Date Published: April 12, 2019
Publisher: Public Library of Science
Author(s): Aisha Rafique, Lars Rejnmark, Lene Heickendorff, Holger Jon Møller, Partha Mukhopadhyay.
We wanted to investigate effects of vitamin D3 (25(OH)D3 and 1.25(OH)2D3) on inflammatory cytokine expression in both activated and non-activated Mφ.
Mononuclear cells, isolated from healthy donor buffy coats were cultured for a 6-day differentiation-period. Fully differentiated Mφ were pre-treated with either 25(OH)D3 or 1.25(OH)2D3 for (4, 12 or 24 hours) +/-LPS challenge for 4 hours. Gene expression analyses of VDR, Cyp27b1 and pro-inflammatory markers TNF-α, IL-6, NF-κB, MCP-1, was performed using RT-quantitative PCR. TNF-α protein levels from Mφ culture media were analysed by ELISA.
Both 25(OH)D3 and 1.25(OH)2D3 significantly inhibited TNF-α expression in both LPS-stimulated and unstimulated Mφ. Also, NF-κB, and to a lesser extend IL-6 and MCP-1 were inhibited. LPS up-regulated Cyp27b1 gene expression which was partly reverted by 1.25(OH)2D3.
These data show anti-inflammatory effects of vitamin D3 (25(OH)D3 and 1.25(OH)2D3) in human macrophages, and support, that means for targeting high dose vitamin D3 to the immune system may have beneficial clinical effect in inflammatory conditions.
Vitamin D3 is a lipid-soluble steroid hormone and the active metabolite 1.25(OH)2D3 is crucial for calcium/phosphate homeostasis and bone metabolism . Pre-vitamin D3 is produced in the skin through rapid isomerization of 7-dehydrocholesterol after exposure to sunlight and is transported by vitamin D binding protein (DBP) to the liver following conversion into 25(OH)D3 by 25-hydroxylase (Cyp27a1). The DBP-25(OH)D3 complex is taken up by megalin and cubilin in the kidney and converted by 1α-hydroxylase (Cyp27b1) into 1.25(OH)2D3 [3, 4]. Besides the classical role, 1.25(OH)2D3 has broad immunoregulatory effects on innate and adaptive immune responses. . The nuclear vitamin D receptor (VDR) and Cyp27b1 are expressed in most immune cells e.g. T and B lymphocytes, monocytes, Mφ, natural killer cells and dendritic cells [6, 7]. Through interaction between VDR and 1.25(OH)2D3 and heterodimerization with retinoic X receptor (RXR), this complex binds to the Vitamin D responsive element (VDRE) in the promoter region of specific genes enabling gene transcription responsible for cell regulation and differentiation [7, 8]. Mφ are plastic, heterogenic immune cells that are able to polarise into specific phenotypes during inflammatory conditions, whether low-grade, autoimmune or infectious [9, 10]. The effects of 25(OH)D3 and 1.25(OH)2D3 on Mφ polarisation have been examined in cell lines e.g. human THP-1 and murine RAW 264.7, however data are not consistent and detailed knowledge about the effects of vitamin D3 on human Mφ is lacking, although current evidence suggests anti-inflammatory effects [11–13]. Supra-physiological concentrations of 1.25(OH)2D3 carry the risk of hypercalcemia, restricting high dose anti-inflammatory treatment. However, technologies for specific targeting of 1.25(OH)2D3 to Mφ may circumvent these obstacles [12, 13]. In this study, we have therefore investigated the anti-inflammatory effects of both physiological and supra-physiological concentrations of 25(OH)D3 and 1.25(OH)2D3 in Mφ.
The main finding of this study was to show a significant inhibition of TNF-α expression in fully differentiated human monocyte-derived Mφ by vitamin D3 both during normal and pro-inflammatory conditions. It has previously been shown that 1.25(OH)2D3 was able to suppress TNF-α expression in murine cell lines   and LPS induced TNF-α gene expression in human monocytes . Di Rosa et al demonstrated, that 1.25(OH)2D3 exerts diverse effects on the inflammatory response in the intermediate phases of monocyte and Mφ differentiation, including TNF-α gene suppression in TNF-α stimulated Mφ . It has been suggested, that 1.25(OH)2D3 induces a switch from an “M1” Mφ phenotype, expressing iNOS, TNF-α and IL-12, to the “M2” Mφ phenotype with higher expression of CD206, Arg-1 and IL-10 and down-regulation of pro-inflammatory markers, via the VDR-PPAR-γ signalling pathway in the mouse . This general shift was supported in our study by a decrease in NF-κB expression and to a lesser extend attenuated MCP-1 and IL-6 expression by both 25(OH)D3 and 1.25(OH)2D3. Attenuation of NF-κB by 1.25(OH)2D3 has in mice been shown to be mediated via reduced degradation of IκBα in co-transfected HEK-293 cells . Suppression of MCP-1 by 1.25(OH)2D3 has previously been reported in THP-1 monocytes and PMA induced, LPS-stimulated THP-1 Mφ . Interestingly, we observed similar effects of 25(OH)D3 and 1.25(OH)2D3 in suppression of pro-inflammatory cytokines in LPS induced Mφ. This emphasises the importance and efficiency of Cyp27b1 in the Mφ for conversion into the active metabolite. In line with this, we show a significant up-regulation of Cyp27b1 in Mφ by LPS, which was partly reverted by 1.25(OH)2D3, but not by 25(OH)D3. Mφ are known for their plasticity and polarisation in accordance to the surrounding microenvironment [21–23] and are known to play important roles in the development and sustaining of chronic inflammatory diseases by the production of pro-inflammatory cytokines. Of notice, TNF-α is a key mediator of inflammation evidenced by the clinical effect of TNF-α blocking biological drugs. It is therefore compelling to explore the use of high-dose vitamin D for anti-inflammatory treatment in e.g. inflammatory liver disease   and metabolic low-grade inflammatory conditions related to insulin resistance and type 2 diabetes, where these pro-inflammatory markers are also involved [26–28]. The use of supra-physiological concentrations of 1.25(OH)2D3 as an anti-inflammatory agent, however, carries the risk of inducing hypercalcemia. To circumvent this, strategies to directly target 1.25(OH)2D3 or 25(OH)D3 to macrophages may be applied . In summary, we have shown that 25(OH)D3 and 1.25(OH)2D3 supress TNF-α in fully differentiated human Mφ, both in resting/non-stimulated cells, and cells challenged by LPS. Our data support further attempts to develop systems for targeted delivery of Vitamin D to Mφ in vivo.