Human T lymphocytes are direct targets of 1,25-dihydroxyvitamin D3 in the immune system

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Abstract

Besides its actions on minerals and bone, the bioactive vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has important immunomodulatory properties. Within the immune system, dendritic cells represent key targets for this hormone and 1,25(OH)2D3-induced changes in their phenotype and function ultimately affects T lymphocytes. However, the presence of vitamin D receptors (VDR) in activated T cells proposes additional mechanisms for 1,25(OH)2D3 to directly regulate T cell responses. Here, we investigated the expression and kinetics of vitamin D-related genes in human activated T lymphocytes. Different activation stimuli elicited increased VDR- and 1-α-hydroxylase expression, with a highly similar kinetic pattern. Addition of 1,25(OH)2D3 effectively triggered VDR signaling, as evidenced by 24-hydroxylase induction, but only when introduced to T lymphocytes expressing high levels of VDR. This enhanced degree of VDR signaling correlated with a stronger inhibition of cytokines (IFN-γ, IL-10) and modulation of homing receptor expression (CCR10, CLA) in long-term T cell cultures. Importantly, chronic 1,25(OH)2D3-exposure further amplified VDR signaling and the concomitant T cell modulating effects. In conclusion, we validate T cells as direct targets for 1,25(OH)2D3 and provide this optimized in vitro model to improve our understanding of the role of vitamin D as a direct regulator of T cell responses.

Introduction

The bioactive form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), was originally described as an essential hormone for mineral and bone homeostasis, but more recently, this hormone has emerged as an important regulator of innate and adaptive immune responses [1], [2], [3]. This notion is supported by the fact that many cell types within the immune system express the vitamin D receptor (VDR). The VDR constitutes a member of the nuclear receptor superfamily, which – upon interaction with its ligand – heterodimerizes with the retinoic X receptor (RXR) [4], [5]. This 1,25(OH)2D3–VDR–RXR-complex subsequently binds to vitamin D responsive elements (VDRE) in the promoter region of target genes to regulate gene transcription in a wide variety of cells [6].

Among the different immune cells, dendritic cells (DCs) are important targets of 1,25(OH)2D3-mediated actions. 1,25(OH)2D3 interferes with the differentiation and maturation of DCs and alters the production of DC-derived cytokines [7], [8], [9], [10]. Consequently, 1,25(OH)2D3-modulated DCs exhibit a markedly decreased T cell stimulatory capacity, while skewing T cell cytokine responses from an inflammatory T helper (Th)1 – and Th17 phenotype towards a Th2 – and a regulatory T cell phenotype [7], [9], [11], [12], [13], [14]. These immunomodulatory actions of 1,25(OH)2D3 have been confirmed in vivo by its ability to prevent and/or cure various autoimmune diseases and to inhibit allograft rejection in experimental models [15], [16].

Thus, combined, these observations have implicated DC-dependent actions of 1,25(OH)2D3 in the regulation of T cell responses. However, the presence of the VDR in activated T cells suggests also direct actions on these cells as a factor contributing to the immunoregulatory properties of 1,25(OH)2D3. Indeed, several groups have demonstrated the ability of 1,25(OH)2D3 to directly modulate human and murine T cell proliferation and cytokine responses [17], [18], [19], [20], [21], [22], [23]. However, varying results have been reported, depending on the activating stimuli, the source of T cells (primary cells versus cell lines, human versus mouse) and the concentration of 1,25(OH)2D3 being used. Some groups have demonstrated anti-proliferative actions of 1,25(OH)2D3 on T cells [17], [22], [24], whereas others reported no or even stimulatory effects of the hormone [20], [25], [26]. Conflicting data were also obtained regarding the modulation of T cell cytokine expression, varying from an overall suppression of both Th1- and Th2 cytokines [20], while others observed a 1,25(OH)2D3-mediated shift from a Th1- to a Th2 profile [19], [27].

In the present study, we investigated the expression and kinetics of vitamin D-related genes in human activated T cells and we evaluated whether activation of these pathways was dependent on the type of T cell activating stimulus. Based on the observed kinetics, we developed an optimized in vitro model to study the direct immunomodulatory effects of 1,25(OH)2D3 and analogs on human primary T lymphocytes.

Section snippets

Materials

RPMI 1640 medium with Glutamax™-I, fetal calf serum and antibiotics (penicillin and streptomycin) were purchased from Invitrogen (Merelbeke, Belgium). Purified anti-CD3 mAb (clone UCHT1) and anti-CD28 mAb (clone 37407) were obtained from R&D Systems (Minneapolis, USA). Phytohaemagglutinin (PHA) was acquired from Remel (Lenexa, USA). Phorbol 12-myristate 13-acetate (PMA) and ionomycin were obtained from Sigma (Saint Louis, USA). Human recombinant IL-2 and IL-12 were purchased from Peprotech

Kinetics of VDR- and vitamin D metabolizing gene expression in human activated CD3+ T cells

To gain more insight into the role of vitamin D as a regulator of T cell responses and to verify whether human T cells would be responsive to the effects of active vitamin D, we analyzed the expression levels of VDR and vitamin D metabolizing genes, such as 1-α-hydroxylase and 24-hydroxylase, at different time points following T cell activation. For this purpose human CD3+ T cells were isolated by negative magnetic cell separation of normal blood donors. In agreement with previous reports,

Discussion

The discovery that receptors for active vitamin D are abundantly present throughout the immune system has stimulated the search for immunomodulatory actions of 1,25(OH)2D3, revealing a plethora of effects targeting different immune cells [1], [3]. Up to now, the well-documented actions of 1,25(OH)2D3 on DCs are believed to be the central mechanism by which adaptive immune responses are regulated by this hormone. Nevertheless, the presence of the VDR and vitamin D metabolizing enzymes, such as

Acknowledgements

The technical experience of Wim Cockx, Nursen Gol and Dirk Valckx is greatly appreciated. This work was supported by the Flemish Research Foundation (FWO) (Grants G.0552.06 and G.0649.08, a postdoctoral fellowship for Conny Gysemans and Hannelie Korf and a clinical fellowship for Chantal Mathieu), the Juvenile Diabetes Research Foundation (a postdoctoral fellowship for Evelyne van Etten (JDRF3-2006-33)), the Belgium Program on Interuniversity Poles of Attraction initiated by the Belgian State

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