1,25-Dihydroxyvitamin D3Is an Autonomous Regulator of the
Transcriptional Changes Leading to a Tolerogenic Dendritic
Lajos Sze ´les,*†Ga ´bor Keresztes,* Da ´niel To ¨ro ¨csik,* Zolta ´n Balajthy,* La ´szlo ´ Krena ´cs,‡
Szila ´rd Po ´liska,* Andreas Steinmeyer,§Ulrich Zuegel,¶Monika Pruenster,?Antal Rot,?
and La ´szlo ´ Nagy3*†
Previous studies suggested that 1,25-vitD could inhibit the changes brought about by differentiation and maturation of DCs. Under-
pinning the described phenotypic and functional alterations, there must be 1,25-vitD-coordinated transcriptional events. However, this
transcriptional program has not been systematically investigated, particularly not in a developmental context. Hence, it has not been
explored how 1,25-vitD-regulated genes, particularly the ones bringing about the tolerogenic phenotype, are connected to differentiation.
We conducted global gene expression analysis followed by comprehensive quantitative PCR validation to clarify the interrelationship
between 1,25-vitD and differentiation-driven gene expression patterns in developing human monocyte-derived and blood myeloid DCs.
In this study we show that 1,25-vitD regulates a large set of genes that are not affected by differentiation. Interestingly, several genes,
impacted both by the ligand and by differentiation, appear to be regulated by 1,25-vitD independently of the developmental context. We
have also characterized the kinetics of generation of 1,25-vitD by using three early and robustly regulated genes, the chemokine CCL22,
the inhibitory receptors CD300LF and CYP24A1. We found that monocyte-derived DCs are able to turn on 1,25-vitD sensitive genes
in early phases of differentiation if the precursor is present. Our data collectively suggest that exogenous or endogenously generated
1,25-vitD regulates a large set of its targets autonomously and not via inhibition of differentiation and maturation, leading to the
previously characterized tolerogenic state. The Journal of Immunology, 2009, 182: 2074–2083.
the tissue they reside in. If DCs sense an abnormal state by detecting
pathogens, endogenous factors released by necrotic cells, or inflam-
matory cytokines, they mature. Maturation is characterized by the
migration of DCs to lymphoid organs, the down-regulation of Ag
uptake, and an enhanced capacity for priming naive T cells (1, 2). The
endritic cells (DCs)4are conductors of the adaptive im-
mune system (1, 2). In their immature form (immature
DCs, IDCs), they act as sentinels and constantly monitor
interaction between DCs, T cells, and environmental cues will dictate
whether an immune response is mounted or whether tolerance is es-
tablished or maintained. DCs are inherently heterogeneous, as they
need to respond to a variety of signals in very different contexts. The
integration of signals will lead to at least two distinct, e.g., immuno-
genic or tolerogenic, DC immunophenotypes. How these stereotypic
immunophenotypes are achieved at the transcriptional level is not well
understood. Nuclear hormone receptors, a group of lipid-activated
transcription factors, have been increasingly implicated in this pro-
A member of this family is the vitamin D receptor (VDR). A num-
ber of studies (4–10) provided evidence that the addition of the active
form of vitamin D3, 1,25-dihydroxyvitamin D3(1,25-vitD), the ligand
for VDR, has an impact on the differentiation, function, and matura-
tion of human and mouse DCs that results in T cell hyporesponsive-
ness. Importantly, DCs differentiated in the presence of 1,25-vitD
share several features with tolerogenic DCs (11, 12). These include
low surface expression of MHC class II and costimulatory molecules
(CD40, CD80, CD86), the up-regulation of inhibitory molecules
(ILT3), decreased production of IL-12, and enhanced secretion of
the maturation of DCs was dependent on VDR (6). Furthermore,
VDR?/?mice have enlarged lymph nodes with an increased propor-
tion of mature DCs (MDCs), implicating 1,25-vitD in the differenti-
ation and/or maturation of DCs in vivo (6). In addition, animal studies
have demonstrated that treatment with 1,25-vitD arrests the develop-
ment of autoimmune diabetes (15) and mediates tolerance to trans-
hormone or its analogues (17).
The ways how immunogenic and tolerogenic DCs develop
and the mechanisms immunosuppressive drugs can modify the
*Department of Biochemistry and Molecular Biology and†Apoptosis and Genomics
Research Group of the Hungarian Academy of Sciences, Research Center for Mo-
lecular Medicine, Medical and Health Science Center, University of Debrecen, De-
brecen, Hungary;‡Institute for Biotechnology, Bay Zolta ´n Foundation for Applied
Research, Szeged, Hungary;§Medicinal Chemistry, Bayer Schering Pharma, Berlin,
Germany;¶Common Mechanism Research Berlin, Bayer Schering Pharma, Berlin,
Germany; and?Novartis Institutes for Biomedical Research, Vienna, Austria
Received for publication October 6, 2008. Accepted for publication December
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1The work is supported by grants RET-06/2004 and OTKA NK72730 (to L.N.). L.N.
is an International Scholar of the Howard Hughes Medical Institute and holds a
Wellcome Trust Senior Research Fellowship in Biomedical Sciences in Central Eu-
2The microarray data presented in this article have been submitted to the Gene Ex-
pression Omnibus (GEO) under GEO accession number GSE13762.
3Address correspondence and reprint requests to Dr. La ´szlo ´ Nagy, Department of
Biochemistry and Molecular Biology, University of Debrecen, Medical and Health
Science Center, H4012 Debrecen, Hungary. E-mail address: firstname.lastname@example.org
4Abbreviations used in this paper: DC, dendritic cell; IDC, immature DC; MDC,
mature DC; VDR, vitamin D receptor; 1,25-vitD, 1,25-dihydroxyvitamin D3; 25-vitD,
25-hydroxyvitamin D3; qPCR, real-time quantitative RT-PCR.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
function, differentiation and maturation of DCs are central is-
sues in DC biology (18–20). 1,25-vitD induces the tolerogenic
DC phenotype by various independent pathways or via a com-
bination of those pathways such as inhibition of differentiation
and/or maturation, interference with NF-?B signaling, or by
direct transcriptional events. It is an important issue to deter-
mine whether 1,25-vitD acts via inhibition of immunogenic
mechanisms or whether it acts autonomously. In particular, it is
not clear what is the interrelationship between the complex pro-
cesses of differentiation and the development of tolerance at the
This prompted us to investigate the impact of 1,25-vitD treat-
ment on the transcriptome of differentiating DCs. Our studies pre-
sented here suggest that ligand-bound VDR acts to a very large
degree autonomously, independently of the transcriptional changes
dictated by the differentiation and maturation program, leading to
a distinct tolerogenic phenotype.
Materials and Methods
Isolation and differentiation of DCs
CD14?monocytes and blood myeloid DCs were obtained from platelet-
free buffy coats from healthy donors by Ficoll gradient centrifugation fol-
lowed by immunomagnetic cell separation with anti-CD14-conjugated or
CD1c-conjugated microbeads, respectively (VarioMACS Separation Sys-
tem; Miltenyi Biotec). Blood myeloid DCs were cultured at a density of
3.5 ? 105cells/ml in RPMI 1640 (Sigma-Aldrich) supplemented with 10%
FBS (Invitrogen) and penicillin/streptomycin (Sigma-Aldrich). Monocytes
were cultured in multiwell culture plates or tissue flasks at a density of 106
cells/ml in RPMI 1640 supplemented with 10% FBS, 800 U/ml GM-CSF
(Leucomax; Gentaur Molecular Products), 500 U/ml IL-4 (PeproTech),
and penicillin/streptomycin. IL-4 and GM-CSF were replenished on day 3.
To obtain MDCs, the medium was supplemented with mixture of proin-
flammatory cytokines containing 10 ng/ml TNF-?, 10 ng/ml IL-1?, 1000
U/ml IL-6 (PeproTech), and 1 ?g/ml prostaglandin-E2(Sigma-Aldrich).
Immature DCs (IDCs) were also challenged with various TLR ligands: 100
ng/ml LPS (TLR4 ligand) (Sigma-Aldrich), 2 ?g/ml CL075 (TLR8/7 li-
gand) (InvivoGen), and 20 ?g/ml polyinosinic:polycytidylic acid (TLR3
ligand) (Sigma-Aldrich) for 18 h.
Ligand treatment of DCs
1,25-vitD (Biomol) was used at 10 nM and at 1 pM to 100 nM for dose-
response experiments, 25-hydroxyvitamin D3(25-vitD) (Biomol) was used
at 100 nM, and ZK159222 (Bayer Schering Pharma) was used at 1 ?M.
The vehicle (ethanol:DMSO at 1:1) had no detectable effect on the differ-
entiation (data not shown).
Microarray analysis: sample preparation, labeling, and
Monocytes differentiating into DCs were treated with 10 nM 1,25-vitD or
vehicle 14 h after plating. Cells were harvested 12 h or 5 days thereafter.
Total RNA from 6 ? 106cells was isolated using the RNeasy kit (Qiagen).
Experiments were performed in biological triplicates representing samples
from different donors. Further processing and labeling, hybridization to
GeneChip human genome U133 Plus 2.0 arrays (Affymetrix), and scanning
were conducted at the Microarray Core Facility of European Molecular
Biology Laboratory (Heidelberg, Germany). Microarray data have been
deposited into the Gene Expression Omnibus database under accession
Microarray data analysis
Image files were imported to GeneSpring 7.3 (Agilent). Raw signal intensities
were normalized per chip (to the 50th percentile) and per gene (to the median).
We removed probe sets that failed to reach a signal intensity of at least 200 in
three of 15 samples. To identify significantly regulated genes between two
or down-regulation by eliminating probe sets with a ratio of signal intensity
filtered for values of p ? 0.05 (samples were normalized to median or to
control samples). For heat map and scatter plot visualization of signal inten-
sities, each probe set was normalized to the signal intensities of vehicle con-
trols (fold change). The PANTHER (Protein Analysis through Evolutionary
jsp) was used for the functional classification of genes.
Real-time quantitative RT-PCR
Real-time quantitative RT-PCR (qPCR) was conducted as described earlier
(21, 22) using TaqMan probes (Applied Biosystems). For details see sup-
plemental Table I.5Gene expression was quantified by the comparative
cycle threshold (CT) method and normalized to cyclophilin A expression.
All experiments were conducted as biological triplicates. Values are ex-
pressed as mean ? SD of the mean.
Monocytes were plated at a density of 106cell/ml (total 6.5 ? 106cells/
sample), cultured as described, and treated with 100 nM 25-vitD. Cells
were harvested at days 1–6, washed, and stored at ?20°C. Pellets were
resuspended in saline and sonicated (Bioruptor; Diagenode) for 10 min to
achieve complete lysis. Lysates were cleared by centrifugation and the
1,25-vitD content of supernatant was concentrated by column chromatog-
raphy and measured by ELISA (1,25-(OH)2vitamin D ELISA kit; Immun-
diagnostik) as recommended by the manufacturer.
Monocytes were cultured in 12-well dishes in the presence of 1,25-vitD,
25-vitD, or vehicle as described earlier. CCL22 content of supernatants
was measured by sandwich ELISA specific for CCL22 (human MDC im-
munoassay; R&D Systems) as recommended by the manufacturer.
Surface expression of CD300LF (also known as IREM-1) was detected
with anti-IREM-1 UP-D2 mAb (a gift from M. Lo ´pez-Botet, Universitat
Pompeu Fabra, Barcelona, Spain). Isotype control anti-IgG1 mAb (R&D
Systems) was used as a negative control. FITC-conjugated polyclonal goat
anti-mouse Ig (DakoCytomation) was used as a secondary Ab. Cell surface
staining was measured with a FACSCalibur flow cytometer and analyzed
with the CellQuest software package (BD Pharmingen).
Western blot analysis
Cell lysates (25 ?g protein) were separated on a 8% SDS-polyacrylamide
gel and electroblotted onto a PVDF (Millipore) membrane. The membrane
was probed with a polyclonal Ab against CYP27B1 (1/5000; article no.
HYD001 from Biologo), stripped, reprobed with an Ab to VDR (1/7000;
C-20, sc-1008 from Santa Cruz Biotechnology), stripped again, and re-
probed with mouse anti-GAP3DH (clone 6C5) (1/5000; catalog no. ab8245
from Abcam). The Ag-Ab complexes were labeled with appropriate HRP-
conjugated secondary Abs (Sigma-Aldrich) and visualized by Immobilon
Western HRP substrate kit (Millipore).
Cells were collected by centrifugation, fixed in buffered formalin, and em-
bedded into paraffin. Sections (5 ?m) were immunostained with anti-
human VDR mAb (1/2000; clone H4537 from Perseus Proteomics) after
wet heat-induced Ag retrieval. The EnVision?-HRP system (Dako) was
used to visualize the labeling according to manufacturer’s instructions.
VDR is expressed early in developing monocyte-derived DCs
We isolated CD14?monocytes from the peripheral blood of
healthy donors and cultured them in the presence of GM-CSF and
IL-4. This represents a well-established model (23) in which we
and others have previously mapped nuclear hormone receptor-me-
diated transcriptional events (8, 9, 21, 22, 24).
First, we determined the kinetics of VDR expression. Mono-
cytes expressed VDR at low but detectable levels. Upon culturing
monocytes in GM-CSF and IL-4, transcription of the VDR gene
increased rapidly. The amount of VDR transcript detected peaked
after 18–24 h. Interestingly, the VDR transcription rate decreased
to lower levels in later phases of the differentiation process (Fig.
1A). Western blotting experiments showed that the VDR protein
level rapidly increased in the first few days and remained at a high
5The online version of this article contains supplemental material.
2075 The Journal of Immunology
level in later phases of the differentiation (Fig. 1B). The VDR
protein was located in the nuclei of IDCs as determined by
immunohistochemistry (Fig. 1A, insert). We thus demonstrated
that VDR is expressed rapidly and at high levels in
1,25-vitD directs the transcription of a large set of genes
independently of the differentiation program
The receptor expression peaks ?18 h after the induction of dif-
ferentiation; we therefore added 1,25-vitD or vehicle 14 h after
plating. Our goal was to assess both the early and the late tran-
scriptional changes caused by 1,25-vitD; we therefore harvested
differentiating DCs at 12 h or IDCs at 5 days after the addition of
ligand. CD14?monocytes served as reference. The transcriptomes
of these cells were studied using Affymetrix GeneChip Arrays. This
experimental setup (Fig. 1C) allowed us to compare the transcrip-
tomes of monocytes, differentiating DCs, and IDCs to define genes
that are differentially expressed during differentiation and upon 1,25-
vitD treatment at early (12 h) and late (5 day) time points.
We and others have already shown (22) that the differentiation of
myeloid cells from precursors will lead to differential expression of
several thousand genes. Consistent with this, we found 4,364 differ-
entially expressed probe sets (representing 2,766 genes) between the
transcriptomes of monocytes and IDCs. Comparing the transcrip-
tomes of 1,25-vitD-treated and vehicle-treated samples, we detected
899 and 1,384 differentially expressed probe sets (representing 578
and 918 genes) at 12 h and 5 days, respectively (Fig. 1, D and E).
If VDR ligands exert their effect mainly by inhibiting the differen-
tiation and maturation program, this would imply that 1,25-vitD tran-
scriptionally regulates part of the gene set that is also developmentally
regulated. Remarkably, 3,511 probe sets affected by differentiation
were not regulated by 1,25-vitD (Fig. 1E). Furthermore, only 853 of
also among the probe sets that were differentially expressed in mono-
cytes and IDCs (Fig. 1E and supplemental Fig. 1). These results thus
strongly suggest that 1,25-vitD should not be simply viewed as a
general and global inhibitor of differentiation.
1,25-vitD treatment leads to the regulation of many immunity
and defense genes in differentiating and IDCs
The tolerogenic phenotype is likely to be brought about by tran-
scriptional modulation of immune function-related genes. We used
the PANTHER classification system that utilizes an unbiased gene
ontology classification to assign function to the affected genes
(supplemental Table II.). We found 41 ? 63 ? 104 and 63 ? 86 ?
149 genes that fell into the functional category “immunity and
defense” regulated at the 12 h and 5 day time points, respectively
(Fig. 2A and supplemental Table III). Importantly, this category
was significantly over-represented among the functional classes
(supplemental Table II) at both time points.
We chose two approaches to validate these findings. First, we
selected a set of genes previously reported (4, 7–9, 13, 14, 25–27)
to be 1,25-vitD regulated in DCs with the caveat that in most
studies the effect of 1,25-vitD was not tested at the transcriptional
level. We investigated whether these genes were regulated by
1,25-vitD in our experimental model at the mRNA level. The ma-
jority was indeed regulated as expected (Fig. 2B), with the notable
exceptions of IL-10, IL-12, and mannose receptor C type 1 (no
regulation) and CD86 and CCL-18 (opposite regulation). It is
likely that 1,25-vitD can modulate the expression of IL-10 and
IL-12 only during maturation. We also looked at the direct VDR
target genes (28–31) falling into the immunity and defense cate-
gory. As anticipated, all direct VDR targets expressed in this cell
type were regulated, most of them at both time points (Fig. 2C).
Second, we also validated our microarray results on selected tar-
gets (choosing genes expressed at low and high levels) using
qPCR. We found that the results of the two independent methods
for measuring gene expression at the transcriptional level showed
good agreement (Fig. 2D). The validation of our expression data
thus gave us confidence to further investigate the relationship of
differentiation and 1,25-vitD treatment at the transcriptional level.
DCs and directs the transcription of a large set of genes independently of
the differentiation program. A, Expression of VDR in differentiating and
maturing human monocyte-derived DCs as determined by qPCR (Mo,
monocyte). To obtain MDCs, cells were treated with a mixture of proin-
flammatory cytokines. VDR is strongly expressed and is localized to the
nucleus in IDCs as demonstrated by immunohistochemical staining. B,
VDR protein is accumulated in an early phase during differentiation as
shown by Western blotting analysis (A, inset). C, CD14?monocytes were
isolated from peripheral blood and cultured in the presence of IL-4 and
GM-CSF. Fourteen hours after setting up the culture, the differentiating
cells were treated with 10 nM 1,25-vitD or vehicle for 12 h or 5 days (d).
The transcriptomes of monocytes, differentiating DCs (difDC), or 5-day
IDCs treated with 10 nM 1,25-vitD or vehicle were analyzed by Affymetrix
microarrays. D, Probe sets regulated by 1,25-vitD treatment in differenti-
ating DCs (12 h) and IDCs (5 days) were identified and compared. The
results are visualized as a Venn diagram. The regulated probe sets were cat-
egorized as early (regulated at 12 h only), sustained (regulated both at 12 h and
are regulated already at 12 h. E, Probe sets differentially expressed during the
monocyte to IDC differentiation as well as probe sets regulated by the 5-day
1,25-vitD treatment were identified and compared. The results are visualized
as a Venn diagram. Note the significant number of probe sets regulated by
1,25-vitD, but not the differentiation process itself.
VDR is expressed early in developing monocyte-derived
2076 VITAMIN D ACTS AUTONOMOUSLY IN DCs
1,25-vitD and differentiation regulated immunity and defense
gene sets only partially overlap
The suggestion that VDR ligands inhibit the differentiation and
maturation program comes from the investigation of individual
immunity and defense genes (CD1A, CD14, etc.). No system-level
analysis involving hundreds of genes was ever conducted to in-
vestigate this issue. To reveal the relationship of the effects of
1,25-vitD and differentiation on immunity and defense genes we
first derived and compared the gene sets regulated by 1,25-vitD
and/or differentiation belonging to this category (Fig. 3A). This
comparison proved that 1,25-vitD and the differentiation-regulated
immunity and defense gene sets only partially overlap, similarly as
the entire gene sets (Fig. 1E). To get a more complex view, we also
determined the ratios of gene expression in DCs vs monocytes
(differentiation effect) and 1,25-vitD-treated vs vehicle-treated
DCs (1,25-vitD effect) and plotted these for both the early (sup-
plemental Fig. 2) and late (Fig. 3B) time points. The majority of
genes (clusters 1 and 6) showed opposite regulation during differ-
entiation and upon 1,25-vitD treatment, as one could have pre-
dicted. Importantly, we also found a smaller number of genes
where the effect of differentiation and 1,25-vitD treatment pointed
to the same direction (clusters 3 and 4). In addition, many 1,25-
vitD-regulated genes were not affected by differentiation at all
(clusters 2 and 5,) providing evidence to suggest that the differ-
entiation program and the 1,25-vitD-induced program are, indeed,
Many early immunity and defense genes are autonomously
regulated in differentiating DCs and blood myeloid DCs
Interestingly, the expression profile of arachidonate 5-lipoxygen-
ase (ALOX5; in cluster 6 in Fig. 3) suggested that genes showing
opposite regulation during differentiation and upon 1,25-vitD treat-
ment may also be regulated independently of differentiation. The
fact that ALOX5 is a direct VDR target (31) made it likely that the
1,25-vitD treatment altered its expression directly. This suggests
that 1,25-vitD and differentiation can regulate the expression of
certain genes independently of one another. Autonomous regula-
tion by 1,25-vitD would thus imply that 1,25-vitD changes the rate
of transcription of its target genes independently of the differen-
tiation state of DCs. To test this hypothesis, we chose a number of
genes that were up-regulated by 1,25-vitD at 12 h and inhibited
during differentiation (cluster 6; Fig. 3) and tested whether they
can be up-regulated in a later phase of DC differentiation by 1,25-
vitD. We treated monocytes with GM-CSF and IL-4 for 4 days and
then added 10 nM 1,25-vitD or vehicle for an additional 24 h and
determined gene expression by qPCR. Our results showed that
1,25-vitD treatment led to the induction of CD14, THBD,
CD300LF, and ALOX5 but not IRF8 (Fig. 4A), suggesting that a
large fraction of genes is indeed regulated autonomously by
To test whether the genes regulated oppositely by differentiation
and the ligand (clusters 1 and 6; Fig. 3) can also be regulated
autonomously by 1,25-vitD, we aimed to validate our results in a
belonging to the early, sustained, and late groups were functionally categorized using the PANTHER classification system. Genes falling to the
functional category immunity and defense are shown as a Venn diagram and in heat maps. In the heat maps the columns represent expression profiles
of independent donors for the two time points studied (12 h and 5 days (d)). Color intensities reflect the ratios of signal intensities as shown. B,
Known 1,25-vitD-regulated immunity and defense genes in DCs as observed by our microarray analysis shown as a heat map. Note that in the vast
majority of cases our results were concordant with the previously published literature. Exceptions are shown with asterisks. C, The effect of 12-h
and 5-day 1,25-vitD treatment on the expression of immunity and defense direct VDR target genes regulated by well-characterized positive and
negative vitamin D response elements shown as a heat map. D, Validation of the microarray results (on the left) by qPCR (on the right) on a list
of genes connected to immunity and defense properties of DC. Bars show the signal intensities of the mean of biological triplicates for vehicle-treated
and 1,25-vitD-treated samples. Note that the axis showing the qPCR signal intensities is twice broken. Fold changes (1,25-vitD-treated/vehicle-
treated) are indicated by numbers next to the bars.
1,25-vitD treatment leads to the regulation of many immunity and defense genes in differentiating and immature DCs. A, Genes
2077 The Journal of Immunology
distinct ex vivo DC type. We isolated myeloid blood DCs from the
peripheral blood of healthy human donors and cultured them in the
absence of cytokines and in the presence of 1,25-vitD for 24 h. Our
qPCR experiments showed that 11 of the 12 studied genes showed
similar regulation in blood DCs and ex vivo differentiating DCs
(Fig. 4B), providing further evidence that many genes are regu-
lated by 1,25-vitD treatment independently of differentiation
Characterization of 1,25-vitD-dependent regulation of CYP24A1,
CCL22, and CD300LF
We selected three genes induced by 1,25-vitD but differentially
regulated by differentiation for further characterization: 24-hy-
droxylase (CYP24A1; a direct target of VDR) (32); CCL22 (a
chemokine attracting regulatory T cells) (14, 33); and CD300LF
(an inhibitory receptor also known as IREM-1) (34). These three
genes were all shown to be regulated by 1,25-vitD treatment by our
expression study but were either up-regulated (CCL22), not regu-
lated (CYPP24A1), or down-regulated (CD300LF) during differ-
entiation (Fig. 3), therefore representing different clusters in our
We first determined the time courses of the expression of the
three genes using qPCR. CYP24A1, CCL22, and CD300LF were
induced as early as 3 h after the start of 1,25-vitD treatment, and
the expression of all three genes remained up-regulated after 6, 12,
and 24 h of treatment (Fig. 5A).
The early and sustained up-regulation of CYP24A1, CCL22,
and CD300LF suggested a direct regulation by ligand-bound VDR.
To show that the effect of 1,25-vitD is indeed mediated through
VDR, we used ZK159222, a partial VDR antagonist (35), and
demonstrated that treatment with ZK159222 significantly re-
pressed the 1,25-vitD-elicited transcriptional response of all three
genes (Fig. 5B).
Next, we determined dose-response curves of CYP24A1,
CCL22, and CD300LF gene expression upon 1,25-vitD treat-
ment and calculated the EC50values for 1,25-vitD. The EC50
value for 1,25-vitD for CD300LF was very similar to that of
CYP24A1 (2–3 nM) whereas the EC50of CCL22 was an order
of magnitude lower, suggesting that this gene is more sensitive
to VDR activation (Fig. 5C).
We next reasoned that if the effect of 1,25-vitD is really inde-
pendent of differentiation, it should induce these three genes at
different time points during the monocyte to IDC differentiation
program and also during the maturation of IDC to MDC provoked
by a mixture of proinflammatory cytokines. The obtained data sup-
ported our hypothesis as shown in Fig. 5D. However, CCL22 was
lated immunity and defense gene-sets only partially
overlap. A, Annotated genes of the immunity and
defense category differentially expressed during the
Mo to IDC differentiation as well as probe sets reg-
ulated by the 5-day (d) 1,25-vitD treatment were
identified and compared. The results are visualized
as a Venn diagram. B, For each probe set represent-
ing an immunity and defense gene, the ratios of tran-
script levels in 5-day IDC vs monocyte (effect of
5-day differentiation) were determined. Similarly,
the ratios of transcript levels of 5-day 1,25-vitD-
treated IDC vs vehicle-treated DCs (effect of 5 day
1,25-vitD) were calculated. The two ratios of tran-
script levels were plotted against each other, result-
ing in a scatter plot that shows the relationship of
transcriptional changes caused by the differentiation
process and the 1,25-vitD treatment. Genes relevant
for this study are marked. Genes already regulated at
the 12-h time point are marked in bold. Known di-
rect VDR targets are shown with asterisks (?). For
clarity only one HLA gene is indicated and the
probe sets not affected by the 1,25-vitD treatment
are omitted from the scatter plot.
1,25-vitD and differentiation regu-
2078 VITAMIN D ACTS AUTONOMOUSLY IN DCs
not induced significantly in MDCs, probably due to its already
high expression level.
Endogenously produced 1,25-vitD regulates the expression of
CYP24A1, CCL22, and CD300LF
The physiological serum levels of 1,25-vitD (?40–130 pM) (36)
are unlikely to be sufficient to turn on 1,25-vitD signaling in DCs
(Fig. 5C). However, previous studies revealed that 1,25-vitD can
be generated endogenously (37–40). We therefore sought to de-
termine whether and when the ?1,000 times more abundant (36,
41, 42) precursor, 25-hydroxyvitamin D3(25-vitD), is actively
converted to 1,25-vitD in DCs. The hydroxylation step of the con-
version process of inactive 25-vitD to 1,25-vitD is catalyzed by
CYP27B1, a cytochrome p450 hydroxylase (43, 44). We therefore
investigated the expression pattern of CYP27B1 by qPCR and
found that it closely matched that of the VDR (Fig. 6A). We also
investigated the expression of CYP27B1 at the protein level by
Western blotting and found that the protein accumulated during the
monocyte to IDC differentiation process (Fig. 6B).
regulated in differentiating DCs and blood myeloid DCs. A, 1,25-vitD changes
the expression levels of its target genes independently of the differentiation
state of DCs. Monocytes (Mo) were cultured with GM-CSF and IL-4 for 4
days (d), and then differentiating DCs were treated with 10 nM 1,25-vitD or
vehicle for 24 h. The expressions of selected immunity and defense genes,
which are oppositely regulated by differentiation and 1,25-vitD treatment
(cluster 6; Fig. 3) were measured by qPCR. B, The 1,25-vitD sensitivity of
selected immunity and defense genes was verified in monocyte-derived DCs
and blood myeloid DCs by qPCR. Genes tested included a selected panel of
by differentiation and 1,25-vitD treatment (clusters 1 and 6; Fig. 3) and CCL22
and CYP24A1 (belonging to clusters 4 and 5, respectively). Gene expression
levels were measured by qPCR. Bars show the mRNA levels of the indicated
genes for vehicle-treated and 1,25-vitD-treated samples. Note that the axes
showing the qPCR signal intensities are each twice broken to accommodate
the large range of expression levels. If applicable, fold changes (1,25-vitD-
treated/vehicle-treated) are indicated by numbers next to the bars (NA, not
Many early immunity and defense genes are autonomously
CYP24A1, CCL22, and CD300LF. CD14?monocytes were isolated
from peripheral blood and cultured in the presence of IL-4 and GM-
CSF. Differentiating cells were treated with 1,25-vitD (VD), VDR an-
tagonist ZK159222 (ZK), or vehicle (Veh) 14 h after setting up the
culture (A–C) or as shown (D). Expression of CYP24A1, CCL22, and
CD300LF was determined by qPCR. A, CYP24A1, CCL22, and
CD300LF expression of differentiating DCs treated with 10 nM 1,25-
vitD for various times. B, The effect of 10 nM 1,25-vitD, 1 ?M
ZK159222, or the combination of them on the transcription of
CYP24A1, CCL22, and CD300LF after 12 h. Vehicle was used as a
negative control. C, CYP24A1, CCL22, and CD300LF expression after
a 12-h treatment with varying concentrations of 1,25-vitD. EC50values
for 1,25-vitD are indicated. D, 1,25-vitD sensitivity is retained during
differentiation and partly during maturation. Effects of 24-h ligand
treatment were tested by adding 1,25-vitD at subsequent days from day
0 (monocyte) through day 5 and harvested 24 h later. The day (d) of
harvest is indicated on the graphs. The 5-day IDCs were treated with
mixture of proinflammatory cytokines (TNF-?, IL-1?, IL-6, and PGE2)
Characterization of 1,25-vitD-dependent regulation of
2079 The Journal of Immunology
Maturation of DCs is induced by many different stimuli (1, 2),
including proinflammatory cytokines and TLR ligands. We were
interested to see how these various stimuli influence the expression
of the receptor and the ligand-producing enzyme. Consistent with
previous reports (37, 39), we found that proinflammatory cyto-
kines and TLR ligands proved to be potent activators of CYP27B1
expression. Interestingly, LPS and the TLR8/7 ligand CL075
also induced VDR expression, suggesting that upon specific
maturation stimuli, VDR and CYP27B1 are likely to be coregu-
lated (Fig. 6C).
To test whether 25-vitD may indeed be actively converted by
differentiating DCs to the active form, we cultured differenti-
ating DCs in the presence of 100 nM 25-vitD and measured the
concentration of the produced 1,25-vitD by ELISA (37). We
could detect increasing amounts of 1,25-vitD in differentiating
DCs (Fig. 6D).
Consistent with a previous report (39) we found that endog-
enously produced 1,25-vitD was effective in regulating key mark-
ers such as CD14, CD1A, and HLA-DR in IDCs and CD83 and
HLA-DR in MDCs (supplemental Fig. 3.).
We then investigatedwhether
CD300LF are induced by not only 1,25-vitD but also by 25-
vitD treatment. As expected 25-vitD treatment resulted in in-
creased transcription of all three genes, showing that the cells con-
verted 25-vitD in a sufficient amount to induce these genes (Fig.
6E). We also demonstrated by ELISA that transcriptional up-reg-
ulation of CCL22 in both 1,25-vitD- and 25-vitD-treated cells re-
sults in a higher concentration of secreted CCL22 by day 5 (Fig.
6F). Similarly, increased CD300LF transcription was manifested
as increased cell surface expression of CD300LF on 1,25-vitD-
and 25-vitD-treated cells as determined by flow cytometry (Fig.
6G). Collectively, these data showed that the endogenously
shows similar kinetics in differentiating human monocyte (Mo)-derived DCs as determined by qPCR. B, CYP27B1 protein is accumulated in an early phase
during differentiation as shown by Western blot analysis. C, VDR and CYP27B1 gene expression is induced by a partially overlapping set of stimuli in
human monocyte-derived DCs. (Stimuli were as follows: LPS; TNF-?; a mixture of the proinflammatory cytokines TNF-?, IL-1?, IL-6, and PGE2;
polyinosinic:polycytidylic acid (polyI:C); and CL075, a TLR8/7 agonist.) D, DCs were incubated with 100 nM 25-vitD and the produced 1,25-vitD content
of the cells was measured by ELISA. For positive and negative controls, cells were incubated for 24 h with 10 nM 1,25-vitD and vehicle, respectively. ND
(not detectable) indicates that 1,25-vitD concentration was under the detection limit. E, Cells were cultured with 10 nM 1,25-vitD or 100 nM 25-vitD for
various times. MDCs were generated with a mixture of proinflammatory cytokines. The expressions of CYP24A1, CCL22, and CD300LF in differentiating,
immature and mature DCs were determined by qPCR. F, Elevated level of secreted CCL22 was detected by ELISA in supernatants of IDCs differentiated
from monocytes obtained from five different donors after a 5-day (d) 10 nM 1,25-vitD or 100 nM 25-vitD treatment. G, Up-regulation of CD300LF (gray
histograms) in the presence of 1,25-vitD and 25-vitD was confirmed by flow cytometry. An irrelevant isotype-matched Ig was used as a negative control
(shown by white histograms). One representative experiment of four performed is shown.
Endogenously produced 1,25-vitD regulates the expression of CYP24A1, CCL22, and CD300LF. A, Transcription of VDR and CYP27B1
2080 VITAMIN D ACTS AUTONOMOUSLY IN DCs
produced 1,25-vitD appeared to be sufficient to regulate the iden-
Complex patterns of gene expression as determined by microarray
analysis have been previously used to map interactions between
biological processes. These investigations revealed new aspects in
the regulation of immune functions by nuclear hormone receptors.
Glucocorticoids acting through a glucocorticoid receptor direct
monocyte-derived macrophage differentiation toward an anti-
inflammatory type macrophage (45). Likewise the primary role of
peroxisome proliferator-activated receptor-? in regulating lipid
metabolism was established in monocyte-derived DCs (22). In this
article we provide an analysis of the 1,25-vitD-induced changes in
differentiating DCs and establish that this receptor regulates the
tolerogenic program largely autonomously, e.g., independently of
differentiation and maturation.
An ex vivo model of in vivo DC development
The in vivo relevance of 1,25-vitD signaling is clearly demon-
strated by studies on VDR?/?mice (6). In this study we deter-
mined the transcriptional targets of 1,25-vitD in ex vivo differen-
tiating primary human DCs. The combined treatment of CD14?
monocytes with GM-CSF and IL-4 in vitro (8, 9, 21–24) results in
a nonproliferating and very homogenous population of cells, an
ideal subject of transcriptome analysis. These cells have DC mor-
phology and share functional characteristics for IDCs. Although a
recent study documented that Langerhans cells arise from mono-
cytes in vivo during inflammation (46), we need to acknowledge
that it is not known to what extent ex vivo differentiation of mono-
cyte-derived DCs recapitulates the in vivo differentiation of DCs.
Nonetheless, monocyte-derived DCs are successfully introduced in
clinical studies (47, 48), underscoring the in vivo relevance of the
cell type of our choice. Our model and experimental approach
were further validated by the concordance of data from monocyte-
derived DCs and blood myeloid DCs (Fig. 4B).
A key issue in analyzing the activity of a nuclear hormone re-
ceptor is the source of the endogenous ligand. Previous studies
documented that 1,25-vitD can be generated in DCs, particularly
after maturation induced by LPS and other maturation stimuli (37,
39). We demonstrated here that 25-vitD is converted to 1,25-vitD
even in differentiating DCs, and the produced ligand appeared to
be sufficient to regulate the identified program (Fig. 6). Further
studies are needed to clarify whether the polarization of DCs to a
more tolerogenic direction by 1,25-vitD or its precursor(s) may
occur during differentiation or whether it is restricted to the mat-
uration phase in vivo.
1,25-vitD treatment leads to the transcriptional regulation of
many genes implicated in the tolerogenic phenotype of DCs
By using monocyte-derived DCs, we determined the transcrip-
tional targets of 1,25-vitD treatment by microarrays. Our aim was
not to characterize the potential targets in detail; that will be the
1,25-vitD-regulated clusters of genes implicated in the tolerogenic phenotype (leukocyte Ig-like receptor B (LILRB) inhibitory receptors, HLA, and CD1
molecules) identified by microarray analysis. The heat maps show the expression levels of the indicated genes from three donors at 12 h and 5 days (d).
The chromosomal locations of the genes are also indicated. The effect of 1,25-vitD is only partially mediated by suppression of target genes (e.g., HLA,
CD1, and costimulatory molecules), and the induction of target genes (inhibitory receptors, chemokines, and cytokines) is likely more important than
previously thought to achieve the tolerogenic phenotype. B, Previous studies investigated the 1,25-vitD effect in developmental context and documented
that 1,25-vitD leads to transcriptional changes that are opposite to those brought upon by differentiation and maturation. RXR, Retinoid X receptor. C, Our
data suggest a complex role for 1,25-vitD in inducing the tolerogenic phenotype. The majority of genes changing during differentiation are not targets of
1,25-vitD regulation (a). A fraction of genes are likely to be regulated, especially in maturing DCs, as suggested earlier via inhibition of other signaling
pathways by controlling master regulators, antagonizing the action of other transcription factors (b). A significant fraction of 1,25-vitD-regulated genes
appear to be regulated independently by 1,25-vitD and the differentiation program, even if the effect of 1,25-vitD and the differentiation programs are
opposite (c). There are several 1,25-vitD-regulated genes not affected during differentiation (d).
The proposed model of how exogenous or endogenously produced 1,25-vitD regulates gene expression in developing DCs is shown. A,
2081 The Journal of Immunology
subject of future studies. However, by studying the 1,25-vitD-reg-
ulated gene set we made two noteworthy observations. First, our
microarray data suggest that up-regulation of target genes appears
to be more prevalent for the tolerogenic phenotype than was pre-
viously thought (Fig. 7A). These changes include the induction of
inhibitory receptors and secreted cytokines and chemokines. This
observation is consistent with a recent report (45) on the role of
the glucocorticoid receptor in macrophage differentiation,
where the authors provided evidence to question the long-held the-
ory that the immunosuppressive glucocorticoid action is primarily
mediated as transrepression of inflammatory genes. Second, many
1,25-vitD-regulated genes with similar functions appear to be co-
ordinately controlled or coregulated (e.g., Ag presentation, co-
stimulation, cytokines, and chemokines contributing to the en-
hancement of regulatory T cells and inhibitory receptors).
Interestingly, some of these genes form clusters or are located on
the same chromosomal region in the genome (e.g., MHC class II,
CD1, and LILRB clusters) (Fig. 7A). These data suggest that entire
gene clusters or even large genomic regions may be coregulated by
1,25-vitD-bound VDR. Deciphering the molecular background of
this phenomenon requires further work.
1,25-vitD initiates an autonomous transcriptional program
Previous studies documented that 1,25-vitD treatment suppressed
the induction of DC differentiation and maturation markers
(CD1A, MHC class II molecules, CD83, costimulatory molecules,
etc.) and suppressed the down-regulation of the monocyte marker
CD14 (7–10). If 1,25-vitD mainly acted through the inhibition of
the differentiation and the maturation program, it would most
likely act through suppressing/antagonizing the effect of transcrip-
tion factors driving DC differentiation and maturation. In this way,
the sets of genes regulated by differentiation and 1,25-vitD would
overlap to a very large degree. Our data, however, do not support
the scenario that the effect of 1,25-vitD is mostly restricted to the
transcriptional regulation of “master transcription factors” or an-
tagonism of transcription factors activated during maturation.
Our comparative analysis of the transcriptomes of monocytes,
differentiating DCs, and IDCs differentiated in the presence or ab-
sence of 1,25-vitD suggest that the 1,25-vitD-elicited transcrip-
tional program is an autonomous one that runs parallel or as a
module with the differentiation and/or maturation transcriptional
program as soon as cells become 1,25-vitD responsive and are
exposed to the ligand. Several lines of evidence presented in this
study support this claim.
First, the set of genes regulated by differentiation and by 1,25-
vitD overlap only partially, e.g., there are many genes that are
regulated by 1,25-vitD, but not the differentiation and vice versa
(Fig. 1E). If the effect of 1,25-vitD were mediated through the
suppression of the differentiation program, we would expect that
1,25-vitD mostly regulates a subset of the genes regulated by the
differentiation process. Our data did not support this hypothesis.
Furthermore, this is not only true for the whole regulated gene set,
but also for the immunity and defense subset (Fig. 3A).
Second, the set of genes regulated both by the differentiation and
by 1,25-vitD are not necessarily regulated in opposite manner. If
1,25-vitD acted through suppression of the differentiation program,
it would regulate gene expression into the opposite direction than
differentiation. A large fraction of the genes are indeed regulated in
opposing directions by the two programs (e.g., CD1A and CD14)
as predicted by the earlier results, but another significant fraction
(clusters 3 and 4; Fig. 3B) did not comply with this rule.
Third, the set of genes regulated in opposite directions by the
differentiation and by 1,25-vitD (clusters 1 and 6; Fig. 3B) contains
genes that can be autonomously regulated by 1,25-vitD. We
showed that many genes that are up-regulated early by 1,25-vitD
are also up-regulated by late application of the hormone (Fig. 4A).
The developmental context therefore did not prove to be essential
for most investigated genes. Very importantly, the differentiation
independent regulation was also demonstrated in blood myeloid
DCs for several genes (Fig. 4B). A more detailed characteriza-
tion of CD300LF, a gene oppositely regulated by differentiation
and 1,25-vitD, showed that this gene is regulated similarly as
CYP24A1 and CCL22 (Fig. 5).
Finally, 1,25-vitD is capable of initiating the 1,25-vitD-depen-
dent transcription program in the absence of maturation signals.
There are several inflammatory stimuli, including LPS and many
proinflammatory cytokines, that trigger DC maturation. 1,25-vitD
has been reported to inhibit this maturation. 1,25-vitD is indeed
documented to antagonize the “inflammatory” transcription fac-
tors, like NFAT/AP-1 and NF-?B, that results in inhibited expres-
sion of IL-2 (49) and IL-12 (50), respectively. Yet, in our system
1,25-vitD could regulate almost 200 immunity and defense genes
(Fig. 2A), including many previously identified targets (Fig. 2, B
and C) in IDCs, demonstrating that the 1,25-vitD-induced tran-
scriptional program can be initiated in the absence of (inflamma-
These arguments collectively imply a more complex role for
1,25-vitD in the regulation of transcriptional targets in DCs than
was previously thought (Fig. 7, B and C). According to our data,
?80% of probe sets representing genes playing a role in IDC dif-
ferentiation are not targets of 1,25-vitD regulation and 40% of
probe sets regulated by 1,25-vitD are not affected by differentiation
(Fig. 1E). A fraction of genes is likely regulated, especially in
maturing DCs, as suggested earlier via inhibition of other signaling
pathways. A significant fraction of genes is likely to be regulated
independently by 1,25-vitD and differentiation, even if the effects
of 1,25-vitD and the differentiation program are opposite.
Several lines of evidence suggest that IDCs can give rise to
distinct types of MDCs, depending on stimuli from the environ-
ment and/or other cell types to become tolerogenic or immuno-
genic. Our interpretation of the presented data is that 1,25-vitD
initiates an autonomous transcriptional program that is to a large
part independent of differentiation and maturation. These findings
also let us propose that the tolerogenic phenotype is the result of an
active process and is unlikely to be a consequence of the inhibition
of differentiation and maturation. Thus, an independent DC differ-
entiation/maturation program could be complemented by either of
two competing transcriptional programs, an immunogenic one ini-
tiated by TLR-receptors, proinflammatory cytokines or other im-
munogenic signals and a tolerogenic one initiated by tolerogenic
signals, including 1,25-vitD or other immunosuppressive agents.
This finding is providing further support to the recent shift in par-
adigm concerning tolerogenic and immunogenic DCs.
We are indebted to Ibolya Fu ¨rto ¨s for outstanding technical help, Prof.
Miguel Lo ´pez-Botet for the IREM-1 (CD300LF) Ab, Sa ´ndor Sze ´les and
Zolta ´n Magyarics for help with data analyses, Drs. Lloyd Sparks, Attila
Ba ´csi, Ma ´te ´ Deme ´ny, and members of the Nagy Laboratory for discussions
and comments on the manuscript.
The authors have no financial conflict of interest.
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2083 The Journal of Immunology