Regulation of CYP27B1 and CYP24A1 hydroxylases limits cell‐autonomous activation of vitamin D in dendritic cells

Abstract

Keywords: CYP27B1 r CYP24A1 r DCs r Macrophages r Vitamin D Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe capacity of vitamin D metabolites to modulate macrophage and DC function has been subject to extensive research interest in light of the association of vitamin D deficiency with a broad spectrum of immunological and infectious diseases. This gives Correspondence: Dr. Mahdad Noursadeghi e-mail: m.noursadeghi@ucl.ac.uk way to the potential for vitamin D supplementation [1][2][3][4] or ex vivo conditioning of DCs with vitamin D for cell therapy strategies [5]. Vitamin D is synthesized from 7-dehydrocholesterol in the skin following exposure to ultraviolet B radiation and thermal isomerisation, or replenished by dietary intake. It is 25-hydroxylated in the liver to form 25-hydroxyvitamin D (25[OH]D), which is in * These authors contributed equally to this work. Results 25[OH]D is not functionally activated during monocyte differentiation to immature DCsWe first compared the activation of 25[OH]D during monocyte differentiation into macrophages and immature DCs. Conventional cell culture media in this model are vitamin D deficient (Fig. 1A) (Fig. 1B). This was also associated with upregulation of the prototypic vitamin D-inducible gene cathelicidin (CAMP) in macrophages (Fig. 1C) (Fig. 1C). Importantly, upregulation of DC-SIGN during monocyte differentiation to DCs was not affected by the presence of 1,25[OH] 2 D (Fig. 1D) DCs show time-dependent increase in activation of 25[OH]D independent of CYP27B1 expression levelsWe confirmed previous observations [16] that expression of the vitamin D-activating hydroxylase CYP27B1 is significantly lower in DCs compared with MDMs at both transcript and protein levels ( Fig. 2A and B). In contrast, vitamin D receptor (VDR) expression was higher in DCs than MDMs ( Fig. 2A). Taken together, these data further support the hypothesis that 25[OH]D has no effect on DCs in the experiments described above, because of a failure to generate 1,25[OH] 2 D rather than an inability to respond to 1,25[OH] 2 D. We also confirmed that 24-h stimulation of DCs with LPS significantly increases transcript and protein levels of CYP27B1 ( Fig. 2B and C (Fig. 2D). However, there was no concomitant upregulation of CAMP (Fig. 2C) . Monocytes are typically differentiated to DCs over 4-7 days, therefore we considered the possibility that time in culture may confound comparisons between different studies. Interestingly, we did find 25[OH]D-dependent upregulation of CAMP gene expression in DCs that increased with time after at least 6 days in culture (Fig. 2E). This was not associated with any significant time-dependent increase in CYP27B1 expression ( DCs express truncated CYP27B1 transcriptsWe found that LPS stimulation was able to upregulate DC expression of CYP27B1 transcript to levels higher than that in MDMs ( Figs. 2A and C). However, the expression of CYP27B1 protein remains much lower in DCs compared with ...

Vitamin D3Metabolites Enhance the NLRP3-Dependent Secretion of IL-1β From Human THP-1 Monocytic Cells

“…The autocrine activation of 25(OH)D 3 to 1,25(OH) 2 D 3 via CYP27B1 by macrophages is proposed to be important for host defense against infection. (Liu et al, 2006;Kundu et al, 2014). In our study, PMA-differentiated THP-1 cells do exhibit basal expression of both CYP27A1 and CYPT27B1 hydroxylases.…”

“…The potent up-regulation of CYP24A1 transcript during 1,25(OH) 2 D 3 exposure has been prevously reported for immune cells, including monocyte-derived dendritic cells and macrophages (Kundu et al, 2014). This upregulation of the inactivating enzyme CYP24A1 may act to suppress the functional effects of vitamin D on inflammasome signaling and innate immune responses.…”

“…Further emphasizing the emerging importance of 1,25(OH) 2 D 3 in innate immune function, peripheral blood macrophages can produce local supplies of 1,25(OH) 2 D 3 from 25(OH)D 3 (Gottfried et al, 2006). Thus, there is an emerging role for vitamin D 3 in the innate immune system wherein it may influence cellular sensing and responses of innate immune cells such as macrophages (Prietl et al, 2013;Ryynänen and Carlberg, 2013;Verma et al, 2014) and dendritic cells (Karthaus et al 2013;Kundu et al, 2014).…”

Cell‐type‐specific modulation of innate immune signalling by vitamin D in human mononuclear phagocytes

“…Monocyte-derived dendritic cells (MDDC) were derived from magnetically sorted CD14-positive monocytes differentiated in RPMI-1640 culture media (Sigma, St Louis, MO) with 10% fetal calf serum, 100 ng/ ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 ng/ml IL-4 for 4 days. 2 Monocytederived macrophages (MDM) were derived after adherence to tissue-culture plastic and culture for 6 days in RPMI-1640 with 10% autologous serum and 20 ng/ml macrophage colony-stimulating factor (M-CSF). 2 The PBMC, MDDC and MDM were stimulated with either 100 ng/ml ultrapure LPS (Invivogen, San Diego, CA) as Toll-like receptor 4 (TLR4) ligand, or with 1 lg/ml Pam 2 CSK 4 (PCSK, Invivogen) as a TLR2 ligand for 30 min to measure phosphorylation of p38 and NF-jB p65 by flow cytometry, 60 min to undertake quantitative confocal microscopy to evaluate nuclear translocation of NF-jB p65, and 4 hr to measure transcriptional responses.…”

Vitamin D immunoregulation through dendritic cells

“…In this way, 1,25‐OH‐VD3‐induced autocrine Cyp24a1 induction can form a negative feedback loop of local VD3 activity in vivo . Although differential Cyp24a1 levels have been reported for in‐vitro ‐differentiated moDC versus macrophages,68 the relevance of this loop is unclear for the control of immune cell trafficking. Future studies will be required to elucidate potential region‐specific T‐cell trafficking patterns induced by VD3 generation and degradation, similar to those outlined for VitA/RA and gut trafficking in recent years.…”