders, such as type 1 diabetes. This review aims to summarize the data as they stand at the present time on the role of vitamin D in the pathogenesis of immune-mediated disorders, with special focus on type 1 diabetes, and on the therapeutic opportunities for vitamin D in the prevention and treatment of this autoimmune disease in mouse models and humans.

Blockade of CTLA-4 enhances allergic sensitization and eosinophilic airway inflammation in genetically predisposed mice.

CTLA‐4 (CD152) expression is restricted to subsets of activated T lymphocytes and shares homology with CD28. CTLA‐4 and CD28 molecules both bind to B7 molecules on antigen‐presenting cells. Whereas CD28‐B7 interaction enhances T cell activation, cytokine production and survival, CTLA‐4 signaling down‐regulates T cell responses. Here, we studied the involvement of CTLA‐4 triggering in thepathogenesis of allergen‐induced airway inflammation in mice. Anti‐CTLA‐4 mAb were injected during i.p. sensitization with ovalbumin (OVA). This treatment favored OVA‐specific IgE production and augmented blood eosinophilia in BALB/c mice. In BALB/c mice, enhanced Th2 sensitization after anti‐CTLA‐4 mAb injections resulted in more severe airway inflammation, and increased airway hyperresponsiveness to metacholine, bronchial eosinophilia and IL‐4 and IL‐5 levels in broncho‐alveolar lavage (BAL) fluid following repeated allergen inhalations. Importantly, aggravation of airway inflammationand enhancement of Th2 responses were accompanied by a significant reduction of pulmonary TGF‐β levels at protein level in BAL fluid as well as on mRNA level in inflamed lung tissue. In contrast to BALB/c mice, blockade of CTLA‐4 did not alter IgE production nor the phenotype of airway inflammation or TGF‐β production in C57BL/6 mice. Our data suggest that CTLA‐4 triggering represents an important regulatory mechanism for Th2 sensitization in genetically predisposed mice by modulating TGF‐β production.

Citrullinated glucose-regulated protein 78 is an autoantigen in type 1 diabetes.

Posttranslational modifications of self-proteins play a substantial role in the initiation or propagation of the autoimmune attack in several autoimmune diseases, but their contribution to type 1 diabetes is only recently emerging. In the current study, we demonstrate that inflammatory stress, induced by the cytokines interleukin-1β and interferon-γ, leads to citrullination of GRP78 in β-cells. This is coupled with translocation of this endoplasmic reticulum chaperone to the β-cell plasma membrane and subsequent secretion. Importantly, expression and activity of peptidylarginine deiminase 2, one of the five enzymes responsible for citrullination and a candidate gene for type 1 diabetes in mice, is increased in islets from diabetes-prone nonobese diabetic (NOD) mice. Finally, (pre)diabetic NOD mice have autoantibodies and effector T cells that react against citrullinated GRP78, indicating that inflammation-induced citrullination of GRP78 in β-cells generates a novel autoantigen in type 1 diabetes, opening new avenues for biomarker development and therapeutic intervention.

Cytokine signalling in the β-cell: a dual role for IFNγ

IFNgamma (interferon gamma), a cytokine typically secreted by infiltrating immune cells in insulitis in Type 1 diabetes, is by itself not detrimental to beta-cells, but, together with other cytokines, such as IL-1beta (interleukin 1beta) and TNFalpha (tumour necrosis factor alpha), or dsRNA (double-stranded RNA), it induces beta-cell apoptosis. The complex gene and protein networks that are altered by the combination of cytokines clearly point towards synergisms between these agents. IFNgamma acts mostly via JAK (Janus kinase) activation, with the transcription factors STAT-1 (signal transducer and activator of transcription-1) and IRF-1 (IFNgamma regulatory factor-1) playing a central role in the downstream pathway. The study of mice with a disruption of these transcription factors has revealed a possible dual role for IFNgamma in beta-cell destruction by cytokines or dsRNA. We demonstrated that the absence of STAT-1 from beta-cells completely protects against IFNgamma+IL-1beta- and IFNgamma+dsRNA-mediated beta-cell death in vitro, whereas absence of IRF-1 does not prevent cytokine-induced beta-cell apoptosis. In vivo, a lack of the IRF-1 gene in pancreatic islets even promotes low-dose streptozotocin-induced diabetes, whereas lack of STAT-1 confers resistance against beta-cell death following low-dose streptozotocin-induced diabetes. Additionally, IRF-1(-/-) islets are more sensitive to PNF (primary islet non-function) after transplantation in spontaneously diabetic NOD (non-obese diabetic) mice, whereas STAT-1(-/-) islets are fully protected. Moreover, proteomic analysis of beta-cells exposed to IFNgamma or IFNgamma+IL-1beta confirms that very different pathways are activated by IFNgamma alone compared with the combination. We conclude that IFNgamma may play a dual role in immune-induced beta-cell destruction. Transcription factors drive this dual role, with STAT-1 driving beta-cell destruction and IRF-1 possibly playing a role in up-regulation of protective pathways induced by IFNgamma.

Defect in activation-induced cell death in non-obese diabetic (NOD) T lymphocytes

Activation-induced cell death (AICD) represents a major means of peripheral tolerance induction, eliminating effector cells. NOD mice, a widely used model for autoimmune diabetes, are characterized by high levels of circulating T lymphocytes and by resistance to several apoptosis-inducing signals. The aim of this study was to analyse AICD in peripheral NOD T lymphocytes. First, we demonstrated in an in vitro AICD model that NOD T lymphocytes are more resistant to AICD (64+/-2%) compared to non-autoimmune C57BL/6 T lymphocytes (73+/-2%), but also diabetes-resistant NOR T lymphocytes (76+/-3%, P<0.05). Moreover, both CD4(+)and CD8(+)subsets were affected. Analysis of the cellular and molecular pathways revealed lower caspase 8 levels, a central caspase proximally involved in the AICD-pathway (fluorescence of 258+/-47 in NOD vs. 441+/-16 in NOR and 414+/-61 in C57BL/6 T lymphocytes, P<0.05). Gene expression analysis using real-time RT-PCR additionally revealed low expression of Fas and FasL, the death receptor system activating caspase 8 and contributing to AICD. Additionally, low IL-2 levels, together with high TGFbeta and Bclx-L levels, confirm the presence of a NOD-specific AICD-resistance profile. In conclusion, we present cellular and molecular evidence for disturbed AICD mechanisms in NOD T lymphocytes. This resistance in AICD may contribute to defective tolerance induction to autoantigens in NOD mice.

Novel insights in the immune function of the vitamin D system: Synergism with interferon-beta

The 1,25(OH)(2)D(3) analog, TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)), has an interesting dissociation profile between its potent immunomodulatory and its calcemic effects in vivo. The strong immunomodulatory potency of TX527 is reflected by its ability to attenuate experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). At present most MS patients are being treated with systemic IFN-beta administration. The aim of this study was to investigate whether combining IFN-beta with TX527 could empower its EAE-protective effects. We evaluated also combinations with the standard immunosuppressant cyclosporin A (CsA). EAE was induced in SJL mice by PLP immunization, treatment was started 3 days before disease induction. The TX527+IFN-beta combination resulted in significant disease protection which was superior to the effect of both treatment separately. No disease amelioration, even aggravation, was obtained with the IFN-beta+CsA combination. By adding TX527 to the IFN-beta+CsA combination near complete protection from EAE was achieved (100% protection from paralysis, mean maximal score of 1.8+/-1.5, both p<0.05 versus controls and all individual treatments). From these data we conclude that adding TX527 to an IFN-beta and/or CsA treatment results in clear additional immunomodulatory effects in EAE prevention and is therefore a potentially interesting candidate to be considered in clinical intervention trials in MS.

Role of interleukin‐12 in the induction of mucosal inflammation and abrogation of regulatory T cell function in chronic experimental colitis

IL‐12 promotes Th1 cell differentiation and cell‐mediated immunity. In the present study, the potential role of IL‐12 was analyzed in an experimental colitis model in scid mice reconstituted with syngeneic CD45RBhighCD4+ T cells. Real‐time reverse transcription‐PCR studies demonstrated that IL‐12 p40 mRNA in inflamed colon is induced shortly after T cell transfer and maintained at a stable level after weeku20044, at the time when wasting disease starts. Administration of anti‐IL‐12 on daysu20040, 14, and 28 (early treatment) or on daysu200428, 42, and 56 (delayed treatment) after T cell transfer, effectively prevented or, respectively cured wasting disease and colitis in scid recipients. Anti‐IL‐12 treatment abrogated mucosal inflammation with significantly diminished leukocyte infiltration (CD4 cells, macrophages) and CD54 expression, and down‐regulated proinflammatory cytokines IFN‐γ and IL‐2. Of note, although splenic CD4+ T cells are unable to induce disease as a result of the presence of regulatory CD45RBlow cells, splenic CD4+ T cells, preactivated by IL‐12 and anti‐CD3 in vitro, were highly pathogenic in inducing severe mucosal inflammation, suggesting that IL‐12 and anti‐CD3 abrogated regulatory T cell function. These findings indicate that IL‐12 is important for the induction of experimental colitis through effects on proinflammatory cytokine production and on regulatory T cell function.

Vitamin D3 and the immune system: maintaining the balance in health and disease.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D3, is a central player in Ca and bone metabolism. More recently, important immunomodulatory effects have been attributed to this hormone. By binding to its receptor, the vitamin D receptor, 1,25(OH)2D3 regulates the expression of various genes and consequently affects the behaviour of different cell types within the immune system. 1,25(OH)2D3 can potently inhibit pathogenic T cells and gives rise to elevated numbers of regulatory T cells via the induction of tolerogenic dendritic cells. These immunomodulatory activities of 1,25(OH)2D3 have also been proven useful in vivo: administration of 1,25(OH)2D3 in several animal models can prevent or cure different autoimmune diseases and graft rejection. To overcome the dose-limiting side effects of 1,25(OH)2D3 on Ca and bone, less calcaemic structural analogues (alone or in combination with synergistically acting drugs or bone-resorption inhibitors) have been successfully used in animal models. Furthermore, as 1,25(OH)2D3 also contributes to host defence against infectious agents by the induction of antimicrobial responses, this molecule might provide a new strategy to deal with drug-resistant infections. According to the pleiotropic effects of 1,25(OH)2D3 in the immune system, increasing epidemiological data underline the importance of adequate vitamin D intakes in reducing the risk of several autoimmune diseases and infections such as tuberculosis.