Katinka Stoffels

Immune Regulation of 25‐Hydroxyvitamin‐D3‐1α‐Hydroxylase in Human Monocytes

Monocytes express 1α‐hydroxylase, the enzyme responsible for final hydroxylation of vitamin D3, in response to IFNγ and CD14/TLR4 activation. Cross‐talk between the JAK‐STAT, the NF‐κB, and the p38 MAPK pathways is necessary, and direct binding of C/EBPβ to its recognition sites in the promoter of the 1α‐hydroxylase gene is a prerequisite.

Immune regulation of 1α-hydroxylase in murine peritoneal macrophages: Unravelling the IFNγ pathway

The activated form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), plays an important role in the immune system. Indeed, receptors for 1,25(OH)(2)D(3) are found on most immune cells, and 1alpha-hydroxylase, the enzyme responsible for final activation of vitamin D(3), is expressed by monocytes/macrophages, resulting in secretion of 1,25(OH)(2)D(3) after immune stimulation. We have previously shown that in murine peritoneal macrophages 1alpha-hydroxylase is highly regulated by immune signals such as IFNgamma and LPS. In the present study we made use of two different knock-out mouse models with disruptions in two key transcription factors in the IFNgamma-signalling cascade (STAT1alpha and IRF1), to evaluate their role in the regulation of 1alpha-hydroxylase. This was performed by culturing peritoneal macrophages from these knock-out mice in the presence of IFNgamma and LPS, and evaluating the impact of the absence of the respective transcription factors on 1alpha-hydroxylase mRNA expression by real-time RT-PCR. In addition also the mRNA expression profiles of the essential transcription factors STAT1alpha, IRF1 and C/EBPbeta were investigated. The data confirm a crucial role for STAT1alpha as well as for C/EBPbeta in the regulation of 1alpha-hydroxylase in monocytes.

Monocytic Expression Behavior of Cytokines in Diabetic Patients upon Inflammatory Stimulation

Abstract: Cytokines are involved in the pathogenesis of type 1 diabetes. The disease is characterized by T cell‐mediated β cell destruction and a biased Th1 cytokine pattern. Type 2 diabetes also presents an inflammatory cytokine imbalance. In this study, mRNA expression of cytokines IL‐12, TNF‐α, IL‐1, and IL‐6 was studied in monocytes from diabetic patients after in vitro immune stimulation. Whereas IL‐12p40 was highly expressed in type 1 diabetic patients, TNF‐α, IL‐1, and IL‐6 transcripts were elevated in type 1 but especially type 2 diabetic patients compared with healthy controls, suggesting an important proinflammatory milieu. We conclude that circulating monocytes from type 1 as well as type 2 diabetic patients have an aberrant cytokine profile when stimulated by an immune stimulus such as IFNγ. This condition not only is likely to be involved in disease pathogenesis, but may contribute to its later complications.

Regulation of 25-hydroxyvitamin d-1α-hydroxylase by IFNγ in human monocytic THP1 cells☆

1,25-DihydroxyVitamin D 3 (1,25(OH) 2 D 3 ), a molecule with well-known actions in bone and mineral homeostasis, also plays a role in the immune system. Indeed, the receptor for 1,25(OH) 2 D 3 is found in most immune cells and important immunological effects have been described in vitro, reflected by its capacity to prevent autoimmunity and to prolong graft survival. The aim of this study was to elucidate the intracellular pathways used by the immune system to regulate 1,25(OH) 2 D 3 production. Therefore we studied the regulation of 25-hydroxyvitamin-D-1α-hydroxylase (1α hydroxylase) in THP 1 cells by IFNγ, demonstrating that its induction is highly dependent on the activation/differentiation by PMA and occurred at a late time point (140-fold at 72 h, P < 0.05). Complete inhibition with actinomycin D indicated that the observed induction was, at least in part, a transcriptional event. Dose-dependent inhibition with cycloheximide demonstrated that the induction was dependent on de novo protein synthesis, a finding that correlates with the late time point of up-regulation. The data presented indicate a role for 1,25(OH) 2 D 3 , activated by la hydroxylase, as a late down-tapering signal in the immune cascade.